the 2nd symposium of the canadian society for …...the 2nd symposium of the canadian society for...

CSV2018:The2ndSymposiumofthe

CanadianSocietyforVirology

June13-15,2018DalhousieUniversity

SirCharlesTupperMedicalBuilding5850CollegeStreet

Halifax,NovaScotia,Canada

The2ndSymposiumoftheCanadianSocietyforVirology page1


ORGANIZERS Dr.NathalieGrandvauxUniversitédeMontréalandCRCHUM

Dr.CraigMcCormickDalhousieUniversity

SCIENTIFICADVISORYCOMMITTEE

Dr.NathalieGrandvauxUniversitédeMontréalandCRCHUM

Dr.MatthiasGötteUniversityofAlberta

Dr.CraigMcCormickDalhousieUniversity

Dr.KarenMaxwellUniversityofToronto

Dr.MarcelineCôtéUniversityofOttawa

Dr.NellyPanteUniversityofBritishColumbia

Dr.DarrylFalzaranoVIDO-InterVac

Dr.AndrewWhiteYorkUniversity

LOCALORGANIZINGCOMMITTEE Dr.CraigMcCormickDalhousieUniversity

Dr.ToddHatchetteNovaScotiaHealthAuthority&DalhousieUniversity

Dr.LisaBarrettNovaScotiaHealthAuthority

Dr.AlysonKelvinDalhousieUniversity

Dr.JeanetteBoudreauDalhousieUniversity

Dr.DenysKhaperskyyDalhousieUniversity

Dr.JennCorcoranDalhousieUniversity

Dr.JasonLeBlancNovaScotiaHealthAuthority&DalhousieUniversity

Dr.RoyDuncanDalhousieUniversity

Dr.ChrisRichardsonDalhousieUniversity

Dr.MayEl-SherifNovaScotiaHealthAuthority&DalhousieUniversity

Dr.RodRussellMemorialUniversity

STUDENTCOMMITTEE(DalhousieUniversity) QuinelleBoudreauKatrinaBouzanisBethCastleBeccaChilversBrettDuguayLucasJarcheDenysKhaperskyy(lead)MarielKleerGrantMacNeilAndreaMonjo

RoryMulloyJacobNearingCarolynRobinsonEricPringleJacobSicheriGillSinghPatrickSlaineIonaStylianidesMackenzieThornbury


MAPS:Halifax&Carletoncampus


SYMPOSIUMPROGRAM

Wednesday,June13th15:00 Registrationandrefreshments(TupperLink,Ol'SchoolDonutBus(TupperCourtyard))

Postersetup(CHEB170)

17:00-17:20 Welcomeremarks(TheatreB) CraigMcCormick(DalhousieU)

CSVco-founder CSV2018co-organizer

17:20-18:00 KeynoteLecturesponsoredbyCIHR(TheatreB)DavidKelvin(DalhousieU.)“1918to2018,OneHundredYearsonthePandemicRoad”

18:00-18:40 KeynoteLecturesponsoredbyDMRF(TheatreB)SylvainMoineau(U.Laval)“CRISPR-CasSystemsandPhages:TheOngoingBattle”

19:00-22:00 PosterSession#1–posters#1to72(CHEB170)WineandCheese,seafoodandantipastoservedduringpostersession

Thursday,June14th6:30 Student-ledFunRun DepartingfromLeMarchantPlaceandLordNelsonHotel,touringPointPleasantPark

8:00-9:00 MeetthePIs-Breakfast(CHEB170)

9:00-10:35 Topic#1:EmergingViruses(TheatreB)SessionChair:DarrylFalzarano(VIDO-InterVac)

9:00 VikramMisra(U.ofSaskatchewan) “Bats:Thesourceofthenextpandemicorcurefordeadlyviraldiseases”

9:25 MichaelDrebot(PublicHealthAgencyofCanada)“Fromthefieldtothelabandbackagain:On-goingLassavirusresearchatNML”

9:50 MdNiazRahim(U.ofManitoba)“Pan-filovirusT-cellvaccineprovidedcompleteprotectionagainstEbolaandMarburgviruslethalchallenges”

10:05 CorinaWarkentin(U.ofOttawa)“Inhibitionofreceptortyrosinekinasesignalingblocksentryoffiloviruses”

10:20 NedaBarjesteh(McMasterU.)“ElucidatingtheroleofviralinfectiononthepathogenesisofAmyotrophicLateralSclerosis”

10:35 HealthBreak(TupperLink)

11:00-12:35 Topic#2:ViralSubversionofHostCellProcesses(TheatreB) SessionChair:NellyPante(UBC)

11:00 BruceBanfield(Queen’sU.) “EarlyStagesintheMorphogenesisofHerpesSimplexVirus”11:25 MayaShmulevitz(U.ofAlberta) “Niche-to-nichevariationsinreovirus-hostinteractions”11:50 NicholeMcMullen(DalhousieU.) “Unconventionalreleaseoffusogenicnonenvelopedreoviruses”

12:05 JustineSitz(U.Laval)“InteractionofhumanpapillomavirusE7withDNArepair-specificE3ligaseRNF168”


12:20 QuentinOsseman(U.deMontréal)“RegulationofRespiratorySyncytialVirusinfectionbytheautophagyreceptorSQSTM1/p62inairwayepithelialcells”

12:45-13:45 CSVBusinessMeeting(TheatreB) Or

WritingWorkshop(TheatreA)Co-sponsoredby:PLoSPathogensandPLoSONECoordinatedby: EileenClancy,PLoSONESeniorEditor

KarenMossman,PLoSPathogensEditor

Contributor: AlanDove,Sciencewriter

Boxluncheswillbeavailable

14:00-15:10 Topic#3:VirusesofMicrobes(TheatreB) SessionChair:KarenMaxwell(U.ofToronto)

14:00 KarenMaxwell(U.ofToronto)“Achemicaldefenceagainstphageinfection”

14:25 AlexanderHynes(McMasterU.) “FollowingPhagesthroughFecalTransplants”14:40 CaseyJones(DalhousieU.)

“ThegutviromeofpediatricCrohn’sDiseasefollowingexclusiveenteralnutrition”14:55 JaclynMcCutcheon(U.ofAlberta)

“TypeIVpili-specificbacteriophagesofStenotrophomonasmaltophilia”15:10 NikhilGeorge(U.ofWaterloo)

“Examiningviralpopulations,CRISPR-resolvedvirus-hostinteractions,andCRISPR-Cassystemdiversityinamunicipallandfill”

15:50 HealthBreak(TupperLink)16:15-17:25 Topic#4:AntiviralsandVaccines(TheatreB) SessionChair:MatthiasGötte(U.ofAlberta)

16:15 3-MinuteThesispresentationsfromtrainees:1. AliZhang(McMasterU.)

“NeuraminidaseInhibitorsEnhanceFc-DependentEffectorFunctionsElicitedbyHemagglutininStalk-BindingBroadly-NeutralizingAntibodies”2. BritiSaha(UofOttawa)“Identificationofpharmacologicaltargetsandsmallmoleculesinhibitingadenovirusreplication”3. MarielKleer(DalhousieU.)

“CharacterizationofaNovelHost-TargetedAntiviralMolecule”4. AndreaMonjo(DalhousieU.)“PhotodynamicInactivationofHerpesSimplexViruses”

16:30 PanelDiscussiononthefutureofvaccinesandantiviraldrugs(TheatreB)PanelMembers:LisaBarrett(NovaScotiaHealthAuthority),MarianneStanford(VPResearch,Immunovaccine),MarcelineCôté(U.ofOttawa),AlysonKelvin(DalhousieU.),DavidWiller(GlobalScientificAffairsLead-Zoster,GSK)


17:25 SoapboxScienceLaunchEvent(TupperCourtyard) Dr Maya Shmulevitz, University of Alberta

“Refurbishing viruses to seek, reveal, and destroy cancer cells” Dr Sarah M. Wells (@sarahwellsDal), Dalhousie University “Engineering replacements for the body” Dr Krysta Coyle (@microbialkrysta), Dalhousie University “Stacking the Deck for Cancer Treatment” Dr Alyson A. Kelvin (@KelvinAly), IWK Health Centre and Dalhousie University “Virus Hunter – Tracking the Origin of Disease”

18:00-18:40 KeynoteLecturesponsoredbyCIHR(TheatreB)KateO’Brien(JohnsHopkinsU.)“ChasingthecausesofseriouspneumoniaamongchildreninAfricaandAsia:PERCHStudyResults”

19:00-22:00 PosterSession#2–posters#73to127WineandCheese,seafoodandantipastoservedduringpostersession

Friday,June15th8:00-9:00 MeetthePIs-Breakfast(CHEB170)

9:00-10:35 Topic#5:EmergingMethodsinVirology SessionChair:MarcelineCôté(U.ofOttawa)

9:00 RogerLippé(U.deMontréal) Flowvirometry:Anovelandpowerfulapproachtodecipherviralegress

9:25 RyanNoyce(U.ofAlberta) “SynViro:Buildingthenextgenerationoftherapeuticpoxvirusvectors”

9:50 VanessaMeier-Stephenson(U.ofCalgary;U.ofLethbridge)“TargetingPersistentHepatitisBVirus:IdentificationofaG4-QuadruplexStructureMotifinaKeyRegionofHBV’sGenome”

10:05 YumikoKomatsu(KyotoU.)“DevelopmentofiPSC-derivedneuralstemcellstransducedwithBornadiseasevirusvectorexpressingthymidinekinaseforcancergenetherapy”

10:20 BrennanDirk(WesternU.)“Seeingisbelieving:Visualizingmulti-proteincomplexesmediatingHIV-1immuneevasion”


11:00-12:35 Topic#6:RNAinVirusInfection(TheatreB) SessionChair:AndrewWhite(YorkU.)

11:00 SelenaSagan(McGillU.) “miR-122-mediatedprotectionoftheHCVgenomefrompyrophosphataseactivity”

11:25 MartinPelchat(U.ofOttawa) “InsightintoinfluenzaAviruscap-snatching”11:50 CarolynRobinson(DalhousieU.) “ViralModulationofRNAGranulesisReliantonAutophagicFlux”12:05 EricPringle(DalhousieU.)

“AlternativetranslationinitiationduringKSHVlyticreplication”12:20 TylerMrozowich(U.ofLethbridge)

“TowardsObtainingaNanoscaleStructureofthe3ʹTerminalRegionofJapaneseEncephalitisVirusGenome”


12:45-13:45 LiveTapingofThisWeekinVirology(TheatreB)withVincentRacaniello(PlanetEarth’sVirologyProfessor)andScienceWriterAlanDove,sponsoredbyCIHRandDMRFGuests: NathalieGrandvaux(U.deMontréal)&CraigMcCormick(DalhousieU.)discussingCSVKateO'Brien(JohnsHopkinsU.)discussingvaccineresearchRyanNoyce(U.ofAlberta)discussinghorsepoxvirusengineering

Lunchboxeswillbeavailable

14:00-14:35 Topic#7:VirusesofFloraandFauna(TheatreB) SessionChair:EricJan(UBC)

14:00 EricJan(UBC)“Translationinitiationofalternativereadingframesbyviralinternalribosomeentrysites”

14:25 HélèneSanfaçon(AgricultureCanada)“Expandingrepertoireofplant(+)-strandRNAvirusproteases:beyondthecysteineandserineproteases”

14:50 ShuangYang(UBC)“Theunderlyingmechanismofauniquenuclearentrypathwayusedbytheparvovirusminutevirusofmice”

15:05 GerardGaspard(DalhousieU.)“AtypicalTransmembraneDomainofReptilianReovirusp14Fusion-AssociatedSmallTransmembrane(FAST)ProteinMediatesit’sTrafficking”

15:25 JaredRowell(U.ofCalgary) “Porcinecircovirus2(PCV2)activatedapoptosisofnon-infectedcells”


16:05 Topic#8:AntiviralInnateImmunity(TheatreB) SessionChair:KarenMossman(McMasterU.)

16:05 StephanieDeWitte-Orr(WilfridLaurierU.)“ClassAscavengerreceptors:theirroleininnateantiviralimmunityinlowervertebrates”

16:30 KatharineMagor(U.ofAlberta)IFNandcytokineresponsesinduckstogeneticallysimilarH5N1influenzaavirusesofvaryingpathogenicity

16:55 JiangyiHe(MemorialU.)“MurineCytomegalovirusImmuneEvasionTargetingtheMEK-IRF1Connection”

17:10 DacquinKasumba(U.deMontréal)“TheNADPHoxidaseDUOX2differentiallymodulatestheproductionofrespiratoryvirus-inducedcytokinesbyairwayepithelialcells”

17:25 HannahStacey(McMasterU.)“IGAimmunecomplexesstimulatenetosisduringviralinfectionandautoimmunity”

17:45 ClosingRemarksandAwardingofPrizesNathalieGrandvaux(U.deMontréal)CSVco-founder

CSV2018co-organizer

18:30-19:30 TourofHalifaxCitadelNationalHistoricSite Citadeltoursby78thHighlanders

20:00-23:00 ReceptionatAlexanderKeith’sBrewery


Saturday,June16th10:00-13:00 SoapboxScienceEventatHalifaxSeaportFarmers’Market

Departure


KEYNOTELECTURE/Biography-1

Dr.DavidKelvinDepartmentofMicrobiology&ImmunologyDalhousieUniversityCanadianCentreforVaccinologyShantouUniversityMedicalCollegeBiography

Dr.David Kelvin’s research interests are focussed on emerging infectious diseases. HeconductsresearchontheemergenceofnovelinfluenzastrainsinChina.HehasworkedonH7N9,H5N1, SARSCoV,WestNileVirus, andNipahVirus infections.He also has aresearchprojectontheimpactofinfectiousdiseaseonhumanimmunegeneevolutionin Sardinia, Italy. Dr. Kelvin is Professor in the Department of Microbiology andImmunology at Dalhousie University in Halifax. He is the Canada Research Chair inTranslational Vaccinology and Inflammation. Dr. Kelvin is Director of Immunity at theInternationalInstituteofInfectionandImmunity,Shantou,China.

“1918to2018,onehundredyearsonthepandemicroad”Abstract:

Inthemoderneraofmedicineatleast6welldocumentedinfluenzapandemicshavebeenidentified.Eventhough1898,1918,1957,1968,1977,and2009arewellrecognizedaspandemicyears,oftenapandemicoccursinwaveslasting years leading to significant human mortality and morbidity. The origins of influenza viruses andenvironmental factors often impact the emergence of pandemic influenza viruses. The course of an influenzapandemicandevolutionofpandemicvirusesisunpredictableandcanenterintocirculationasanendemicvirus,orasinthecaseofthe1957H2N2pandemic,suddenlydisappear.Wewillexploreorigins,mysteries,andanecdotesofinfluenzapandemicsin“1918to2018,OneHundredYearsonthePandemicRoad.”



Dr.SylvainMoineauDepartmentofBiochemistry,MicrobiologyandBiologyFacultyofScienceandEngineering,UniversitéLavalBiography

Sylvain Moineau is a Professor in the Department of Biochemistry,Microbiology and Bioinformatics at the Université Laval. He also holds theCanada Research Chair in Bacteriophages and is the Curator of the Félixd’HérelleReferenceCenterforBacterialViruses,theworldlargestcollectionof reference phages (www.phage.ulaval.ca). His research group uses anintegrativeapproachtostudyphagebiologyaswellastobetterunderstandthe phage-bacteria interactions. He has characterizedmechanisms used bybacteria to resist phage infections, including his landmark discoveries onCRISPR-Cas systems that is at the heart of the current widely acclaimed

genome-editing technology. Prof. Moineau has won several teaching and research awards. In 2017, he wasawarded the NSERC John C. Polanyi Award, the Canadian Society of Microbiologists Murray Award for CareerAchievementandtheDuPontExcellenceMedal.LastFall,hewaselectedtotheAcademyofSciencesoftheRoyalSociety of Canada and appointed an Officer of the Order of Canada. His research work has also received theattention of Thomson Reuters and Clarivate Analytics who ranked him amongst the most cited and influentialmicrobiologistsontheplanetforthepastfouryears(2014-2017).“CRISPR-Cassystemsandphages:theongoingbattle”Abstract:

Fighting viruses is no easy task. Bacterial cells have survived phage attacks by evolving sophisticateddefence strategies that enable them to thrive even in virus-rich ecosystems. Phages have also evolvedcounter-tactics to thwart such mechanisms, leading to a biological arms race. Four decades after thediscovery of restriction enzymes, another bacterial anti-phage system that cleaves foreign DNA wasidentified—one that acts as an adaptive immune system. Clustered regularly interspaced shortpalindromic repeats (CRISPR) and their associated cas genes protect microbial cells against infection byforeign nucleic acids, including phage genomes and plasmids. Bacterial CRISPR-Cas type II systemsfunction by first incorporating short DNA ‘spacers’, derived from invading phage genomes or plasmidsequences,intoaCRISPRarraylocatedintheirgenome.TheCRISPRarrayisthentranscribedandmaturedintoshortRNAs,which,byrecruitingaCasendonuclease,actassurveillancecomplexesthatrecognizeandcleave invadingmatchingsequences.Thecleavageoccursnearashortmotif,calledthePAM,adjacenttothe sequence targetedby the spacer. Exploiting this systemhas also resulted in thedevelopment of themuch-publicized CRISPR-Cas9 technology for precise genome manipulation of various organisms.However, phages canbypass theprotectionprovidedby theCRISPR-Cas system throughpointmutationsordeletionoftheCRISPRtargetintheirgenomeorbytheproductionofanti-CRISPRproteins(Acrs).TheseAcrs can also be used to fine-tune the activity of CRISPR-based genome editing tools. This lecture willrecall the roles played by phages in the understanding of CRISPR-Cas systems. I will also highlight therecentdiscoveryofnewfamiliesofanti-CRISPRproteinsandtheofuseofCRISPR-Cas9technologyforviralgenomeediting.



Dr.KatherineO’BrienJohnsHopkinsUniversityOnbehalfofPERCHStudyTeamBiography

KateO’BrienisProfessorofInternationalHealthandEpidemiologyattheJohnsHopkinsBloombergSchoolofPublicHealthinBaltimore,Maryland.SheistheExecutiveDirectorof the International Vaccine Access Center at the Johns Hopkins Bloomberg School ofPublic Health. Her scientific and policywork domestically and globally has focused onvaccine preventable illnesses, among both children and adults. Thework has includedsurveillance,epidemiology,andvaccineclinicaltrialsofpneumococcaldisease;rotavirus;Haemophilus influenzae typeb; respiratory syncytial virusand influenzavaccines. Shehas worked extensively with American Indian populations and in Africa and Asia,partneringwith local scientists todevelop rigorous scientificevidenceandbring it intothevaccinepolicyarena,therebyacceleratingtheuseandaccesstolife-savingvaccinesfor children living in low resource countries and settings. Dr. O’Brien trained as apediatric infectious disease physician, epidemiologist and vaccinologist with a BSc inchemistryfromUniversityofToronto(Canada),anMDfromMcGillUniversity(Canada),

andanMPHfromJohnsHopkinsBloombergSchoolofPublicHealth(US)beforecompletinghertrainingattheUSCentersforDiseaseControlandPreventionasanEpidemicIntelligenceOfficer,intheRespiratoryDiseasesBranch.She has worked in close partnerships with the Gavi, the Vaccine Alliance, with the World Health Organization(WHO)andwithcountriestoadvanceprogramsandpoliciesonchildhealth.ShehasbeenhonoredwiththeU.S.Department of Health and Human Services Secretary’s Distinguished Service Award, the Sabin Vaccine InstituteYoungInvestigatorAward,theU.S.PresidentialEarlyCareerAwardinScienceandEngineering(PECASE).SheservesontheWHOStrategicAdvisoryGroupofExperts(SAGE)onImmunization,whichdevelopsglobalpolicyonvaccinesandimmunizationsandontheGaviBoard.

“ChasingthecausesofseriouspneumoniaamongchildreninAfricaandAsia:PERCHstudyresults”

Abstract:

Understandingtheaetiologyofpneumoniaiscriticalforacceleratinginterventionstotreatandpreventthisleadingcauseofchildmortality.ThePneumoniaEtiologyResearch forChildHealth (PERCH)study isa7countrystudy inAfrica and Asia designed to determine the aetiology of pneumonia in this era ofwidespread conjugate Hib andpneumococcal vaccination. PERCH is a highly standardized case-control study of World Health Organization(WHO)-definedsevereandveryseverepneumoniaamongover4000hospitalizedchildren1to59monthsofageand over 5000 age-matched controls. Cases and controls hadmultiple clinical specimens collectedwhichwereanalysed bymolecular and culturemethods; cases had chest x-rays obtained and read using standardizedWHOcriteria. Important limitations of standard approaches to descriptive aetiology analysis were addressed using aBayesian,partiallatentclassanalysisapproachtocombinetheresultsfromthepathogentestingandquantifytheetiologic probability in individual cases and in the population. The description of the cases and controls, theprimaryetiologyresults,andaclinicalandetiologicdescriptionofmortalityinPERCHwillbepresented.Further,thepresentationwill highlight the value of post-mortemminimal invasive tissue sampling to better characterise thecomplexityinattributingpathogenspecificcausesofdeathinchildrendyingfrompneumonia.


ABSTRACTS:ORALPRESENTATION (inorderofappearance,presenterisunderlined)

Topic#1:EmergingViruses

Bats:ThesourceofthenextpandemicorcurefordeadlyviraldiseasesVikramMisra1,ArinjayBanerjee1,SonuSubudhi1,NoreenRapin1,DarrylFalzarano1,21DepartmentofMicrobiology,WesternCollegeofVeterinaryMedicine,VaccineandInfectiousDiseasesOrganization/InternationalVaccineCentre,UniversityofSaskatchewan,Saskatoon,Saskatchewan,S7N5B4

Severalviruses,thatappeartocausenoovertdisease intheirnaturalbathosts,havespilledover intoothermammalscausingseriousandoftenfataldisease.Theseincludevirusesthatcause:Severeacuterespiratorysyndrome(SARS)andMiddleeastrespiratorysyndrome(MERS);MarburgandEbolahaemorrhagicdiseases;HendraandNipahrespiratoryandneurologicaldiseases;andporcineepidemicdiarrheaandswineacutediarrhoea.Anoverblowninflammatoryresponseexacerbatesthepathologyinthesediseases.Whiletherearemanystudiesontheinteractionoftheseviruseswiththeirspilloverhostsorsurrogatelaboratoryanimals,thereis little informationontherelationshipoftheseorsimilarviruseswith theirnaturalbathosts.Ourobjective is toexamine theapparentlybenignrelationshipbetweenvirusesand theirnaturalbathostsaswellasthefactorsthatupsetthisrelationshipleadingtoincreasedvirusreplicationand,potentially,tospillovertootherspecies.WehaveidentifieduniqueCoronaandHerpesvirusesthatpersistentlyinfectmostmembersoftwoCanadianbatspecieswithnoovertsignsofillnessorpathology.Secondaryfungalinfections,however,causeanincreaseinvirusreplication.Wehavealsodiscoveredthatwhilebothbatandhumancellsreacttoviralinfectionwithanantiviral response, pro-inflammatory pathways are actively suppressed in bat cells. Bat cells are also resistant to theabilityof viruses to subverthostantiviral responses.Our studieswill notonlyprovide fundamental informationaboutvirus-hostrelationshipsinthesespecializedmammalsbutwillalsogivecluesaboutmitigatingtheseriousconsequencesthatoftenaccompanyviralspill-overfrombatstohumansandotherspecies.Fromthefieldtothelabandbackagain:On-goingLassavirusresearchatNMLMichaelDrebot11NationalMicrobiologyLaboratory,PublicHealthAgencyofCanadaThe recent deployment of the rVSV-Ebola vaccine has renewed interest in the VSV vaccine platform for theprevention of viral hemorrhagic fevers as well asmany other infectious diseases. At the same time that theEbola vaccinewas created and characterized at the NationalMicrobiology Laboratory inWinnipeg, a secondVSV-based vaccine against Lassa fever was also created. Lassa fever is caused by the Lassa virus which is arodent-borne pathogen endemic to many Western African countries including Guinea, Liberia, Nigeria, andSierraLeone.Thevirushasanestimated incidence rateof200,000-500,000 infectionseachyear. Inaddition,Lassa virus has been recently associated with prolonged outbreaks in Nigerian where mortality rates haveexceeded 20% in confirmed cases. Despite this, to date no approved treatment or vaccine exists for Lassafever.Thistalkwillsummarizethepre-clinicalstudiesconductedtodateonthe“made-inCanada”Lassafevervaccineandhighlighttheimportantnextstepstowardsclinicaltrials.


Pan-filovirusT-cellvaccineprovidedcompleteprotectionagainstEbolaandMarburgviruslethalchallengesMdNiazRahim1,2,EdmundG.Wee3,ShihuaHe1,JonathanAudet1,2,KevinTierney1,TomášHanke3,4,XiangguoQiu1,21NationalMicrobiologyLaboratory,PublicHealthAgencyofCanada,Winnipeg,MBR3E3R2Canada,2DepartmentofMedicalMicrobiology,UniversityofManitoba,Winnipeg,MBR3E0J9,Canada,3TheJennerInstitute,NuffieldDepartmentofMedicine,UniversityofOxford,OxfordOX37DQ,UnitedKingdom,4InternationalResearchCenterforMedicalSciences,KumamotoUniversity,Kumamoto,Japan

FilovirusessuchasEbola(EBOV)andMarburg(MARV)virusescausedeadlyviralhemorrhagicfeverinhumanswithhighcasefatalityrates.Todate,nolicencedtherapeuticorvaccinehasbeenclinicallyapprovedtopreventinfection.Severalvaccine candidates are under development against the fewmost common filoviruses targeting the virus glycoprotein(GP).However,protectiveantibodiesinducedbysuchGPvaccinesareusuallylimitedtothesamespecies.Incontrast,T-cellvaccinesofferanopportunitytodesignasinglepan-filovirusvaccineprotectingagainstallmembersoftheFiloviridaefamily. Fourmost conserved regionswere identified among filoviruses by epigraph analysis in previous study. In thisstudyFILOcepXvaccineswereconstructed targeting the fourmostconserved regionsamong theviralproteomeswiththeaimtoinduceprotectiveT-cellresponsesagainstdifferentfiloviruses.BALB/cmicewereimmunizedwithFILOcep1and 2 vaccines vectored by non-replicating engineered simian adenovirus and poxvirus MVA. Vaccinated mousesplenocyteswerestimulatedwith12poolsofpeptidesderivedfromtheFILOcepXepigraphs,andFilocepXspecificT-cellresponsesweredeterminedbyIFN-ϒELISPOTassay.Inthenextstep,groupsof20BALB/cmicewereprimedandboostedwitheithertheFILOcep1andFILOcep2vaccinesorcontrolChAdOx1-andMVA-vectoredvaccines.Fouranimalsineachgroupweresacrificedafter1weekofboostingtodetectT-cellresponsefortheFILOcepXantigen.HighfrequencyTcellsspecificresponsesweredetectedinmicereceivingthetestvaccinesbyIFN-γELISPOTkits.Oftheremaining16animalsineach group, 8 were challenged with mouse-adapted EBOV and 8 were challenged with mouse adapted MARV inContainmentLevel4(CL4) laboratory.Allthemiceinthecontrolgroupeitherdiedorhadtobeeuthanizedbetween4and6dayspostchallenge.OntheotherhandalltheFILOcepXvaccinatedmicemaintainedtheirnormalbodymassandsurvived till the end of the scheduled protocol on day 29 post challenge. These FILOcepX vaccines provided 100%protectionagainstthelethalchallengeswithfilovirusesoftwodifferentgenera.Currentlyweareassessingtheefficacyofthisvaccineinnon-humanprimate(NHP).InhibitionofreceptortyrosinekinasesignalingblocksentryoffilovirusesCorinaWarkentin1,3,MarcelineCôté1,31UniversityofOttawa,2UniversityofOttawa,3OttawaInstituteofSystemsBiology

Ebolavirus(EBOV)isanenveloped,negativestrandedRNAvirusoftheFiloviridaefamilythatcausessevereoutbreaksofhemorrhagicfever.Currently,therearenoFDA-approvedantiviraltherapiesagainstEBOVdisease.EBOVentryinvolvesinternalizationandtraffickingwithinhostcellsforthevirustoreachits intracellularreceptor,Niemann-PickC1(NPC1),which is localized in late endosomes/lysosomes. Given the requirements for internalization and endolysosomaltrafficking, we hypothesized that EBOV activates signaling pathways to induce its uptake and regulate trafficking tofacilitateitsdeliverytoNPC1+cellularcompartments.Todetermineifsignalingeventsinducedbythevirusplayaroleininfection,wescreeneda libraryofkinase inhibitors for theireffecton infectionbyMLVpseudotypesbearingEBOVorvesicularstomatitisvirus(VSV)glycoproteins.WeidentifiedcompoundsthateitherspecificallyinhibitedEBOVinfection,orblocked infectionbybothviruses.However, results revealed thatno compoundexclusively inhibitedVSV infection,suggesting that EBOV GP-mediated infection is dependent on specific signaling pathways. We additionally screenedMarburg virus (MARV) and Lassa fever virus (LFV) and obtained similar results.One class of inhibitors thatwas over-represented in our hits for the filoviruses (EBOV andMARV) were inhibitors of receptor tyrosine kinases (RTKs).Wefurthercharacterizedspecific inhibitorsofepidermalgrowthfactorreceptor (EGFR)andtyrosineproteinkinaseMet inboth Vero cells and bone-marrow derived macrophages, and found that they inhibited infection of all pathogenicfiloviruses.Interestingly,theseinhibitorsalsoactsynergistically,suggestingthatEBOVutilizesmultipleRTKstopromoteentry. Furthermore,experimentsonmodeofaction suggested thatEGFR signalingoccursearlyduring infection, likelyduringattachmentorinternalization,whileMetsignalingoccursatapost-internalizationstage.OurstudieshighlighttheimportantroleofsignalingpathwaysinEBOVinfection, inadditiontotheidentificationofRTKactivationasapotentialmechanismthroughwhichsignalingisinitiated.


ElucidatingtheroleofviralinfectionontheparthenogenesisofAmyotrophicLateralSclerosisNedaBarjesteh1,2,JonathanMapletoft1,2,BraedenCowbrough1,2,DanielCeleste1,2,MatthewMiller1,21McMasterUniversity,2MichaelG.DeGrooteInstituteofInfectiousDiseasesResearch,DepartmentofBiochemistryandBiomedicalSciences,McMasterImmunologyResearchCentre,McMasterUniversity,,3McMasterUniversity

Amyotrophiclateralsclerosis(ALS)isadevastatingneurodegenerativediseaseforwhichtherearecurrentlynoeffectivetreatments. While several genes have been associated with ALS, environmental triggers have long been thought tocontributetotheonsetandprogressionofALS.ViruseshavereceivedlongstandingattentionaspotentialenvironmentaltriggersofALS.However,noconclusivecausativerelationshipbetweenaspecificpathogenandonsetorprogressionofALS have been identified to-date.We hypothesized that viral infection triggers the onset and progression of ALS byperturbing pathophysiological pathways associated with the disease. To assess the role of viral infection on ALS weutilizedtheSOD1G93AmurinemodelofALSandtestedtheeffectofinfluenzaAvirus(IAV)infectionondiseaseonsetandprogression.Wedemonstrated that IAVexacerbatedALSsymptomsandacceleratedmortality inSOD1G93Amice.UsingmouseembryonicfibroblastsderivedfromeitherSOD1G93Aorwild-typemice,weassessedtheaccumulationofmisfoldedSOD1following IAV infection. We have shown that infection caused accumulation of misfolded SOD1. We have alsodemonstrated thatSOD1G93AMEFsproduceelevated levelsof inflammatory cytokines/chemokinesupon infection.Ourfuture work will focus on characterizing neuroinflammation within the CNS following viral infection. Additionally, todeterminetheroleoftheimmunesysteminALS,wewillidentifythesource(s)ofvirus-inducedneuroinflammation.Wewill also define the direct effects of viral replication on pathogenic mechanisms associated with ALS. This studydemonstratesthatcommonviralinfectionsmayacceleratediseaseonsetandprogressionintheSOD1G93AmurinemodelofALS.Topic#2:ViralSubversionofHostCellProcesses

EarlyStagesintheMorphogenesisofHerpesSimplexVirusJ.Gao1,M.A.Gulak1,M.R.Sherry1,A.Nassiri1,R.L.Finnen1,H.Burrows1,B.W.Banfield11DepartmentofBiomedicalandMolecularSciences,Queen'sUniversity

HerpesvirusassemblystartsinthenucleuswheretheviralDNAgenomeispackagedintoapreformedcapsid.Asherpescapsids are too large to pass through nuclear pores, capsids must pass through both the inner and outer nuclearmembranes(INM,ONM)toaccessthecytoplasmwherefinalvirusassemblyoccurs.Thisprocess,termednuclearegress,isconventionallydividedinto3phases:1)recruitmentofcapsidstotheviralnuclearegresscomplex(NEC)residingintheINM;2)primaryenvelopmentofcapsidsattheINM;and3)fusionofenvelopedcapsidsintheperinuclearspacewiththeONMtoreleasecapsidsintothecytoplasm.TheHSV-encodedproteinsUL31andUL34areconservedNECcomponentsthat play a central role in the primary envelopment of DNA containing capsids at the INM. Others have shown thatexpression of UL31 and UL34 in the absence of other viral proteins leads to vesiculation of the INM at the nuclearperipheryandtheinteractionofpurifiedUL31andUL34withmembranesinvitrocaninducevesiculation.WhiletheNECconstitutes the minimal requirement for membrane vesiculation, this activity must be regulated in infected cells toprevent unproductive primary envelopment, thus other host/viral proteins likely play regulatory roles in nuclearegress.We investigated nuclearmembrane remodeling byHSV-2UL31 andUL34 and found that, in addition to INMvesiculation, cells expressing both proteins form long membranous tubules that extend inwards from the nuclearperiphery deep into the nucleoplasm. These tubules are dynamic, contain cellular INM components, are devoid ofnuclearpores,lacknuclearlaminaontheirnucleoplasmicfaceandformattimespostinfectionconsistentwitharoleinvirus assembly. Extending the INM into the nucleoplasmwould evade barriers to primary envelopment, such as thenuclear lamina and marginalized cellular chromatin, and thus might present an attractive surface for primaryenvelopment ofHSV-2 capsids located in thenucleoplasm.Wehypothesize that, rather than capsid recruitment to aperipheralINM,theINMisdirectedtothenucleoplasmwherenuclearegresscanbeinitiated.OurrecentstudiesontheHSV-2UL16proteinprovide support for thishypothesis.HSV-2 lackingUL16 showdefects inprimaryenvelopmentofcapsidsandseverelyreducedvirusreplicationintheabsenceofcomplementation.Replicationofthisviruswasmarkedlyimproved in HeLa cells stably expressing a version of UL31 with enhanced ability to form tubules compared to itsreplicationinparentalHeLacells.IfartificiallyevokingINMtubuleformationcancompensateforthelackofUL16,thismay suggest a role for UL16 in directing the INM to capsids located in the nucleoplasm. We are currently testingpredictionsstemmingfromthisalternateviewofnuclearegressaswelldefiningrolesforotherHSV-2proteinsinnuclearegress.


Niche-to-nichevariationsinreovirus-hostinteractionsAdilMohamed1,2,JasonFernandez1,2,WanKongYip1,2,FranciscaCristi1,2,PatriciaChen1,2,MaryHitt2,3,MayaShmulevitz1,21UniversityofAlberta,MedicalMicrobiologyandImmunology,2LiKaShingInstituteforVirology,3UniversityofAlberta,Oncology

Mammalianorthoreovirus(reovirus)naturallycausesbenigninfectionoftheintestinaltract.Itwaspreviouslyestablishedthatwhenadministeredtotumor-bearinganimalsorhumans,reovirusspecificallyreplicatesintumorswithlimitedharmto non-transformed cells. Reovirus is therefore a candidate oncolytic therapy. In an effort to improve the oncolyticpotency of reovirus, we askedwhether specific virus characteristics that evolved towards the enteric niche could bemodifiedtopromotevirusreplicationanddose-escalationintumorenvironments.Ourfindingsshowthatmodificationstoreoviruscanpromotebothentryandpost-entrystepsofreplicationintumorcells,withoutjeopardizingrestrictionbynon-transformedcells.Forexample,reovirusvariantswithfewercellattachmentσ1trimersrelativetowild-typereovirus(which typically has twelve σ1 trimers) exhibit four-fold better uncoating on a panel of cancer cells in-vitro anddemonstrateimprovedoncolyticpotencyinanin-vivomelanomamodel.Specifically,wefoundthatthreeσ1trimersaresufficientforreovirusbindingtotumorcells,andthatprogressivelyincreasinglevelsofσ1trimersresultsindecreasingproportionsof virions that successfully shedσ1duringentry (aprerequisite toestablish infection).Conversely, havingfewerthanwildtypelevelsofσ1trimersmakesreovirionsadverselyunstableunderentericconditions,suggestingthatoptimal reovirus structure may depend on the niche of infection. As a second example, reovirus capsid cleavagemediated by intestinal and intracellular proteases has been well defined. However, the role of proteases in theextracellulartumorenvironmentduringreovirusoncolysisislessunderstood.Wediscoveredthatbreasttumorproteasescan lowerreovirus infectivityby100-fold.Specifically,metalloproteasessecretedbyapanelofbreastcancercells,andpresent in extracellular environments of in-vivomousebreast tumors,were found to cleave reovirus σ1proteins andreducevirusbindingtotumorcells.Throughmutagenesisoftheσ1proteasesensitivesite,wewereabletoovercomecleavage and inactivation of reovirus σ1 by breast tumor metalloproteases. Our findings not only suggest that virustherapieswouldbenefit from full repurposing towards their target niche, but also exemplify howdistinct cellular andenvironmentalfactorsindifferentnichescanimpacttheoutcomeofreovirus-hostinteractions. UnconventionalreleaseoffusogenicnonenvelopedreovirusesNicholeM.McMullen1,ChungenPan1,RobertodeAntueno1,GerardJ.Gaspard1,DuncanMacKenzie1,KathleenM.Proudfoot1,RoyDuncan1

1DalhousieUniversity

Fusogenic reoviruses induce cell-cell fusion and syncytium formation mediated by a virus-encoded fusion-associatedsmalltransmembrane(FAST)protein.FASTproteinsenhancelocalizeddirectcell-cellspreadoftheinfectionviacell-cellfusion, followed by syncytial lysis and release of infectious virus particles for systemic dissemination. As with mostnonenvelopedviruses,virus-inducedcell lysis istheprimarymechanismforreovirusreleasefromcells.Recentfindingssuggestvariousnonenvelopedviruses,includinghepatitisAvirus(HAV)andpoliovirus,canalsobereleasednon-lyticallyfrom cells inside extracellular vesicles (ECVs). Western blotting, transmission and cryo-electron microscopy revealedsignificantlevelsofnon-envelopedvirionsandFASTprotein-containingECVsincellculturesupernatants.Kineticanalysisof virus release was coupled with live-cell imaging using Sytox to assess plasma membrane integrity (a membraneimpermeable dye and only fluoresceswhen bound to nucleic acid inside cells). Results indicate release of substantialquantitiesofinfectiousvirusparticlesfromcellspriortoanyevidenceofreducedplasmamembraneintegrity,indicativeofnon-lyticvirusrelease.Unlikenon-lyticHAVrelease,silencingofESCRTproteinsTsg101andAlixdidnotdecreaseFASTinducedsyncytiumformationnornon-lyticreovirusrelease.Previousresults,usingyeast2-hybridanalysis,indicatedaninteraction between FAST proteins and autophagy related 5 (Atg5) protein. Given recent evidence that showsinvolvement of Avian reovirus and Muscovy duck reovirus proteins in inducing autophagy and the release ofautophagosomelikestructures,geneticandpharmacologicapproachesarecurrentlybeingusedtoassessthepotentialinvolvementofautophagypathwaysinthemechanismunderlyingthisnon-lyticvirusrelease.


InteractionofhumanpapillomavirusE7withDNArepair-specificE3ligaseRNF168J.Sitz1,A.Blondeau1,M.FuchsandA.Fradet-Turcotte11CHUdeQuébecResearchCenter-UniversitéLaval,CancerResearchCenter

HumanpapillomavirusesaresmallDNAviruses that replicate indifferentiatingepitheliumof theskinandthemucosa,and they are classified as low-risk or high-risk according to their respective ability to cause condylomas or cancers.Althoughhigh-riskHPVscontributetothedevelopmentofcervicalcancerandasubsetofheadandneckcancers,onlyasmallproportionofinfectedpatientsdevelopsthedisease,underliningtherequirementofadditionalgeneticalterationsfor the progression of the infected cells to carcinoma. Currently, HPV is known to interfere with at least two of thepathways thatguardgenomic stability inhost cellsby:1) sequestering specific factorsof theDSB signalingand repairpathwaystoreplicateitsgenome,and2)byactivelyblockingcellcyclecheckpoints.InHPV+cancercells,twofactorsthatplayscentralroleincellularresponsetoDNAdouble-strandbreaks(DSBs),theE3-ligaseRNF168and53BP1,accumulateinatypicallargenuclearbodies.HowHPVinterfereswithDSBsignalingpathwaysandhowitaffectstheabilityofthecelltodealwithgenomicinstabilityisstillunknown.Here,wereportourprogressindelineatingtheinterplaybetweenthevirus and DSB responses. First, we found that the large nuclear bodies observed in HPV+ cancer cells are 53BP1 G1bodies,astructurethatassemblearoundDNAdamagethatoccurredinthepreviousS-G2phaseofthecellcycle.ThesefindingssuggestthatHPV+cancercellsundergo,andadaptedtolivewithhighlevelsofreplicationstress.Consistently,wefoundthattheyareresistanttotreatmentwithPARPiandetoposide,twomoleculesthatenhancereplicationstressbyinducingdamageinS-phase.TodeterminewhetherthevirusprovidescellswithabetterabilitytofacespecifictypesofDNAdamage,weinvestigatedwhetherviralproteinsimpactDSBsignalling.Usingasystembasedonthelacoperator(lacO)/lac repressor (LacI),we found that twoLacI-HPV fusionproteinspromote the recruitmentofRNF168 toa LacOarray,independentlyofupstreamDSBsignallingfactorssuchasγ-H2AXandRNF8.TheseresultsraisethepossibilitythatHPVproteinsinterferewithDNArepairprocessesbyinteractingwiththeE3-ligase.Altogether,ourresultsshowthatHPVproteinscanrewirethecellularlocalizationofDSBsignallingproteinsandinterferewiththeabilityofcellstocopewithDNAdamagingagents.FurtherstudieswillaimatdefiningtheinteractionoftheviraloncogeneswithRNF168andhowitimpacts their respective functions. Thiswill contribute toelucidate themechanismbywhichHPV factorsperturbDSBrepair pathways. As HPV extensively relies on the activation of DSB signaling to replicate its genome in infectedepithelium,ourstudiesmayrevealimportantinsightonthemoleculareventsunderlyingtheseprocesses.


RegulationofRespiratorySyncytialVirusinfectionbytheautophagyreceptorSQSTM1/p62inairwayepithelialcells.QuentinOsseman1,2,SandraCervantes1,2,EliseCaron1,EricS.Pringle3,AlexaRobitaille1,2,XioachunGuan1,2,CraigMcCormick3,NathalieGrandvaux1,21CRCHUM,2UniversitédeMontréal,3DalhousieUniversity

Respiratorysyncytialvirus (RSV)causesahighrateofmorbidityandmortality in infants,childrenandelderly,butalsoadults of all ages with a compromised immune system, cardiopulmonary diseases or who have undergonetransplantation,worldwide.RSVvaccinedevelopmentfacesnumerouschallengesandcurrentverycostlyprophylacticsare restricted to high-risk children. Antivirals targeting host factors required for virus replication are an emergingalternative to traditional direct-acting antivirals. RSV primarily replicates in airway epithelial cells (AECs). Therefore,understandingRSV:hostinteractioninhumanAECsiscrucialforfuturedevelopmentofdrugstargetingRSVreplication.Autophagy is critical for intracellularhomeostasis in response to stress, including virus infection.However, compellingevidence demonstrate that a number of viruses have evolved mechanisms to exploit and modulate the function ofautophagycomponentstousethemfortheirbenefit.Selectivityinautophagyisconferredbycargoreceptors,includingp62/SQSTM1,which tether ubiquitylated (Ub) cargo to a autophagosomes for degradation into autophagolysosomes.Previous data have highlighted an important role for p62-dependent autophagy in the bacteria and virus clearance,althoughp62wasalsoshowntopromotereplicationofsomeviruses.Very little information isavailableregardingtheinterplaybetweenRSVandautophagy. Importantly, theroleofautophagy inRSV infection inhumanAECshasnotyetbeenaddressed.Ourgoalistodeciphertheroleofp62-mediatedautophagyintheregulationofRSVinfectiouscycleinhuman AECs. We observed that RSV triggers autophagy in A549 cells, a model of AECs. Replication of RSV wassignificantlydecreasedincellswithincreasedautophagicactivitysupportingananti-RSVactivityofautophagy.Wealsoaccumulateddatasupportingactivationofthecargoreceptorfunctionofp62duringRSV infection.Phosphorylationofp62onSer403,amodificationknowntoenhancebindingofp62toitscargo,wasinducedinaTBK1-dependentmanner.Accordingly,p62bindingtopoly-UbproteinswasincreasedduringRSVinfection.InhibitionoflysosomalenzymesusingBafilomycin confirmed that autophagy was responsible for p62 degradation during RSV infection. The finding of RSVproteinsamongstpulleddownpolyUbproteinssuggestseitherdirectpolyUbofRSVprotein(s)ortheir interactionwithpolyUbhostprotein(s).Moreover,weobservedcolocalizationbetweenRSVFandLC3.Currentproteomics-basedstudiesareaimedatidentifyingp62poly-UbcargothataretargetedtoautophagosomesandhavearoleintheregulationofRSVreplication cycle. Altogether, our results imply that selective autophagy involving the p62 receptor exhibits antiviralactivityagainstRSVinhumanAECs.Topic#3:VirusesofMicrobes

AchemicaldefenceagainstphageinfectionKarenMaxwell11DepartmentofBiochemistry,UniversityofToronto

Thebattleforsurvivalbetweenbacteriaandthevirusesthatinfectthem,knownasphages,hasresultedintheevolutionofawidevarietyofanti-phagedefences.Theseincludecellsurfacemodifications,restrictionenzymes,abortiveinfectionsystems,andtheCRISPR-Casadaptive immunesystem.While thesedefencesystemsarehighlyvariedmechanistically,theyallrelyonproteinsorprotein-RNAcomplexestomediatetheirfunctions.Werecentlydiscoveredanewchemicallybasedanti-phagedefencesysteminthesoildwellingbacteriaStreptomyces.Thesebacteriaarerenownedforproducingspecialized metabolites that kill bacteria and other competitors that share their environment. We show that theproduction of anti-phage specialized metabolites is widespread in Streptomyces and that these compounds target asingle step early in the phage infection process that would broadly inhibit the majority of phages. As bacteria areoutnumberedbyphagesinallenvironmentsandtheproductionofspecializedmetabolitesbybacteriaisubiquitous,thismechanismofanti-phagedefencelikelyplaysamajorevolutionaryroleinshapingbacterialcommunities.


FollowingPhagesthroughFecalTransplantsHibaShareefdeen1,ShahrohkShekarriz1,MichaelGSurette1,AlexanderPHynes11McMasterUniversity

Fecal microbiota transplants/transfers (FMTs) have been used to treat human intestinal diseases ranging fromClostridiumdifficileinfectionstoulcerativecolitis.ThecompositionalchangesofthegutmicrobiomefollowingFMTareofconsiderableinterest.Surprisingly,despitethesuccessfuloutcomesofthistherapy,evidencefortransplantationofthedonor’smicrobiomeremainsinconclusive.Anoften-overlookedcomponentofthistransferisthebacteriophage(phage)population,whichcould impacttherecipient’smicrobiome.Wetrackedthehighlyprevalentgutphage,crAssphage, insamplescollectedfromdonorsandrecipientsofFMTs.Thisphage,originallyidentifiedbymetagenomics,ispresentin~40%ofhumans,althoughitsbacterialhostisnotyetknown.CombiningPCRandmetagenomicanalysis,wewereabletoobservetemporalvariabilityincrAssphageprevalenceaswellascorrelatetransferofthephagewithFMTsfromphage-containingdonors.Toassesscausality,wealsodemonstratedengraftmentofcrAssphageintogerm-freemicereceivinghuman fecal matter.We confirmed the presence of an established, replicating phage population. Identifying speciestransferredintomicealongsidecrAssphagehasenabledustoembarkonatargetedapproachtoculturetheelusivehostofthisremarkablephage.ThegutviromeofpediatricCrohn’sDiseasefollowingexclusiveenteralnutritionCaseyJones1,JessicaConnors2,BradMacIntyre2,ScottWhitehouse2,HanaJames2,GamalMahdi2,MohsinRashid2,3,AngelaNoble2,3,JohanVanLimbergen2,3,MorganGILangille11DepartmentofPharmacology,DalhousieUniversity,2DivisionofGastroenterology,IWKHealthCentre,Halifax,NS,3DepartmentofPediatrics,DalhousieUniversity,Halifax,NS,Canada

Background: Inflammatory bowel disease (IBD), is an increasingly common disorder that causes inflammation in thegastrointestinal tract. Although causes are elusive, the gut microbiome is known to play a prominent role in IBDpathogenesis.Additionally,theviralcomponentofthegutmicrobiome,thegutvirome,hasbeenshowntobeabnormalin IBDpatients.Exclusiveenteralnutrition (EEN) isusedasa first-line induction therapy forpediatric IBDpatients,buthowitaffectsthegutmicrobiotaisstillnotfullyunderstood.Inthisstudy,weaimedtooptimizecharacterizationofthegutviromeinresponsetoEENtreatmentandtoidentifylongitudinalviral-bacterialinteractions.Methods:Metagenomicshotgunsequencing(MGS)wascarriedouton40longitudinallycollectedstoolsamplesenrichedforfreevirionsfrom6pediatric patients with CD. Viral taxonomy of each sample was determined by aligning sequencing reads against theIMG/VRproteindatabase.Bacterial taxonomicprofilesweregeneratedusing16S rRNAdataandwere integratedwithviralprofilestoanalyzeinteractionswithtreatmentprogression.Results:Anaverageof5,024,873readsweresequencedineachsample,withanaverageof8.0%of the reads (SD:8.2%)havingsignificanthits to the referencedatabase.Weidentified661viraltaxa,withthemajoritybelongingtobacteriophages.Annotatedreadswerealsocollapsedinto44531unique viral clusters. Additionally, presence of Enterococcus phage EF62phi was moderately correlated with relativeabundanceofitsbacterialhostduringthetwo-yearstudyperiodinonepatient(Spearmanr:0.54;p=0.039).Significance:OurstudyisthefirstattemptatintegratingviralMGSand16SmicrobiomedatainpediatricCDpatients.Thispreliminaryanalysiswarrantsfurtherworktogaininsightintothebacteriophage-bacteriainteractionduringtreatment.


TypeIVpili-specificbacteriophagesofStenotrophomonasmaltophiliaJaclynG.McCutcheon1,DanielleL.Peters1,AndreaLin1,JonathanJ.Dennis11UniversityofAlberta

Theemergingbacterial pathogen Stenotrophomonasmaltophilia is rapidly increasing inprevalence innosocomial andcommunity-acquired infections. This bacterium is ubiquitous in the environment and is easily transmitted betweenimmunocompromisedpatientsandhealthcareproviders.TreatmentofS.maltophiliainfectionsisdifficult,however,dueto its innate resistance to a broad range of antibiotics and assortment of virulence factors. A possible alternativetreatment is phage therapy, the clinical application of bacteriophages to eradicate target bacteria. The specificity ofphagestotheirhostsreliesonthepresenceofthecorrectcellsurfacereceptor. Identifyingthesephagereceptorsandcharacterizing themechanism of phage-host interactionwill help create effective phage cocktails containingmultiplephageswithdifferentreceptorsfortherapeuticuse.Ourlabhasisolatedanumberofbroadhostrangebacteriophagesfrom soil samples obtained across Alberta, Canada that are capable of infecting 24 out of 27 S. maltophilia strainscollectively.Previous studyof thebacteriophageDLP1, capableof infecting twostrainsofPseudomonasaeruginosa inadditiontoeightS.maltophiliastrains, identifiedthetype IVpilusas theprimaryreceptor for infectionacross itshostrange.ThetypeIVpilusisavirulencefactoronthesurfaceofthebacteriumthatplaysanimportantroleinattachmenttohosts as well as biofilm formation and twitching motility in many bacterial pathogens. DLP1 along with five otherbacteriophagesisolatedinourlabinfecttheS.maltophiliastrainD1585astheirmainhost.Cleandeletionofthemajorpilinsubunit,encodedbypilA, inD1585prevents infectionbyallsixphageandsubsequentcomplementationwiththeendogenouspilAgenerestoresinfection.Thisindicatesthatthesesixuniquebacteriophagesharethesamereceptorforinfection of their major host, however their host ranges are vastly different. Deletion of pilA in a second host, 280,infectedby fourof thesixbacteriophageproduced identical results, supporting the identityof thetype IVpilusas thereceptor for these phages. Examination of genome sequencing data of the strain D1571 that is susceptible to only asingle bacteriophage of the six revealed that this strain does not encode the PilA major subunit for type IVa pilusassembly. Interestingly, expressingbothD1585and280pilA genes inD1571broadens thephage susceptibilityof thisstrain,allowinginfectionbyallsixphages.TheseresultsidentifythetypeIVpilusinS.maltophiliaasthereceptorforsixunique bacteriophages and suggest that phages may adopt evolutionarily conserved bacterial surface structures asprimaryreceptorsforinitialhostcellinteraction.Futureresearchwillexaminedifferingphagebindingsitesonthemajorpilinsubunitaswellasdifferencesandsimilaritiesbetweenphagereceptorbindingproteinstoexplainthedifferenthostrangesofthesephages.ThisresearchfurthercharacterizesthesephagesascandidatesforphagetherapycocktailsinthetreatmentofmultidrugresistantS.maltophiliainfections.


Examiningviralpopulations,CRISPR-resolvedvirus-hostinteractions,andCRISPR-CassystemdiversityinamunicipallandfillNikhilA.George1,AngusS.Hilts1,LauraA.Hug11UniversityofWaterloo

Recent culture-independent approaches have unveiled a staggering diversity of viruses from a wide variety ofenvironments. The comparatively poor representation of viruses within reference databases, despite their numericalabundance inecosystems, clearly illustrateshowcharacterizing thevirospherehashistoricallybeenamethods-limitedendeavor.Understudied environments, likemunicipalwaste sites, are likely to house novel, diverse viral populations,whosehostinteractionsmayimpactnutrientcyclingandcontaminantdegradation.Usingmetagenomics,weprobedtheviraldiversitywithinalandfillandadjacentaquiferinSouthernOntario.Metagenomicsequencesweregeneratedfromsamplescollectedfromthree leachatewells,a leachatecollectioncistern,andfromanaquiferadjacenttothe landfill.DNAwasextractedfromfilteredbiomass,andsequencedbytheJointGenomeInstitute(JGI).UsingFastViromeExplorer,avirusandphageidentificationpipeline,alongwithNCBI’sRefSeqdatabaseandtheJGI’sEarthViromeProjectdatabase,weidentifiedtheviralsignaturesinallsixmetagenomes.Thedifferentsitesvariedintheirviralpresence,withthecisternsamplescontainingthehighestviralabundancesanddiversities,andtheaquifersampleshowingthelowest.Toestablishvirus-prokaryotic host interactions within the landfill, we used Crass to first identify CRISPR (Clustered RegularlyInterspacedShortPalindromicRepeats)withinourmetagenomes.ThespacerswithintheCRISPRarrayswereextracted,and searched againstNCBI’s Refseq database and ourmetagenomes using BLASTn, in order identify the viruses fromwhichthespacersoriginated.ThevastmajorityofidentifiedspacershadnohitsinRefseq,suggestingthatmanyoftheecologicallyimportantviruseswithinoursystemarenotpresentwithinthisreferencedatabase.Thefewspacersthatdidhave hits to Refseq had homology predominantly to Pseudomonad-infecting phage genomes. Spacer hits to ourmetagenomes were identified as spacer-viral element matches or spacer-host CRISPR array matches, allowingconnectionsbasedonprior infectionstobedetermined.CRISPR-Cassystemsareuseful inelucidatingvirus-prokaryotichost interactions, and are of huge value in the agricultural and health industries. Current biotechnology applicationsutilize CRISPR-Cas systems adapted from isolated bacteria, while novel Cas proteins may offer improvements oralternativestocurrentgene-editingtools.WesoughttoidentifytheCasproteindiversitywithinourmetagenomes.UsingprofileHiddenMarkovModels(HMMs)generatedfromspecificCasproteinalignments,wescreenedourmetagenomesfor the presence of Cas proteins. Numerous Cas proteins types and subtypes were detected, aligned, and homologymodeled in order to identify novel Cas proteinswithin the landfillmicrobial communitywith potential biotechnologyapplications.Topic#4:AntiviralsandVaccines

NeuraminidaseInhibitorsEnhanceFc-DependentEffectorFunctionsElicitedbyHemagglutininStalk-BindingBroadly-NeutralizingAntibodiesAliZhang1,MatthewMiller21McMasterUniversity,2McMasterUniversity

InfluenzaAviruses (IAV)cause3-5millionserious illnessesandhalfamilliondeathseachyear.Vaccination is thebestway toprevent infection,butprotectionprovided isnarrowand ineffectiveagainstpandemic strainsdue toantigenicdrift in the immunodominant hemagglutinin (HA) head domain. Recently discovered broadly-neutralizing antibodies(bNAbs) that target the conserved HA stalk domain provide great promise towards development of a “universal”influenzavaccine.ThesebNAbsrequireFcreceptorbindingandeffectorcellfunctiontoconfermaximalprotection.Wenowshowthatenzymaticinhibitionofneuraminidase(NA)enhancesFc-dependenteffectorfunctionselicitedbybNAbsandmayreducethedevelopmentoftherapeuticresistance.Usinganinvitroantibody-dependentcellcytotoxicity(ADCC)assay,wedemonstratethatoseltamivir,anNAinhibitor,causesadose-dependentincreaseinbNAb-mediatedADCCofIAV infected cells. Using mouse models, we also show that oseltamivir/bNAb combination therapy is superior atprotectingagainstlethalIAVinfectioncomparedtousingeithertreatmentaloneinvivo.Furthermore,weareperformingexperiments to determine if oseltamivir/bNAb combination therapy is effective at preventing or delaying therapeuticresistance. Themechanismbywhich bNAbs protect against IAVpropagation is incompletely understood.Our findingsmay explain the variable efficacy of oseltamivir in patients, and also guide design of universal influenza vaccines andotherbNAb-basedanti-viraltherapeutics.


IdentificationofpharmacologicaltargetsandsmallmoleculesinhibitingadenovirusreplicationBritiSaha1,2,OliverVarette2,3,Jean-SimonDiallo2,3,RobinJ.Parks1,2,41RegenerativeMedicineProgram,OttawaHospitalResearchInstitute2DepartmentofBiochemistry,MicrobiologyandImmunology,UniversityofOttawa3CancerTherapeuticsProgram,OttawaHospitalResearchInstitute4CenterforNeuromuscularDiseases,UniversityofOttawa

Thehumanadenovirus(Ad)causesminorrespiratoryillnessesinmostpatients,butcanleadtoseverediseaseand death in pediatric, geriatric and immunocompromised patients. No approved antiviral therapy currentlyexists for the treatment of severe Ad-induced diseases.Within the first few hours of infection, the Ad DNAenters the host cell nuclei and associateswith cellular proteins including histones, adopting a nucleoproteinstructure similar to the cellular DNA. Assembly of the viral genome into this repeating nucleosome-likestructure is required for efficient expressionof virus-encodedgenes. Consequently, one approach to treatingAd-induced diseasemay be to prevent the viral DNA from transitioning to this transcriptionally active state.Thus, our objective is to identify novel small-molecule inhibitors of Ad replication and to investigate themolecular mechanism underlying the inhibition, including effects on epigenetic regulation of the viralgenome.We havegenerated a wild-type-like Ad construct encoding the red fluorescent protein (RFP) withinthe viral genome. RFP from this construct is only expressed followingAdDNA replication,which allowsus toeffectivelymonitorvirusreplicationbyfluorescencemicroscopy.Usingthisconstruct,wedesignedanefficientmethod for screening small-molecule libraries. We have screened two small molecule libraries consisting ofover1300compounds to identifydrugs thatexhibitanti-Adactivity.Severalpositivehitshavebeenvalidatedto either inhibit or considerably delay Ad gene expression, and reduce virus yield. In particular, the pan-histone deacetylase (HDAC) inhibitor vorinostat was found to significantly reduce RFP expression. Follow-upstudiesrevealedthatvorinostatdelaystheonsetofviralgeneexpressionandreplication,anddecreasesvirusyield from infected cells. Vorinostat was also effective against more virulent and clinically relevant Adserotypes. The drug’s inhibitory effectswere found to bemediated through the inhibition ofHDAC2 activity.Furtherelucidationof theunderlyingmechanismand in vivo studiesareunderway.The costs associatedwithAd-induced disease are significant in terms of medical expenses, lost work hours and loss of life in somepopulations. Identification of novel Ad inhibitors will allow the design and development of more effectiveantivirals,ultimately leading todecreaseddiseasepathogenesisandhighersurvival rates insevere infections.Investigation of themechanism bywhich these compounds impact Ad replicationwill provide useful insightsonvirus-cellinteractions,andallowustoidentifynewpharmacologicaltargetsfortherapeuticintervention. CharacterizationofaNovelHost-TargetedAntiviralMoleculeMarielKleer1,PatrickD.Slaine1,DenysA.Khaperskyy1,2,CraigMcCormick1,2.1DepartmentofMicrobiologyandImmunology,DalhousieUniversity,2CanadianCenterforVaccinology

Introduction: Current front-line influenza antivirals rely on targeting specific viral proteins to limit replication. Thesedrugsareknownasdirect-actingantivirals(DAAs).However,rapidviralevolutionallowsfortheemergenceofmutationsthat confer resistance to these typesof antivirals. Because influenza virusesdependonmanyhost processes for viralreplication, host-targeted antivirals (HTAs), provide an attractive alternative to limit the emergence of drug-resistantviruses.Here,wecharacterizeanovelhost-directedantiviral,SG3,thateffectivelylimitsinfluenzaAvirus(IAV)replicationin vitro.Methods:A549 lungepithelial cellswere infectedwith IAVand treatedwith SG3at4hpi.Releaseofprogenyvirionswas analyzed by plaque assays and protein expressionwas assessed via immunoblotting. Results: SG3-treatedA549cellsexhibiteda2-logreductionininfectiousvirionscomparedtocontrolcells.SG3treatmentcausedactivationofboththePERKandIRE1αarmsoftheunfoldedproteinresponse(UPR),andselectivelyreducedaccumulationofviralHAandNAglycoproteinswhilehavingminimaleffectsonNPandM1expression.Conclusions:SG3treatmentreduces IAVreplication and accumulation of viral glycoproteins HA and NA, while activating the unfolded protein response. Ourfindings demonstrate that SG3 is a promising candidate HTA for influenza. These in vitro findings will have to becorroboratedinanappropriatepre-clinicalinvivomodel.


PhotodynamicInactivationofHerpesSimplexVirusesAndreaMonjo1,EricPringle1,MackenzieThornbury1,SherriMcFarland3,4,CraigMcCormick11DalhousieUniversity,2DalhousieUniversity,3DepartmentofChemistryandBiochemistry,UniversityofNorthCarolinaGreensboro,4PhotoDynamic,Inc.

Introduction:Photodynamicinactivation(PDI)employsaphotosensitizer,light,andoxygentoinactivatemicroorganismsthroughtheproductionofreactiveoxygenspecies.PDI’smechanismofactionisnonspecificbutitseffect is immediateandconfinedtoregionswhereaphotosensitizer, light,andoxygenoverlapinspaceandtime.OrthoquinTM,abotanicalplant extract developed by PhotoDynamic Inc., is a powerful photosensitizer with antimicrobial properties that areamplified by exposure to visible light. OrthoquinTM-mediated PDI has been shown to kill bacteria and destroy biofilmwithoutcausinginflammation,makingitanattractivecandidatefortreatingoralbacterialdiseases.Herpessimplexvirusinfections are commonglobally (affecting67%of peopleunder age50) and symptoms includepainful oral (HSV-1) orgenital/anal (HSV-2) lesions. OrthoquinTM-mediated PDI was investigated as a potential treatment for symptomsassociated with these two viral infections.Methods: OrthoquinTM-mediated PDI was tested against HSV-1 and HSV-2infectionofHeLacellsusingplaquereductionassaysandsinglecycle infectionassays(viaflowcytometry),wherevirusinoculumwas incubatedwith different concentrations of OrthoquinTM and exposed to visible light before inoculatingcells. Results: Upon photoactivation, OrthoquinTM-mediated PDI inhibited HSV-1 and HSV-2 infections in a dose-dependentmanner, thoughwith different efficacies against each virus. Conclusions: PDI is a promising alternative totraditional antibiotics and antivirals due to its immediate and selective mechanism of action. We showed thatOrthoquinTMinhibitsherpessimplexvirusinfection. Topic#5:EmergingMethodsinVirology

Flowvirometry:AnovelandpowerfulapproachtodecipherviralegressBitaKhadivjam1,NablElBilali1,EricBonneil1,PierreThibault1,RogerLippé11UniversitédeMontréal

Flowcytometryhasbeeninstrumentaltocharacterizenormalandinfectedcells.However,ithasuntilrecentlynotbeenpossible to use such approach to analyze small entities such as viruses owing to the 0.5 µm resolution of mostinstruments. To circumvent this limitation, some laboratories decorate viruses with antibodies or nanoparticles. Ourlaboratory instead exploits an alternative approach that relies on the tagging of internal viral constituents withpermeablesytodiesorthefluorescenttaggingofindividualviralproteinaceouscomponents,whethercapsid,tegumentorglycoproteins.Thisopensuparangeofnewresearchavenuesand,forexample,enabledustocharacterizeindividualHSV-1particles,discernandcharacterize theirdifferentnuclear subpopulations,measure theheterogeneityofmaturevirionsintermsofproteincontent,sorttheseviralparticleswith>90%purityand,forthefirsttime,directlyaddresstheimpactofthisheterogeneityonviralfitness.Thisapproach,coinedflowvirometrybyothers,allowsthestudyofawidevarietyofpathogenswithhighstatisticalsignificanceandthepotentialdiscoveryofnovelvirulencefactors.


SynViro:BuildingthenextgenerationoftherapeuticpoxvirusvectorsRyanNoyce11UniversityofAlberta

The power of synthetic biology is revolutionizing the way we build better therapeutics. Given the importance ofmodifyingvectorstoexpress immunodominantantigensagainstaparticularpathogenortumourantigens, it isvital toestablishaplatformtodothisinasystematicandrapidmanner.Poxvirusesarewellsuitedasbiotherapeuticvectorstodeliver foreign antigens, although manipulating their genomes can become time-consuming when one would like tomanipulateanumberofdifferentareasof thegenomesimultaneously.Here,wewilldiscussanovelplatformthatwehavedevelopedtoreactivatepoxvirusesfromoverlappingsyntheticDNA.Asaproofofprinciple,wechosetoreactivatehorsepox virus (HPXV) sincemolecular analyses have shown thatmodern vaccinia virus (VACV) vaccine strains sharecommonancestrywiththisvirus.FragmentsofDNAweresynthesizedbasedonapublishedHPXVsequencealongwithVACV terminal sequences. These fragments were recombined into a live synthetic chimeric HPXV (scHPXV) in cellsinfectedwithahelpervirus.Sequencingofthe212kbpscHPXVconfirmeditencodedafaithfulcopyoftheinputDNA.Webelievethisisthefirstcompletesynthesisofapoxvirus.ThisscHPXVproducedsmallerplaquesinvitroandexhibitedlessvirulence inmice compared toVACV, but still provided vaccine protection against a lethal VACV challenge.Our lab isfurther investigating thismethodology for themanufactureofotherpoxvirus-basedtherapeutics,withapplications forrapid synthesis of synthetic chimeric vaccines and oncolytics. We will also discuss some of the technical challengesassociatedwithusingsyntheticbiologyasatooltodevelopthesenext-generationofpoxvirus-basedtherapeutics.TargetingPersistentHepatitisBVirus:IdentificationofaG4-QuadruplexStructureMotifinaKeyRegionofHBV’sGenomeVanessaMeier-Stephenson1,2,SarahShultz2,Hans-JoachimWieden2,CarlaSCoffin1,TrusharRPatel1,2,31DepartmentofMedicine,CummingSchoolofMedicine,UniversityofCalgary,Calgary,AB,2AlbertaRNAResearchandTrainingInstituteandDepartmentofChemistryandBiochemistry,UniversityofLethbridge,Lethbridge,AB,3DiscoveryLab,FacultyofMedicine&Dentistry,UniversityofAlberta,Edmonton,AB

Background: Approximately 240 million people worldwide are chronic carriers of hepatitis B virus (HBV), and are atincreased risk of developing cirrhosis and hepatocellular carcinoma. The virus persists in the nucleus in a covalentlyclosedcircularDNA(cccDNA),whichactsasthetemplateforallHBVmRNAtranscripts.Whilecurrentantiviraltherapiesare effective at viral suppression, they do not target HBV cccDNA and cannot eradicate infection. Recently, we havediscoveredandprovideevidenceforauniquestructuralmotif inHBV’spre-corepromoterregion—aG4-quadruplex—adistinct, stacked, four-guanosine foldingarrangementofDNA. Suchquadruplexesarebeing foundat key transcriptionandtranslationsitesofnumerousorganismsandarethoughttoregulatetheseprocesses.Methods:Thewild-typeandsingle-nucleotide mutation oligomers of the HBV pre-core promoter region were purified through size-exclusionchromatography.Fractionswereanalysedusingcirculardichroism(CD),electrophoreticmobilityshiftassay(EMSA),andsmall-angle X-ray scattering (SAXS) analyses. Next, a known quadruplex-binding protein, DHX36was expressed via anE.coliexpressionsystemandpurifiedusinghistidine-tagaffinitychromatography.Finally,cccDNAwasextractedfromanHBV-infected liver explant and pull-down assays using DHX36 were performed on whole and fragments of cccDNA.Results: Through CD, EMSA and SAXS data, we demonstrate that thewild-type oligomer forms a parallel quadruplexstructure,whilethemutantoligomerremains linear.Aswell,weshowthequadruplex-bindingprotein,DHX36,tobindthewild-typeoligomer,aswellaspulldowncccDNAfromclinically-relevantsamples.Conclusions:Thisnovelfindingofaquadruplex intheHBVpre-coreregionprovidesauniqueopportunitytostudyacriticalhost-protein interactionsite inHBVcccDNAtranscription.Throughthepursuitofhigh-resolutionstructuraldata,wewillbecreatingtheframeworkfordesigninganovelinhibitoroftheresilientHBVcccDNA,themastertemplateforHBVreplication.


DevelopmentofiPSC-derivedneuralstemcellstransducedwithBornadiseasevirusvectorexpressingthymidinekinaseforcancergenetherapyYumikoKomatsu1,2,KeizoTomonaga1,31LaboratoryofRNAViruses,DepartmentofVirusResearch,InstituteforFrontierLifeandMedicalSciences,KyotoUniversity,Japan,2TheKeihanshinConsortiumforFosteringtheNextGenerationofGlobalLeadersinResearch(K-CONNEX),KyotoUniversity,3MammalianRegulatoryNetwork,GraduateSchoolofBiostudies,KyotoUniversity

Bornadiseasevirus(BoDV)isanRNAvirusthatinfectsawiderangeofvertebrates.AnintriguingcharacteristicofBoDVisitsability toestablishapersistent infection in thenucleuswithout requiring to integrate into thehostgenome. Inourpreviousstudy,weexploitedbiologicalcharacteristicsofBoDVanddevelopedanon-integratingvectorsystemcapableofsustained transgene expression in humanpluripotent stem cells (hiPSCs). Recently,we found that neuroblastoma celllinesarehighly susceptible to infectionbyBoDVvector,whichpromptedus toexplorepotential applicationsofBoDVvectorforcancergenetherapy.Inthisstudy,wedevelopedaBoDVvectorencodingthymidinekinase(TK)andevaluatedanti-tumoreffectsofBoDV-TKvectorinvitro.HerpessimplexvirusTKwasinsertedbetweentheviralphosphoprotein(P)andmatrix (M) genes, and the recombinant vectorwas rescuedusing reverse genetics systemofBoDV. Todevelopasystem to target BoDV vector to cancer cells, we used neural stem cells (NSCs) as carrier cells, which have beendemonstratedtopossessinherenttumortropicproperties.hiPSCstransducedwithBoDV-TKvectorweredifferentiatedintoNSCsbyculturinginneuralinductionmedium.ExpressionlevelsofNSCmarkersNestinandPax6weresimilarinbothmock transduced and BoDV-TK vector transduced cells, indicating that BoDV vector does not interfere withdifferentiation of iPSCs into NSCs. Migration of NSCs loaded with BoDV-TK vector to cancer cells was analyzed bymodifiedboyden chamber assay.Ongoing experiments evaluating anti-tumor effects of BoDV-TKNSCs and ganciclovir(GCV)againstneuroblastomawillbepresented.Seeingisbelieving:Visualizingmulti-proteincomplexesmediatingHIV-1immuneevasionBrennanDirk1,ChristopherEnd1,JimmyDikeakos11UniversityofWesternOntario

AmajorfactorpreventingtheeliminationofHIV-1 lieswithintheabilityofthevirustohidefromthe immunesystem.This inherent ability of HIV-1 to confuse the immune system is accomplished by the small accessory protein Nef.Specifically,MHC-IdownregulationbytheHIV-1NefisofcriticalimportanceinpreventinginfectedcellsfromcytotoxicT-cellmediatedkilling.NefdownregulatesMHC-Ibymodulatingthehostmembranetraffickingmachinery,resultingintheendocytosisandeventualsequestrationofMHC-Iwithinthecell.Here,weexpandonourpreviousworkwhichidentifieda molecular map underlying the subcellular compartments mediating viral immune evasion. Utilized the intracellularprotein-protein interaction reporter system, bimolecular fluorescence complementation (BiFC), in combination withsuper-resolution microscopy, we identified the membrane trafficking regulator PACS-1 to be critical in MHC-Isequestration. The interaction between PACS-1 and Nef was found to be critical in the recruitment of the clathrinadaptor AP-1 to the Nef:MHC-I complex. Through super-resolution microscopy, we were able to determine that themutation of the Nef:PACS-1 interface doubled the molecular distance between AP-1 and MHC-I (30nm to 75nm),highlighting the role of PACS-1 as a critical mediator of Nef-mediated MHC-I downregulation. Furthermore, wedemonstrate that mutation of the Nef:PACS-1 interaction drastically changes the localization of MHC-I to the earlyendosomes.Overall,wehighlighttheabilityofsuper-resolutionmicroscopyandfluorescentimagingtoidentifyandtrackmulti-proteincomplexesmediatingHIV-1immuneevasion.UnderstandingthesecomplexmolecularpathwayswillaidinthedevelopmentofnewinhibitorstargetedatcripplingHIV-1’sabilitytoevadeourownimmunesurveillancesystem.


Topic#6:RNAinVirusInfection miR-122-mediatedprotectionoftheHCVgenomefrompyrophosphataseactivityAnnieBernier1,JasminChahal1,YalenaAmador-Cañizares2,JoyceWilson2,SelenaSagan1

1McGillUniversity,2UniversityofSaskatchewan

HepatitisCvirus(HCV)recruitstwomoleculesoftheliver-specificmicroRNA-122(miR-122)tothe5’endofitsgenome.Incontrast to the canonical activity of miRNAs, the interaction of miR-122 with the viral genome promotes viral RNAaccumulation in cultured cells and animalmodels of HCV infection. Although this interaction has been the subject ofseveral studies, the precise mechanism of miR-122-mediated viral RNA accumulation remains incomplete. PreviousstudiessuggestthatmiR-122isabletoprotecttheHCVgenomefrom5’exonucleases(Xrn1/2),butthisprotectionisnotsufficient toaccount for theeffectofmiR-122onHCVRNAaccumulation.Wehypothesized thatmiR-122 interactionswiththe5’ terminusof theHCVgenomepreventsrecognitionof theviral5’ triphosphateby innatesensorsofRNAorcellular pyrophosphatases. We thus explored miR-122-dependent and miR-122-independent viral RNA accumulationduringknockdownoroverexpressionofseveralinnatesensorsofRNAorpyrophosphatases.WefoundthatmiR-122doesnotplayaprotectiveroleagainstrecognitionbyproteinkinaseR(PKR),RetinoicacidinducibleI(RIG-I)-likereceptors,orIFN-inducedproteinwith tetratricopeptiderepeats (IFITs)1and5.However,we foundthatknockdownof twocellularpyrophosphatases, DOM3Z and DUSP11, was able to restore viral RNA accumulation of subgenomic replicons in theabsence ofmiR-122. However, pyrophosphatase knockdownwas not able to rescue viral RNA replication inmiR-122knockout cells. Thus,weprovideamodelwherebymiR-122mediatesprotectionof the viral 5’ terminus fromcellularpyrophosphataseactivityandsubsequentturnoverbyXrn1/2.However,miR-122mustplayanadditionalrole(s) intheHCVlifecyclebeyondgenomestabilitysincepyrophosphataseknockdownwasnotabletorescueviralRNAaccumulationin the absence of miR-122. In addition, we have used Selective 2’ Hydroxyl Acylation analyzed by Primer Extension(SHAPE)toinvestigatehowmiR-122bindingaltersthestructureofthe5’non-codingregionoftheHCVgenome.Wealsoprovidepreliminarydatasuggestingthatmutations inthe5’non-codingregionthatrelieveHCV’sdependenceonmiR-122alterthestructureofthe5’non-codingregioninamannerthatpromotesHCVgenomestability.InsightintoinfluenzaAviruscap-snatchingMartinPelchat11DepartmentofBiochemistry,MicrobiologyandImmunology,FacultyofMedicine,UniversityofOttawa,Ottawa,Ontario,K1H8M5,Canada

Influenza A Virus (IAV) causes yearly epidemics and significant worldwide mortality. IAV has a segmented genomecomposedofeightsingle-strandednegativesenseviralRNAs(vRNAs).AttheearlieststepofIAVreplication,acomplexformedbytheviralRNA-dependentRNApolymerase(RdRp)andanyofthevRNAs interactswithhostRNAPII,cleavesthe5’endofhostpre-mRNAs,andusesthesecappedRNAfragmentsasprimersforviralmRNAsynthesis,usingaprocesscalled cap-snatching. Because the capped RNA fragments also contain 10-15 nucleotides downstream of the cap,sequenceheterogeneity isthusfoundatthehost-derived5’endsofviralmRNAs.Profilingofthehostprimersusedbyseveral IAVstrains indicates that theeightRdRp:vRNAcomplexescap-snatchdifferentsubsetsofhostRNAs,andthatvRNA UTRs might provide target specificity during cap-snatching. This suggests that features on vRNA UTRs areresponsiblefortargetspecificity,and/orthattheavailabilityofhostcappedprimersmightbedictatedbythedynamicsofboth IAV transcriptionand thehost response.More importantly,our results indicate thathostRNAsarecap-snatchedbasedon theirabundance,and thatprimersassociatedwithgenes linked to thecellular response to IAV infectionareoverrepresentedinthecap-snatchedpopulation.BytargetingtheseRNAsearlyduringinfection,IAVcouldmodulatetheantiviralresponseearlyafter infection,whichcouldleadtoenhancedreplicationandpathogenesis.FurtherstudiesareunderwaytoelucidatethemolecularmechanismofIAVcap-snatching,andtodeterminetheroleofcap-snatchingasamolecularmechanismusedbythevirustoinhibitthecellularresponseearlyafterinfection.


ViralModulationofRNAGranulesisReliantonAutophagicFluxCarolyn-AnnRobinson1,ElizabethL.Castle1,GillianK.Singh1,BreQ.Boudreau1,JenniferA.Corcoran1,2,31DepartmentofMicrobiologyandImmunology,DalhousieUniversity,2DepartmentofSurgery,DalhousieUniversity,3BeatriceHunterCancerResearchInstitute,Halifax,NovaScotia

Kaposi’s Sarcoma-associated herpesvirus (KSHV) is the etiological agent of Kaposi’s Sarcoma. Kaposin B (KapB) is ahallmarkviralgeneexpressedinboththelatentandlyticlifecyclesofthevirus.KapBcanactivatetheMK2-RhoAaxistopromotetheformationofactinstressfibresandthedisassemblyofprocessingbodies(PBs).PBsaresmall,cytoplasmic,ribonucleoprotein granules found in all cells that regulate gene expression via decay or translational suppression ofmRNA.PBsplayacentralroleintheconstitutivedecayofasubsetofhumantranscriptsthatcontainAU-richelements(AREs)andtypicallycodeformoleculessuchasproinflammatorycytokinesandangiogenicfactors.ThedispersalofPBsbyKapB requiresMK2activation;however, theprecisedetailsof thismechanism remainunclear.MK2activationhasbeen shown to upregulate autophagy through the phosphorylation of Beclin.We hypothesize that KapB is engagingautophagytodisassemblePBsandpreventthedecayofARE-mRNAs,therebyincreasingtheexpressionofinflammatorymolecules.Primaryendothelial cellswere transducedwith lentivirusescontainingKapBoravectorcontrol; cellsweretreatedwithvarious inhibitorsandactivatorsofautophagy; LC3punctaewerevisualizedby immunofluorescence,p62and residentPBproteinexpression levelswereassessedby immunoblot. KapB-expressing cells showed increasedLC3punctae and decreased steady-state levels of p62 protein, indicating that KapB expression increases autophagic flux.KapB-expressingcellsalsoshowedadecreaseintheexpressionofseveralresidentPBproteinscomparedtocontrols,anddroplet digital PCR revealed increased steady state levels of ARE-containing inflammatory cytokine mRNAs. Whenautophagic flux was blocked, PB levels increased, while decreased levels of PBs occurred during induction ofautophagy. Furthermore, a luciferase assay for ARE-mRNA stability showed an increase in ARE-mRNA stability duringinductionofautophagyandadecreaseduringinhibitionofautophagy.Collectively,thesedatashowthatthemechanismof KapB-induced PB dispersal and concomitant increases in inflammatory gene expression rely on engaging theautophagic pathway. We believe that through thismechanism, KapB creates a pro-inflammatory and pro-angiogenicenvironmentthatbenefitsKSHVinfectionandKStumourprogression.AlternativetranslationinitationduringKSHVlyticreplicationEricPringle1,CraigMcCormick21DalhousieUniversity,2DalhousieUniversity

Kaposi’s sarcoma-associated herpesvirus (KSHV) the causative agent of Kaposi’s Sarcoma and primary effusionlymphoma.As aherpesvirus, KSHV replicates in thenucleuswhere it has access tohostmRNAprocessingmachinery.However,wehavefoundthattranslationofcapped,poly-adenylatedKSHVmRNAsdoesnotrequirethecanonicaleIF4Ftranslation-initiationmachinery.Here theexamine the roleof stress-responsive, alternative initiation systems inKSHVreplicationandassess the compositionof translatingmRNA-ribonucleoprotein complexes (mRNPs) in viral latencyandlytic replication. We isolated translating mRNPs using ultracentrifugation and sucrose-gradient fractionation. Proteincomponentsof translatingmRNPswere identifiedbyquantitative LC-MS/MSandconfirmedwith immunoblotting.WeusedRNAsilencingtolimitactivityofdifferenttranslationinitiationsystemsandassesstheirroleinvirusreplication.Inlytic replication, eIF4F is functional but not required for virus replication, while methyl-6-adenosine-dependenttranslationisrequired.LC-MS/MSstudiesidentifythreeviralproteinsthatspecificallyassociatewithactivelytranslatingmRNAs. KSHV usurps distinct host translation initiation systems during latent and lytic phases of infection. Thesedifferent systems likely have important roles in altering the translatome to support viral latency or promote virionproduction.


TowardsObtainingaNanoscaleStructureofthe3ʹTerminalRegionofJapaneseEncephalitisVirusGenomeTylerMrozowich1,VanessaMeier-Stephenson2,JustinVigar1,Astha3,JanuszBujnicki3,Hans-JoachimWieden1,TrusharRPatel41UniversityofLethbridge,2UniversityofCalgaryandUniversityofLethbridge,3LaboratoryofBioinformaticsandProteinEngineering,InternationalInsititueofMolecularandCellBiologyinWarsaw,4UniversityofLethbridge,UniversityofAlberta,UniversityofCalgary

Background: Japanese encephalitis virus (JEV) elongs to the Flaviviridae family of RNA viruses – a family that includesotherpathogenicviruses suchasDengue,WestNile,andZika.Theseviruseshavea conserved3’ terminal region thatplaysasignificantroleinviralreplicationbymediatinglong-rangeinteractionswiththe5’terminalregionofthegenome.The3’ terminal regionalso interactswithhostproteinsaidingviral replication.Despite thesignificance these terminalregions play in human infection, their structural details are largely unknown.Methods: Plasmidswere designed thatencodefortheentire~600kblong3’terminalregionofJEV,aswellasnumerousfragmentstherein.TheresultingRNAwaspurifiedusingsize-exclusionchromatography(SEC).StructuralstudiesoftheconstructswereperformedusingsmallangleX-rayscattering(SAXS)togeneratelow-resolutionmodelsoftheRNAandselective2'hydroxylacetylationanalyzedbyprimerextension(SHAPE).Results:Usinginvitrotranscription,full-lengthandfragmentsoftheJEV3’terminalregionRNA have been generated. Preliminary SAXS data indicate that one of the segments of the terminal region adoptsmultipleconformationsinsolution,whereasotherregionshaveelongatedshapes.Thedataisbeingcombinedwiththatof SHAPE in an iterative process to obtain high-resolutionmodels of these constructs.Conclusion: Future studieswillfocusonthestructuralanalysisofthe3’terminalregionincomplexwiththe5’terminalregionaswellasvarioushostproteins, includingDEADboxhelicases, togive further insight intoviral replicationand thusgive rise topotentialnewantiviraltargets.Topic#7:VirusesofFloraandFauna

TranslationinitiationofalternativereadingframesbyviralinternalribosomeentrysitesEricJan1

1UniversityofBritishColumbia

RNAstructurescan interactwith the ribosome toalter translational reading framemaintenanceandpromoterecoding that result inalternativeproteinproducts.Weshowedpreviously thatasubsetof internal ribosomeentry sites (IRESs) from thehoneybeedicistroviruses candirect translation in the0and+1 frames.Here,weshow that the IRES from the relateddicistrovirusCricketparalysis virusalsodrives translationof the0-frameviralpolyproteinandanoverlapping+1openreadingframe,calledORFx.Usinganunprecedentedmechanism,theCrPVIRESutilizesamechanismwherebyasubsetofribosomesrecruitedtotheIRESbypassesdownstreamto resumetranslationat the+1-frame13thnon-AUGcodon.AmutantofCrPVcontaininga stopcodon in the+1 frame ORFx sequence, yet synonymous in the 0-frame, is attenuated compared to wild-type virus inaDrosophilainfectionmodel,indicatingtheimportanceof+1ORFxexpressioninpromotingviralpathogenesis.Collectively, this work demonstrates a novel programmed IRES-mediated recoding strategy to increase viralcoding capacity and impact virus infection, highlighting the diversity of RNA-driven translation initiationmechanismsineukaryotes.


Expandingrepertoireofplant(+)-strandRNAvirusproteases:beyondthecysteineandserineproteasesHeleneSanfaçon1,KrinMann1,JoanChisholm11SummerlandResearchandDevelopmentCentre,AgricultureandAgri-FoodCanada

Manyvirusesencodeoneorseveralproteasesthatregulatethereleaseofmatureproteinsfromviralpolyproteins. Inaddition, viral proteases have been shown to cleave specific host proteins to facilitate virus infection. So far,characterizedproteasesfromplantvirusescorrespondtothreeclasses:cysteine,serineorasparticproteases.Cysteineandserineproteasesareencodedby (+)-strandRNAviruses,whileasparticproteasesareencodedbyretrovirusesandpararetroviruses. Members of the family Secoviridae (secovirids) constitute a large group of plant picorna-likeviruses. Like picornaviruses, they encode a 3C-like cysteine protease. So far, no other viral proteases have beencharacterized from secovirids. Strawberry mottle virus (SMoV, family Secoviridae) is one of several viruses found inassociationwithstrawberrydeclinediseaseinEasternCanada.Ithasabipartite(+)-strandRNAgenome,witheachRNAencoding one large polyprotein. RNA1 encodes the 3C-like cysteine protease that cleaves at five sites in the RNA1polyproteinandatasinglesitebetweenthepredictedmovementproteinandcapsidprotein(CP)domainsintheRNA2polyprotein. We detected two additional cleavage events in the RNA2 polyprotein downstream of the CPdomain. Cleavageat these sites isorchestratedbya secondproteasedomainpresent in theC-terminal regionof theRNA2polyprotein.SequencealignmentsofSMoVisolateswithrelatedviruses(blackraspberrynecrosisvirusandlettucesecovirus1)revealedseveralhighlyconservedaminoacids,whichweremutatedtoalanine.Surprisingly,mutationsofconservedserine,cysteine,histidineorasparticacidresiduesdidnotpreventtheproteolyticcleavage.Instead,weshowthat twoglutamicacid residuesare strictly required forpolyproteinprocessing.N-terminal sequencingof thecleavedproductusingEdmandegradationmappedthepredominantcleavagesitetoaP/AFPsequenceimmediatelydownstreamoftheCPdomainandsystematicmutagenesisofaminoacidsaroundthemappedcleavagesiteconfirmedarequirementforthetwoprolinesatthe-1and+3positionsofthecleavagesite.AsecondarycleavagesitewasdetectedatarelatedP/KFPsequence46kDadownstreamoftheprimarycleavageandwasalsoconfirmedbymutagenesis.WeconcludethatprocessingofSMoVpolyproteinsrequirestwoviralproteases,theRNA1-encodedcysteine3C-likeproteaseandasecondRNA2-encodedprotease,whichrepresentsanoveltypeofviralprotease,withputativeglutamicacidcatalyticresidues. TheunderlyingmechanismofauniquenuclearentrypathwayusedbytheparvovirusminutevirusofmiceShuangYang1,NellyPante11DepartmentofZoology,UniversityofBritishColumbia,BritishColumbia,Canada

Parvovirusesaresmall,non-enveloped,single-strandedDNAvirusesthatreplicateinthenucleusofthehostcell.Rodentparvovirusesarenotonlyabletopreferentiallytargetrapidlydividingcancercells,butalsopossessoncolyticproperties.They have thus been evaluated as potential anti-cancer agents, either as unmodified infectious virus, attenuatedmutants,orvectors.Forthedevelopmentofsuchparvoviruses-basedtherapy,weneedtobettercharacterizetheirlifecycle,particularlytheirnuclearentrypathway.Our laboratoryhaspreviouslyfoundthattheparvovirusminutevirusofmice(MVM)andotherparvovirusesenterthenucleusbyauniquemechanismthatinvolvessmall,transientdisruptionsof the nuclear envelope (NE) and partial dismantling of the underlying nuclear lamina.We are now investigating themolecular mechanism by which MVM disrupts the NE. Since N-myristoylation of different viral proteins has beenreportedtoplayrolesinvirus-cellularmembranesinteractionformanyothernon-envelopedviruses,andcapsidproteinsofMVMhaveputativeN-myristoylationsites,wehavebeeninvestigatingtheinvolvementofproteinN-myristoylationinthe nuclear entry mechanism of MVM. We found that the protein N-myristoylation inhibitor 2-Hydroxymyristic acid(HMA) significantly prevented viral gene expression inMVM-infectedmouse fibroblast cells.More importantly,whenaddedatdifferenttimepointpost-infection, the inhibitoryeffectsofHMAonviralgeneexpressioncoincidedwiththetimeofthenuclearentryofMVM.TofurtherillustratetheroleofproteinN-myristoylationduringthisprocess,weusedsemi-permeablizedHeLacellsinwhichtheinfluxofTRITC-dextranintothenucleusisanindicatorforMVM-inducedNEdisruption.WiththissystemwefurtherfoundthatwhenproteinN-myristoylationwasblockedbyHMA,MVM-inducedNE disruptionwas also inhibited. Therefore, we propose that protein N-myristoylation plays an important rolein themechanismofMVM-inducedNEdisruptionduringthenuclearentryofMVM.


AtypicalTransmembraneDomainofReptilianReovirusp14Fusion-AssociatedSmallTransmembrane(FAST)ProteinMediatesit’sTrafficking.GerardGaspard1,MuzaddidSarker1,TimKey1,NicholeMcMullen1,JanK.Rainey1,RoyDuncan1

1DalhousieUniversity

Reovirusfusion-associatedsmalltransmembrane(FAST)proteinsaresolelyre-sponsibleforthesyncytiogenicphenotypeofthefusogenicreoviruses.FASTpro-teinscompriseverysmallN-terminalectodomainsandequal-sizedorconsidera-blylargerC-terminal cytosolicendodomains flankinga single transmembrane (TM)domain.TMdomainsofdifferentFASTproteins do not share sequence similarity but are interchangeable with each other, but not with TM domains fromheterologousviralfusionproteins.ThesefindingssuggesttheFASTproteinTMdomainsareafusionmodulethatplaysamoredirectroleinthefusionprocess,butinsightsintoitsstructuralandfunctionalfeaturesarenotknown.Toaddressthisdeficit, theatomic resolutionstructureofaFASTproteinTMdomainwasdeterminedusingsolutionstatenuclearmagneticresonance(NMR)spectroscopyinthepresenceofdodecylphosphocholinemicelles.The25-residue-helicalTMdomainof thereptilian reovirusp14FASTproteinconsistsofa funnel shapedhe-lixowing toaskeweddistributionofbulkyaromaticresiduestowardsitsC-terminus.Wealsonotedapreponderanceofhydrophobicβ-branchedresiduesatthe N-terminal interfacial region, and what could be a conserved solvent-accessible cleft in the concave face of thecurvedhelical structure.Functionalandbiochemicalanalysis (cell fusionefficiency,proteinabundanceandcell surfaceexpression)of individualpointmutationsaffecting theseatypicalTMdomaincharacteristics supporteda role forallofthesefeaturesinproteintrafficking.Porcinecircovirus2(PCV2)activatedapoptosisofnon-infectedcellsJaredRowell1,JanaHundt1,RkiaDardari1,MarkusCzub11UniversityofCalgary

Porcine circovirus 2 (PCV2) is a pathogen of major importance for swine production around the world. Despitedevelopmentof several vaccinesand robustvaccinationprograms,PCV2continues topersistently transmitwithinandbetween swine herds causing infectionsmarked by immunosuppression via lymphocyte depletion. This study aims toimprovetheunderstandingofthepathogenesisofporcinecircovirusdiseases(PCVD)causedbyPCV2capsidcytotoxicityby revealingacytotoxicmotifwhichactivatesapoptosis innon-infectedbystandercells.Thecapsidprotein is theonlystructural protein of PCV2. Preliminary results show thatmonomeric PCV2Cap and1% formaldehyde inactivated anddialyzed PCV2 are both able to activate apoptosis in porcine kidney (PK15) cells at a rate of ~%30 after a 24-hourexposure;whichissimilartoapoptosiscausedby50µMetoposide,astrongchemicalinducerofapoptosis.Theseresultswere obtained through flow cytometric analysis of apoptosis usingAnnexinV-FITC and 7-AAD tomeasuremarkers ofapoptosis and cell death. The results support the conclusion that both monomeric PCV2 Cap and 1% formaldehydeinactivatedPCV2induceapoptosisinPK15cellsafterexternalexposurewithoutinfection,lendingcredencetotheideaofPCV2Capasaprimaryvirulencefactorduringinfections.Futureexperimentswillutilizetheaboveapoptosisassayflowcytometry experiment using non-pathogenic PCV1 and PCV2 Cap virus-like particles (VLPs) produced in insect cellsthroughabaculovirusexpressionsystem(BES)toinvestigatethecytotoxicityofPCV2Cap.ThisstudymayalsoshedsomelightonthedifferenceinpathogenesisbetweenPCV2andthenon-pathogenicPCV1whichisalsopoorlyunderstood.


Topic#8:AntiviralInnateImmunityClassAscavengerreceptors:theirroleininnateantiviralimmunityinlowervertebratesNguyenT.K.Vo1,SarahJ.Poynter3,AndreaL.Monjo2,JeremyWeleff1,AdamSoares1,GabriellaMicheli1,JoshuaEverson2,StephanieJDeWitte-Orr1,21WilfridLaurierUniversity,DepartmentofHealthSciences,Waterloo,Canada,2WilfridLaurierUniversity,DepartmentofBiology,Waterloo,Canada,3UniversityofWaterloo,DepartmentofBiology,Waterloo,Canada

Class A scavenger receptors (SR-As) are a family of five surface expressed receptors who in mammals play roles inmacrophage-mediatedclearanceofpathogensandapoptoticcells,atherosclerosis,andas surface receptors for innateimmune stimulantsand for virusentry.Very little is knownof SR-As, including their sequence,expressionprofileandfunctioninlowervertebrates.OurgrouphasfocusedinthelastfewyearsonelucidatingtheroleofSR-Asinmediatinginnate antiviral immune responses in lower vertebrates, specifically using teleost fish and frog cell lines.Of particularinterest iswhetherSR-As in lowervertebratesaresimilartotheirmammaliancounterparts intheirabilitytobindviraldouble-stranded (ds)RNA, a potent innate immune stimulant produced by viruses during replication. We havesuccessfullyidentifiedthefullcodingsequenceforMARCO,SCARA3,SCARA4andSCARA5fromrainbowtroutandhavefoundthatcellsexpressingfunctionalSR-AsareabletobinddsRNA.AsopposedtomammalianSR-Aswhereanymemberof the family appears to bind dsRNA, rainbow troutMARCO when over-expressed did not bind dsRNA, but did bindbacteria.We have also characterized CHSE-214, a Chinook salmon cell lines, as a newmodel for studying SR-As anddsRNAbinding,asthiscelllineisunabletobinddsRNAunlessengineeredtoover-expressexogenousSR-As.Finally,wehave begunwork identifying SR-A sequences in newly developed frog cell lines.We have found our frog cell lines allexpressSR-Asatthetranscriptlevel,albeitwithdifferentexpressionpatterns,andthoseexpressingSR-AIareabletobindAcLDL,anSR-AI ligand.Wehavealsofoundthatallofourfrogcell linesareexquisitelysensitivetodsRNA-inducedcelldeathcomparedto fishcell lines.FinallywehaveevidencethatSR-Asmaybe functioningassurfacereceptors for theranavirus, frogvirus3. Thesedataprovideevidence for the significanceof SR-As in innateantiviral immunity in lowervertebratesandtheimportanceofunderstandingthiscomplexfamilyofreceptorsinfishandfrogs.IFNandcytokineresponsesinduckstogeneticallysimilarH5N1influenzaavirusesofvaryingpathogenicityLeinaB.Saito1,LauraDiaz-Satizabal1,DanyelEvseev1,XimenaFleming-Canepa1,SaiMao1,2,RobertG.Webster3,KatharineMagor11UniversityofAlberta,2SichuanAgriculturalUniversity,3StJudeChildren'sResearchHospital,Memphis,TN

Ducks,thereservoirhost,aregenerallypermissivetoinfluenzaAvirusinfectionwithoutdiseasesymptoms.Thisnaturalecology was upset by the emergence of H5N1 strains, which can kill ducks. To better understand the host-virusinteractions in thereservoirhost,and influenzastrain-specificmolecularcontributions tovirulence,we infectedWhitePekinduckswiththreesimilarH5N1viruses,withknowndifferencesinpathogenicityandreplicationrate.WequantifiedviralreplicationandinnateimmunegeneactivationbyqPCR,inlungandspleentissues,isolatedoneachofthreedaysofinfection.Allthreevirusesreplicatedwell,asmeasuredbyaccumulationofmatrixgenetranscript,andviralloaddeclinedovertimeinthespleen.Theducksproducedrapid,buttemporallylimited,interferonandcytokineresponses,peakingonthe first day post-infection. Interferon and proinflammatory cytokine gene induction were greater in response toinfectionwiththemorelethalviruses,comparedtoanattenuatedstrain.Weconcludethatawell-regulatedinterferonresponse, with the ability to overcome early viral immune inhibition, without hyperinflammation, contributes to theabilityofduckstosurviveH5N1influenzareplicationintheirairways,andyetclearsystemicinfectionandlimitdisease.


MurineCytomegalovirusImmuneEvasionTargetingtheMEK-IRF1ConnectionJiangyiHe1,YumikoKomatsu1,SuzetteRutihinda1,KensukeHirasa11MemorialUniversityofNewfoundland

Murine cytomegalovirus (MCMV) modulates host immune system for their propagation. Although MCMV immuneevasion against NK cells or CD8 T cells has been extensively studied, less is known aboutmodulation of host innateimmunity byMCMV. Recently,we found that activation ofmitogen-activated protein kinase kinase (MEK) suppressesantiviralfunctionsofinterferonregulatoryfactor1(IRF1)(ref.1).AsMCMVinfectionisknowntoactivateMEK(ref.2),wehypothesized thatMCMV suppresses antiviral functions of IRF1 by promotingMEK activation during infection.WhenNIH3T3cellsormouseembryonicfibroblasts(MEF),wereinfectedwithMCMV,weobservedincreasedphosphorylationofextracellularsignal-regulatedkinases(ERKs),downstreamelementsofMEK,at1,2,3and4daysafterinfection,anddecreased expression of IRF1 at 2, 3 and 4 days after infection. Treatment of theMEK inhibitorU0126 restored IRF1expressionandfurtherinhibitedMCMVinfection.WearecurrentlyinvestigatingwhetherMEKactivationregulatesIRF1functionsbymodulatingoneofitsposttranslationalmodifications,SUMOylation,duringMCMVinfection.ThisstudywillidentifyanovelstrategyofMCMVimmuneevasionthattargetsthepotenthostantiviralproteinIRF1.1. Oncogenic Ras inhibits IRF1 to promote viral oncolysis. (2015) Komatsu Y, Christian SL, Nhu Ho, Pongnopparat T,

LicursiMandHirasawaK.Oncogene,34(30):3985-93.2. cAMPresponseelementofmurinecytomegalovirusimmediateearlygeneenhanceristransactivatedbyrasoncogene

products.GaboliM,AngerettiA,LemboD,GariglioM,GribaudoG,LandolfoS.JGenVirol.1995Apr;76(Pt4):751-8.TheNADPHoxidaseDUOX2differentiallymodulatestheproductionofrespiratoryvirus-inducedcytokinesbyairwayepithelialcellsDacquinKasumba1,NataliaZamorano1,EspéranceMukawera1,AudrayFortin1,AlexaRobitaille1,KarinFink1,QuentinOsseman1,NathalieGrandvaux11CRCHUMandDepartmentofBiochemistry,UniversitédeMontréal

AirwayEpithelialCells (AECs)constitutethe first lineofdefenseagainst respiratoryviruses.AECsautonomousantiviralresponsereliesonthesecretionofmucus,peptidesandawiderangeofsolublemediators,calledcytokines.Aspartoftheantiviralresponse,efficientproductionofcytokinesbyAECsisanimportantdeterminantoftheoutcomeofinfectionthrough the shaping of the antiviral (innate and adaptive) and inflammatory response. However, an excessiveinflammatoryresponseleadingtoanexaggeratedrecruitmentandactivationofinflammatorycellsoftencontributestovirus-associated pathogenesis and disease severity.We and others have previously documented the induction of theepithelialH2O2-generatingNADPHoxidaseDUOX2expressionandactivityatlatetimeofinfectionbyrespiratoryviruses,includingSendaivirus(SeV),RespiratorySyncytialVirus,andinfluenzavirus.DUOX2activitywasfoundtobeessentialfortheestablishmentofanantiviralresponsethroughthesustainedproductionoftypeIandIIIInterferons.Consideringtherole of various cytokines in shaping the antiviral and inflammatory response against respiratory viruses,we sought todefine the contribution of DUOX2 in the regulation of a panel of cytokines produced by AEC during respiratory virusinfections.DUOX2expressioninA549cells(ahumanalveolarepithelialcelllineage)wassilencedbysiRNAtransfection.Cellswerethen infectedwithSendaivirus (SeV)asmodelof respiratoryvirus infection.Usingmultiplex luminex-basedassays,weprofiledthelevelsofapanelof48cytokines.Amongstthose,30cytokinesareinducedbySeVindependentlyof DUOX2. Interestingly, DUOX2 modulates, either positively or negatively, a limited subset of twelve SeV-inducedcytokines.OurresultsproposearoleofDUOX2inthemodulationoftheintensityorqualityofantiviralandinflammatoryresponses orchestrated by AECs. Current studies are aimed at validating these results in AECs deficient in DUOX2obtainedviagenomeeditingandatstudyingtheimpactontherecruitmentandactivationofimmunecells.TheproposedDUOX2-linkedimmune-modulationofcytokineproductionsuggestsapreviouslyunsuspectedinvolvementofthisH2O2-producingproteinininflammatoryresponsessuchascytokine-drivenrecruitmentandactivationofinflammatorycells.ProjectfundedbyCIHR


IGAimmunecomplexesstimulatenetosisduringviralinfectionandautoimmunityHannahStacey1,3,DianaGolubeva1,3,AlyssaPosca1,3,MuhammedAtifZahoor2,3,CharuKaushic2,3,EwaCairnes4,CaitlinE.Mullarkey1,3,MatthewMiller1,31DepartmentofBiochemistryandBiomedicalSciences,2DepartmentofPathologyandMolecularMedicine,3MichaelG.DeGrooteInstituteforInfectiousDiseasesResearch,McMasterImmunologyResearchCentre,McMasterUniversity,ON,Canada,4DepartmentofMicrobiologyandImmunology,WesternUniversity,ON,Canada

Neutrophil extracellular traps (NETs) are extracellular chromatin structures that play a role in trapping and killing avariety of pathogens. NETs are released in response to a variety of stimuli such as bacteria, fungi and viruses.Immunoglobulin A (IgA) is enriched atmucosal sites and interactswith FcαRI on the surface of neutrophils.Wehavefound that IgA-opsonized antigens stimulate NETosis using a pathway that is distinct from that of immunoglobulin G(IgG). Human neutrophils were first isolated from peripheral blood and stimulated with immune complexes (ICs)containing immunoglobulins and Influenza A virus (IAV) or human immunodeficiency virus (HIV). IgA-containing ICspotentiatedNETosiswhencomparedtoIgGcontainingcomplexes,andthiseffectwasdependentonFcαRI.WehavealsoshownthatIgAantibodiespurifiedfrompatientswithrheumatoidarthritis(RA)potentlyinducedautoantigen-dependentNETosis relative to IgA from healthy controls. Our data suggests that IgA-stimulated NETosis may occur through apreviously uncharacterized signaling pathway, which we are currently elucidating. Since IgA stimulates NET release,mucosalvaccinationscouldenhanceNETproduction,therebyreducingtheseverityofviralinfections.Incontrast,duringautoimmunitytheproductionofNETsmayexacerbatediseasethroughthereleaseofself-antigens.Therefore,inhibitingthispathwaycouldbeoftherapeuticbenefit.


ABSTRACTS:POSTERPRESENTATIONS (byorderofabstractnumber,presenterisunderlined)

Posters1−72:posterSession#1

1-ProteomicsofHerpessimplexvirustype1nuclearcapsidsNabilElBilali1,BitaKhadivjam1,EricBonneil1,PierreThibault1,RogerLippé11UniversitédeMontréalHerpessimplexvirushasacomplexlifecyclethatincludestheproductionofdistinctparticlesinthenucleus,wherenewcapsidsareassembled.Amongthesenuclearcapsids,procapsidsaredevoidofnucleicacidbutcontainlargeamountsofthepreVP22ascaffoldprotein.Thesethermo-unstableparticlesthenmatureintoA-,B-orC-nuclearicosahedralcapsids,dependingontheirabilitytoshedtheproteolyticallyprocessedscaffoldandincorporatetheviralgenome.Interestingly,C-capsidsarepreferentiallyexportedtothecytoplasmandultimatelygiverisetoinfectiousvirions.WhileA-,B-andC-capsids share several components, their distinct fate hints at meaningful differences. To probe them, we performedproteomicsstudiesofhighlyenrichednuclearcapsids,relyinginpartonflowvirometrytopurifyC-nuclearcapsids.Wefoundthatwhilemanyproteinsareindeedcommonamongthesenuclearcapsids,theyexhibituniquesetsofproteins.Thesenaturallyincludeviralproteinsbutalsomanycellularproteins.3-Developmentofpre-pandemicinfluenzavaccineagainsthighlypathogenicH5strainsYaoLu1,2,GuanQun(Leonard)Liu1,3,ShelbyLandreth1,3,AmitGaba1,RobertBrownlie1,YanZhou1,2,31VIDO-InterVac,UniversityofSaskatchewan,2DepartmentofVeterinaryMicrobiology,WesternCollegeofVeterinaryMedicine,UniversityofSaskatchewan,3Vaccinology&Immunotherapeutics,SchoolofPublicHealth,UniversityofSaskatchewanTheemergenceof highly pathogenic avian influenzaA viruses (HPAI) and their spillover intohumanpopulationposessubstantial economic burden and public health threats. AmongHPAI, H5 viruses are of particular concern given theirglobalspreadandpandemicpotential.Inthisstudy,weaimedatdevelopinganovelpre-pandemicH5influenzavaccinethat is of potential importance in improving pandemic preparedness. The first North American HPAI H5N1 strain(A/Alberta/01/2014)wasselectedforrecombinantH5hemagglutinin(HA)design.RecombinantH5HAisexpressedinbothmammalianandbacterialsystemforcomparisonofH5antigenicityfromdistinctexpression hosts. The oligomeric status and functional characterization of purified H5 immunogens from differentexpression systems were determined by native PAGE and hemagglutination assay in vitro. In the meantime, weestablishedthemousediseasesmodelfortheHPAIH5N1virus(A/Alberta/01/2014)with3differentdoses.Inthismodel,viralreplicationandinnateimmunegeneupregulationwerequantifiedbyTCID50andqPCR,respectively,inmouselungandspleentissues. ImmunogenicityandprotectiveefficacyofH5immunogenswill laterbedeterminedbyvaccinationandchallengestudiesinvivo.4-ElucidatingtheRoleofSenataxinDuringClassSwitchRecombinationandSomaticHypermutationJonathanMapletoft1,MatthewMiller21McMasterUniversity,2McMasterUniversity

Senataxin (SETX) is a large, 303kDa, ubiquitously expressedDNA: RNAhelicase that has been shown to play a role inmeioticrecombination,theDNAdamageresponseandtheantiviral response.There issignificantoverlapbetweentheknown functions of SETX and the pathways required for both class switch recombination (CSR) and somatichypermutation(SHM).Forexample,SETXisknowntoresolveR-loopsandbothCSRandSHMrequireR-loopformationtogenerateassDNAsubstrateforAID.Furthermore,SETXco-localizeswithproteinsinvolvedintheDNAdamageresponsethatareknowntoplayacentralroleinCSR,including53BP1andγ-H2AX.ToelucidatetheroleofSETXinCSRandSHMweareutilizingaSETXknockoutmousemodel.WehypothesizedthatSETXwouldplayanimportantroleinbothCSRandSHM via cell intrinsic and extrinsicmechanisms. Currently,we have demonstrated that SETX knockoutmice exhibit aprolonged antigen-specific IgM response, coupled with a delay in the production of antigen specific IgA antibodies,following vaccination with influenza virus. Additionally, we have demonstrated that SETX deficiency results in anincreasedmutation rate in the V(D)J region. Our futureworkwill focus on defining the precisemechanisms throughwhichSETXaffectsbothCSRandSHM.Thesestudieswillexpandourknowledgeoftheprocessesthatregulateantibodygenerationandmayinformrationalvaccinedesignstrategies.


6-NeuraminidaseInhibitorsEnhanceFc-DependentEffectorFunctionsElicitedbyHemagglutininStalk-BindingBroadly-NeutralizingAntibodiesAliZhang1,MatthewMiller21McMasterUniversity,2McMasterUniversity

InfluenzaAviruses (IAV)cause3-5millionserious illnessesandhalfamilliondeathseachyear.Vaccination is thebestway toprevent infection,butprotectionprovided isnarrowand ineffectiveagainstpandemic strainsdue toantigenicdrift in the immunodominant hemagglutinin (HA) head domain. Recently discovered broadly-neutralizing antibodies(bNAbs) that target the conserved HA stalk domain provide great promise towards development of a “universal”influenzavaccine.ThesebNAbsrequireFcreceptorbindingandeffectorcellfunctiontoconfermaximalprotection.Wenowshowthatenzymaticinhibitionofneuraminidase(NA)enhancesFc-dependenteffectorfunctionselicitedbybNAbsandmayreducethedevelopmentoftherapeuticresistance.Usinganinvitroantibody-dependentcellcytotoxicity(ADCC)assay,wedemonstratethatoseltamivir,anNAinhibitor,causesadose-dependentincreaseinbNAb-mediatedADCCofIAV infected cells. Using mouse models, we also show that oseltamivir/bNAb combination therapy is superior atprotectingagainstlethalIAVinfectioncomparedtousingeithertreatmentaloneinvivo.Furthermore,weareperformingexperiments to determine if oseltamivir/bNAb combination therapy is effective at preventing or delaying therapeuticresistance. Themechanismbywhich bNAbs protect against IAVpropagation is incompletely understood.Our findingsmay explain the variable efficacy of oseltamivir in patients, and also guide design of universal influenza vaccines andotherbNAb-basedanti-viraltherapeutics.7-RegulationofRespiratorySyncytialVirusinfectionbytheautophagyreceptorSQSTM1/p62inairwayepithelialcells.QuentinOsseman1,2,SandraCervantes1,2,EliseCaron1,EricS.Pringle3,AlexaRobitaille1,2,XioachunGuan1,2,CraigMcCormick3,NathalieGrandvaux1,21CRCHUM,2UniversitédeMontréal,3DalhousieUniversity

Respiratorysyncytialvirus (RSV)causesahighrateofmorbidityandmortality in infants,childrenandelderly,butalsoadults of all ages with a compromised immune system, cardiopulmonary diseases or who have undergonetransplantation,worldwide.RSVvaccinedevelopmentfacesnumerouschallengesandcurrentverycostlyprophylacticsare restricted to high-risk children. Antivirals targeting host factors required for virus replication are an emergingalternative to traditional direct-acting antivirals. RSV primarily replicates in airway epithelial cells (AECs). Therefore,understandingRSV:hostinteractioninhumanAECsiscrucialforfuturedevelopmentofdrugstargetingRSVreplication.Autophagy is critical for intracellularhomeostasis in response to stress, including virus infection.However, compellingevidence demonstrate that a number of viruses have evolved mechanisms to exploit and modulate the function ofautophagycomponentstousethemfortheirbenefit.Selectivityinautophagyisconferredbycargoreceptors,includingp62/SQSTM1,which tether ubiquitylated (Ub) cargo to a autophagosomes for degradation into autophagolysosomes.Previous data have highlighted an important role for p62-dependent autophagy in the bacteria and virus clearance,althoughp62wasalsoshowntopromotereplicationofsomeviruses.Very little information isavailableregardingtheinterplaybetweenRSVandautophagy. Importantly, theroleofautophagy inRSV infection inhumanAECshasnotyetbeenaddressed.Ourgoalistodeciphertheroleofp62-mediatedautophagyintheregulationofRSVinfectiouscycleinhuman AECs. We observed that RSV triggers autophagy in A549 cells, a model of AECs. Replication of RSV wassignificantlydecreasedincellswithincreasedautophagicactivitysupportingananti-RSVactivityofautophagy.Wealsoaccumulateddatasupportingactivationofthecargoreceptorfunctionofp62duringRSV infection.Phosphorylationofp62onSer403,amodificationknowntoenhancebindingofp62toitscargo,wasinducedinaTBK1-dependentmanner.Accordingly,p62bindingtopoly-UbproteinswasincreasedduringRSVinfection.InhibitionoflysosomalenzymesusingBafilomycin confirmed that autophagy was responsible for p62 degradation during RSV infection. The finding of RSVproteinsamongstpulleddownpolyUbproteinssuggestseitherdirectpolyUbofRSVprotein(s)ortheir interactionwithpolyUbhostprotein(s).Moreover,weobservedcolocalizationbetweenRSVFandLC3.Currentproteomics-basedstudiesareaimedatidentifyingp62poly-UbcargothataretargetedtoautophagosomesandhavearoleintheregulationofRSVreplication cycle. Altogether, our results imply that selective autophagy involving the p62 receptor exhibits antiviralactivityagainstRSVinhumanAECs.


8-TheNADPHOxidaseDUOX2DifferentiallyModulatestheProductionOfRespiratoryVirus-InducedCytokinesByAirwayEpithelialCellsDacquin Kasumba1, Natalia Zamorano1, Espérance Mukawera1, Audray Fortin1, Alexa Robitaille1, Karin Fink1, QuentinOsseman1,NathalieGrandvaux11CRCHUMandDepartmentofBiochemistry,UniversitédeMontréal

AirwayEpithelialCells (AECs)constitutethe first lineofdefenseagainst respiratoryviruses.AECsautonomousantiviralresponsereliesonthesecretionofmucus,peptidesandawiderangeofsolublemediators,calledcytokines.Aspartoftheantiviralresponse,efficientproductionofcytokinesbyAECsisanimportantdeterminantoftheoutcomeofinfectionthrough the shaping of the antiviral (innate and adaptive) and inflammatory response. However, an excessiveinflammatoryresponseleadingtoanexaggeratedrecruitmentandactivationofinflammatorycellsoftencontributestovirus-associated pathogenesis and disease severity.We and others have previously documented the induction of theepithelialH2O2-generatingNADPHoxidaseDUOX2expressionandactivityatlatetimeofinfectionbyrespiratoryviruses,includingSendaivirus(SeV),RespiratorySyncytialVirus,andinfluenzavirus.DUOX2activitywasfoundtobeessentialfortheestablishmentofanantiviralresponsethroughthesustainedproductionoftypeIandIIIInterferons.Consideringtherole of various cytokines in shaping the antiviral and inflammatory response against respiratory viruses,we sought todefine the contribution of DUOX2 in the regulation of a panel of cytokines produced by AEC during respiratory virusinfections.DUOX2expressioninA549cells(ahumanalveolarepithelialcelllineage)wassilencedbysiRNAtransfection.Cellswerethen infectedwithSendaivirus (SeV)asmodelof respiratoryvirus infection.Usingmultiplex luminex-basedassays,weprofiledthelevelsofapanelof48cytokines.Amongstthose,30cytokinesareinducedbySeVindependentlyof DUOX2. Interestingly, DUOX2 modulates, either positively or negatively, a limited subset of twelve SeV-inducedcytokines.OurresultsproposearoleofDUOX2inthemodulationoftheintensityorqualityofantiviralandinflammatoryresponses orchestrated by AECs. Current studies are aimed at validating these results in AECs deficient in DUOX2obtainedviagenomeeditingandatstudyingtheimpactontherecruitmentandactivationofimmunecells.TheproposedDUOX2-linkedimmune-modulationofcytokineproductionsuggestsapreviouslyunsuspectedinvolvementofthisH2O2-producingprotein in inflammatoryresponsessuchascytokine-drivenrecruitmentandactivationof inflammatorycells.ProjectfundedbyCIHR9-EpinephrineandnorepinephrineaugmentEbolavirusreplicationLaurenGarnett1,2,JimStrong1,2,311SpecialPathogens,NationalMicrobiologyLaboratory,PublicHealthAgencyofCanada,2DepartmentofMedicalMicrobiologyandInfectiousDiseasesUniversityofManitoba,Winnipeg,Canada,3DepartmentofPaediatricsandChildHealth,CollegeofMedicine,FacultyofHealthSciences,UniversityofManitoba,Winnipeg,Canada

Ebolavirus (EBOV) causesmultisystem failuremimicking sepsis in humans,with a fatality rate of up to 90%.AlthoughthereareanumberofexperimentalandprophylactictreatmentsforEBOV,therearecurrentlynoapprovedtherapeuticagents.AssuchpatientsdiagnosedwithEBOVaretreatedwithsupportivecaretherapy, includingtheuseoffluidsandvasoactivesubstances(epinephrine/andnorepinephrine)tostabilizebloodpressureinanattempttomaintainadequateorganfunction.PreliminaryevidencefromourlaboratorysuggeststhatthesevasoactiveagentscanaugmentreplicationofEBOVincellculture.Thisimplicatesthesehormones,bothfromsupportivecaretherapy(exogenous)andendogenoussecretions, in playing a role in thepathogenicity of thedisease.Although this is preliminary data in regards to EBOV,otherviral infections,suchasEnterovirus71(EV71)havebeenshowntohaveincreasedviralreplicationandenhanceddiseaseseverityinresponsetoepinephrineandnorepinephrine.TheaimofthecurrentstudyistoexploretheeffectsofvasoactiveagentsonEBOVreplication.Experimentswillbefirstcompletedinanenhancedlevel2biosafety laboratoryusingacellculturemodelwithvariouscelllinesinfectedwithPseudotypedVesicularstomatitisvirus-Ebola(VSV-EBOV),which expresses EBOV transmembrane glycoproteins exposed to varying concentrations of epinephrine andnorepinephrine.LaterstudieswillperforminfectionsofthesamecelllinesundersimilarconditionswithwildEBOVinacontainmentlevel4(CL4)environment.Ifvasoconstrictorsareshowntoaugmenttheviralreplicationashypothesized,thiscouldleadtofuturereconsiderationsofbloodpressuremanagementtechniquesforinfectedpatients.


12-Theinhibitoryeffectofproproteinconvertasesubtilisin/kexintype9onhepatitisCvirusreplicationZhubingLi1,2,QiangLiu1,21VaccineandInfectiousDiseaseOrganization-InternationalVaccineCentre(VIDO-InterVac),2UniversityofSaskatchewan

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a serine protease that plays an important role in lipidhomeostasisthroughfacilitatingthedegradationof low-densitylipoproteinreceptor(LDLR).HepatitisCvirus(HCV)isapositive-sensesingle-strandedRNAvirusthatcanresultinchronichepatitis.NoHCVvaccinehasbeendevelopedandthecurrent antiviral treatments have some limitations. Although PCSK9 has been shown to inhibit HCV replication, theunderlyingmechanismhasnotbeenthoroughlycharacterized. In thisstudy,we firstconfirmedthe inhibitoryeffectofPCSK9 on HCV replication by over-expressing or knocking down PCSK9 in HCV genomic replicon cells. Then wedemonstrated that PCSK9-induced LDLR degradation was not involved in HCV replication regulation using gain-of-function(D374Y)or loss-of-function(Δaa.31-52)PCSK9mutantsforLDLRdegradation.Moreover,theauto-cleavageofPCSK9affectedHCV replication sinceonlyuncleavedproPCSK9 suppressedHCV replicationandcleavedPCSK9didnothaveeffectonHCVreplication.Next,wefoundthatPCSK9couldinteractwithseveralHCVproteinsincludingNS5A.ThePCSK9interactingregionofNS5Awasaa.95-215inDomainI.TheinteractionbetweenPCSK9andNS5AinhibitedNS5AdimerizationandHCVRNAbindingtoNS5A.ConsideringthatNS5Adimerization,RNAbindingactivityofNS5Aisrequiredfor HCV replication, the interaction between PCSK9 and NS5A could be a mechanism of how PCSK9 inhibits HCVreplication.ThisstudymayimprovetheunderstandingoftheantiviraleffectofPCSK9onHCVandhelpoptimizeanti-HCVregimens.13-PrincipalcomponentanalysisasatooltostudytheevolutionaryhistorytheviralAlkBdomainClaytonMoore1,BaozhongMeng1,21UniversityofGuelph,2UniversityofGuelph

AlkBproteinsareubiquitousamongcellularorganismswheretheyacttoreversedamageinRNAandDNAresultingfrommethylation, suchas1-methyladenine, and3-methylcytosine.Recently, it hasbeendiscovered that anAlkBdomain isencodedaspartofthereplicasepolyproteinbyasmallsubsetofsingle-stranded,positivesenseRNAvirusesthatmostlyinfect woody perennial plants. However, the boundary of these viral AlkB domains as well as their function in viralreplication and infection is unknown. Phylogenetic characterization has indicated that this AlkB domain was likelyacquiredrelatively recentlybyanancestorof the familyBetaflexiviridae.Here,weutilizeprincipalcomponentanalysis(PCA) toestablish theboundariesofa functionalviralAlkBdomainataprimarystructure level.Additionally,weapplyPCAandotherclusteringtechniquestoanalyzetherelationshipbetweenviralAlkBdomainsfromdifferentviralfamiliesto examine the hypothesized single acquisition event. Using a similar approach, we compare and group AlkBproteins/domains from virruses, bacteria, plants, and animals into distinct substrate-specific clusters to hypothesize abroadoriginoftheviralAlkBdomain.WehopethisinsilicoanalysiswouldallowustomakepredictionsofthestructureandfunctionofviralAlkBdomainsthatcanbetestedthroughexperimentation.17-Identificationofvirus-hostreceptorsforAtlanticsalmonbafinivirus,acoronavirusoffishAshleyMcKibbon1,FrederickKibenge11UniversityofPrinceEdwardIsland/AtlanticVetrinaryCollege

Aquatic virology is a rapidly expanding field. This is due to the growing global demand for seafood, and subsequentintensificationofaquaculture.Thisintensificationcanservetofacilitatediseasesinfishduetothechronicstresscausedbythehigh-densityconfinement.Theseconditionsprovideopportunitiesfortheemergenceofviralpathogensthatmaybe harmless under natural conditions, such as Atlantic Salmon Bafinivirus (ASBV). ASBV is a recently discoveredcoronavirus, isolated from farmed Atlantic salmon. As ASBV is a recently isolated virus, its clinical significance is stillunknown, and as such it is uncertainhow itmay threatenbothwild, and farmedpopulationsofAtlantic salmon. Theprimary aim of this research is tomap the receptor-binding domain in the S1 subunit of the spike (S) protein of the(ASBV),andthecorrespondinghostcellsurfacereceptor(s)towhichitbinds.ThiswillbeelucidatedusingrecombinantPCRtechniques,andspecificco-purificationofreceptor-bindingsubunit fromlysatesofsusceptiblecells.Thesestudieswillservetoelucidatetheemergence,virulenceandpersistenceofASBV.


18-Involvementofthe90kDaRibosomalS6Kinases(RSKs)inviraloncolysisDanyangXu1,MariaLicursi1,YumikoKomatsu1,KensukeHirasawa11MemorialUniversityofNewfoundland

Oncolytic viruses exploit tumor-specific cellular changes for their selective replication, including p53 deficiency,oncogenicRasactivation, insensitivity to type I interferon (IFN)andviral receptorsuniquelyexpressedoncancercells.OurpreviousresearchshowedthatactivationoftheRas/MEKpathwaysuppressesthehostantiviralresponseinducedbyIFN1.ThisiscausedbysuppressionofthetranscriptionofagroupofIFN-induciblegenes2,3.Ourresearchfurthershowedthatthissuppressioniscausedbydownregulationofinterferonregulatoryfactor1(IRF1),thetranscriptionalregulatorofthe IFN-inducible genes 4,5. Together,we have demonstrated that the Ras/MEK downregulation of IRF1 is one of themajormechanismsunderlyingviraloncolysis.ActivatedMEK1and2phosphorylateERK1and2,whichinturnactivatetheirdownstreamsignalling(MAPK-interactingKinases(MNKs)1-2,Mitogen-andStress-activatedProteinKinases(MSKs)1-2 and the 90kDa Ribosomal S6 Kinases (RSKs) 1-4) to regulate transcription and translation. While we previouslydemonstrated the role of ERK in IRF1 regulation, the elements further downstream of ERKs that are involved in thisregulationstillremaintobeidentified.Inthisstudy,wefirstconductedRNAiscreeningofMNK1-2,MSK1-2andRSK1-4usingRasV12-transformedNIH3T3cells.IRF1expressionwasrestoredbyknockdownofRSK3andRSK4,butnotbythatoftheotherERKdownstreamelementsinthecellswithactivatedRas.OverexpressionofRSK3inNIH3T3cellsreducedIRF1expressionand inhibited transcriptionof IRF1-mediatedantiviral genes.CRISPRknockdownofRSK3also restored IRF1expression inhuman cancer cells. These results suggest thatRSKs are theERKdownstreamelements involved in IRF1downregulationincancercellswithactivatedRasandsubsequentlyinviraloncolysis.1. Battcock, S. M., Collier, T.W., Zu, D. & Hirasawa, K. Negative regulation of the alpha interferon-induced antiviralresponsebytheRas/Raf/MEKpathway.J.Virol.80,4422–4430(2006).2.Christian,S.L.etal.SuppressionofIFN-InducedTranscriptionUnderliesIFNDefectsGeneratedbyActivatedRas/MEKinHumanCancerCells.PLoSOne7,e44267(2012).3.Christian, S. L. et al.ActivatedRas/MEK inhibits theantiviral responseofalpha interferonby reducingSTAT2 levels.J.Virol.83,6717–6726(2009).4.Komatsu,Y.etal.OncogenicRasinhibitsIRF1topromoteviraloncolysis.Oncogene34,3985–3993(2015).5.AbuSara,N.etal.Restorationof IRF1-dependentanticancereffectsbyMEK inhibition inhumancancercells.CancerLett.(2014).20-BovineViralDiarrheaVirus:QuantifyingAntigenicityWilliamBremner1,FrankvanderMeer21UniversityofCalgary,2UniversityofCalgaryVeterinaryMedicine

Bovine viral diarrhea virus (BVDV) is a globally distributed pathogen responsible for a broad spectrum of illnesses incattle.Therapidevolutionandsubstantialdiversityof thevirushavemadebroadlyprotectivevaccinationchallenging.ThevariabilityofBVDViswelldocumented,howevertheimpactofthisdiversityontheantigenicityofthevirusremainsto be fully characterized.We aim to investigate the antigenic components of the E2 surface protein of the virus thatinduceimmuneresponsesincattle.Todoso,anexpressionvectorfortheproductionofawidearrayofvariantsoftheimmunodominantproteinE2wasdesignedusingenzyme-mediated restrictionand ligation.Uniqueenzyme restrictionsites flanking the E2 coding sequencewere included to allow for replacementwith alternate protein sequences. Theresulting constructswere introduced intomammalian cell culture via electroporation in order to produce appreciablequantities of the protein for downstream antigenic analysis using the Bio-layer interferometry platform. Thisexperimentalframeworkwillallowfortheanalysisofantigenicvariantspresentinwildtypeviruses,andtheevaluationoftheaffinitiesandaviditiesofantibodiesinducedthroughvaccinationtowardsfieldviruses.


21-GeneexpressionmodulationwithinNPTr-CD163infectedcellswhichareresistanttocelldeathinducedbyporcinereproductiveandrespiratorysyndromevirus(PRRSV)MarikaKoszegi1,ChantaleProvost1,CarlAGagnon11Facultédemédecinevétérinaire,UniversitédeMontréal

Porcinereproductiveandrespiratorysyndrome(PRRS)isadiseasewidelyspreadinNorthAmericaandisthesourceofhuge economic losses in the porcine industry. The PRRSV is known to trigger the death of permissive infected cellsthroughapoptosis, suchas theporcinealveolarmacrophages (PAM),andMARC-145cells.Recently,aporcinecell linewas genetically modified to study PRRSV co-infection pathogenesis with others swine pathogens. More precisely,newbornpigtracheacells(NPTr)weremodifiedtoexpresstheCD163receptor,toallowPRRSVinfection.Interestingly,PRRSVcan replicates intoNPTr-CD163cellsata similar rate than inMARC-145 infectedcellsbutwithout inducingcelldeath.Thatobservation leadtothefollowingquestion:howdoesPRRSVaffectthegeneexpression inMARC-145cellsand inNPTr-CD163 cells. Our hypothesis is thatNPTr-CD163 cells response against PRRSV infection is better adaptedwhichallowthesurvivalofthecellscomparedtoMARC-145 infectedcells.Therefore,mRNAexpressionmodulationoftheMARC-145cellsandtheNPTr-CD163cellsinfectedcellswasdeterminedbyaRNA-SeqexperimentusingtheIlluminatechnology.ThisexperimentwillgiveusimportantinsightsinregardstothedifferentmechanismsinvolvedintoPRRSVpathogenicity.22-Genomiccharacterizationofporcinereproductiveandrespiratoryvirus(PRRSV)QuebecstrainswithinclinicalsamplesusingwholegenomesequencingChristianLalonde1,2,3,ChantaleProvost1,2,3,CarlA.Gagnon1,2,31Centrederechercheeninfectiologieporcineetavicole,2Groupederecherchesurlesmaladiesinfectieusesenproductionanimale(GREMIP),3Facultédemédecinevétérinairedel'universitédeMontréal

PRRSVisresponsibleofmajoreconomiclossesintheswineindustryworldwide.TherearecurrentlyeffortstosequencetheORF5ofall circulatingPRRSVstrains inQuebec,withacurrentdatabasecontainingover4000ORF5sequences, inordertodomolecularepidemiologicalsurveillancebutnodataisavailableonthefull-lengthgenomeofPRRSVstrainsinQuebec. We hypothesized that full-length genome sequencing of PRRSV would provide a better insight into thepathogenicity of different strains and would allow for a better epidemiological monitoring compared to ORF5sequencing.Serum,salivaandlungtissuesampleswithPRRSVviralloadevaluatedbyRT-qPCRwereused.TheRT-qPCRCtvaluesofthesamplessequencedwerevaryingbetween10and32.Viralgenomeswereconcentratedusingpoly(dt)magnetic beads and librarywere prepared using Nextera XT technology. Theywere sequenced on an IlluminaMiseqsequencer.24full-lengthgenomeswereobtained.ItwasfoundthatthewholePRRSVgenomewasmorevariablethantheORF5(nucleotideidentitybetweenstrains:78.17%-99.63%vs83.06%-100%)whichisofinterestbecausetheORF5isrecognizedasoneofthehypervariableregionofthePRRSVgenome. Itwasfoundthatallwild-typestrainssequencedhad 3 important deletions totalling 513 nucleotides in the ORF1a,more precisely in the NSP2 and NSP7 region, twodifferentlineagesofPRRSVinQuebecweredefinedbasedonthepresenceofthesedeletions.Interestingly,thepresenceof twodifferentPRRSV strains (co-infection)were found in two lung tissue samples suggestingan8.7%prevalenceofPRRSVco-infectionwithinclinicalsamples.Thetwostrainsinoneofthesamplesshared92.12%identityatthenucleotidelevel.Moreover, one recombinant PRRSV strain (i.e. recombination between the two lineages)was also found.Whilemorefull-lengthviralgenomesarestillrequiredtoimproveourPRRSVgenomedatabankandouranalyses,itisexpectedthatwhole-genomesequencingofPRRSVwillallowustobetterunderstandthepathogenesisofthevirusandcontrolthediseaseitcauses.


24-EpidemiologyandmolecularcharacterizationofnovelparvovirusesinCanadianwildlifeMarta Canuti1, EmilyMcDonald1,Hillary E.Doyle1, StephanieM.Graham1, BruceRodrigues2, AnnP. Britton3,HughG.Whitney1,H.DawnMarshall1,AndrewS.Lang11DepartmentofBiology,MemorialUniversityofNewfoundland,2WildlifeDivision,NewfoundlandandLabradorDepartmentofFisheriesandLandResources,3AnimalHealthCentre,BCMinistryofAgriculture

Viruses of the genus Amdoparvovirus, family Parvoviridae, cause an immune-associated and often lethal wastingsyndrome invariouscarnivores.TheAleutianminkdiseasevirus (AMDV)wastheonlymemberof thisgenus formanyyears butmany novel amdoparvoviruses have been discoveredmore recently. Since these viruses can infectmultiplehosts and most of the research so far has been focused on captive animals, there is limited understanding of theirecology, host- and geographic distributions. To fill this knowledge gap, we investigated the presence and molecularfeaturesofamdoparvovirusesinCanadianwildlife.Tissuesamples(spleen,muscleorintestine)collectedovertheyearsfor various studies were screened with a PCR assay designed to detect all known amdoparvoviruses. These effortsallowedustoidentifyamdoparvovirusesinskunk(43/50,86%)andmink(1/2,50%)fromBritishColumbia(BC),inpinemarten(1/58,17.2%)andredfox(7/127,5.5%)fromLabrador,andinmink(8/23,34.8%)andredfox(2/29,6.9%)fromNewfoundland.SamplescollectedfromArcticandredfoxesfromQuebec,Nunavut,NorthwestTerritoriesandManitoba(46, 65, 12 and 105 samples, respectively) were negative. Complete or partial genomeswere obtained from positivesamplesandvirusesweremolecularlycharacterized.Threepotentiallynovelvirusesweredetected.Twooftheseviruseswere identified in samples from BC, one in mink (Aleutianmink disease virus 2, AMDV-2) and one in skunks (skunkamdoparvovirus, SKAV). The non-structural protein sequences (NS1) of these viruses diverge by >15% from otheramdoparvoviralspecies,fulfillingtheICTVmolecularcriterionforspeciesdemarcation.AMDV-2,phylogeneticallyclosetoAMDV(NS1proteinidentity:78-82%)anddifferentfromvirusesoffarmedminkinBC,hassofarbeenidentifiedinonlyoneindividual,anditsdistributioniscurrentlyunderinvestigation.SKAV,nowofficiallyclassifiedbytheICTVasCarnivoreamdoparvovirus4,wasphylogeneticallyclosetotheminkviruses(NS1proteinidentity:77-83%)andanalysesperformedon44sequencesrevealedthepresenceofat least3circulating lineages.Thisviruswasalso identified inskunksandinoneminkinotherdistantareasofNorthAmerica(suchasOntario)andstrainsshowedgeographicsegregation.ThethirddivergentviruswasidentifiedinredfoxesfromLabradorandistentativelynamedLabradorfoxamdoparvovirus(LaFAV).Wehaveonlyobtainedcapsidprotein (VP2)sequences for thisvirusso far,a region that ishighlyconservedbetweenspecies and phylogenetically uninformative, but preliminary data suggest that this virus is likely a novel species andamongthe7LaFAVstrainsweidentifiedtwovariantswithdistinctgeographicorigins.VirusesidentifiedinNewfoundland(frombothmink and red fox) could be classified asAMDVand theywere phylogenetically close to viruses previouslyidentifiedinfarmedmink.However,5oftheseviruses,allfrommink,weregroupedwithinacladethatcontainedonlyvirusesfoundinthewild,providingevidenceforviralperpetuationandmaintenanceinthewild.Overall,ourworkhasprovedthatseveraldifferentamdoparvovirusesarepresentinvariouswildcarnivoresacrossCanada.Theindividualviralspeciesseemtoaffectpredominantlyoneanimalspecies,withonlyrarespilloverevents,andvirusesshowgeographicsegregation.Becauseseveraldifferent (novel)viruseshavebeen identified,webelievethatamdoparvoviraldiversity ishigher than anticipated and many amdoparvoviruses could exist that are yet to be discovered. It is our priority tocomplete the genomic sequencing of the detected viruses and future screening efforts are likely to reveal additionaldiversity.


26-AnalysisofProgrammedCellDeathInducedbyHCVInfectionLingyan Wang1, Hannah Wallace1, Jiangyi He1, Maria Licursi1, Vipin Chelakkot1, Michael Grant1, Kensuke Hirasawa1,RodneyRussell11MemorialUniversity

Virusinfectionresultsinhostcelldeathviavariousmodesofprogrammedcelldeath.Inourpreviousstudy,weshowedthat hepatitis C virus (HCV, JFH-1) infection induces apoptosis and pyroptosis in both infected and bystander cells.Pyroptosisisacaspase-1-dependentformofcelldeaththatisconsideredaninflammatoryformofcelldeath.Therefore,pyroptosis may play roles in the development of liver pathogenesis and hepatocellular carcinoma in HCV-infectedindividuals.TheobjectiveofthisstudyistoidentifycellularmechanismsbywhichHCVinfectioninducesprogrammedcelldeath.WhenHuh-7.5cellswereinfectedwithHCVinvitroatMOI=0.1,weobservedactivationofcaspase-1(pyroptosis)at3 days after infection and activation of caspase-3 (apoptosis) at 4 days after infection. As the nucleotide-bindingoligomerizationdomain-likereceptorcontainingpyrindomain3(NLRP3)isoneoftheupstreammodulatorsofcaspase-1,wegeneratedNLRP3knockoutHuh-7.5cellsbyCRISPR.HCVinfectiondidnotinducepyroptosisinNLRP3knockoutHuh-7.5cells.Surprisingly,apoptosiswasalso inhibited intheabsenceofNLRP3.Furthermore,weobserved lower levelsofviralproteinsandvirustiterinNLRP3knockoutHuh-7.5comparedtocontrolHuh-7.5cells,suggestingthatprogrammedcell death affects HCV replication. As apoptosis is considered a host defense mechanism to inhibit HCVreplication,pyroptosis appearsto be a strategy used by HCV to enhance virus production. The findings from this study have thepotentialtoidentifymechanismsunderlyingchronicliverinflammationandviralspreadinHCVpatients.27-TheRoleoftheNLRP3ImflammasomeinHCV-InducedPyroptosisHannah L.Wallace1, LingyanWang1, LouisaWiede1, Vipin Chelakkot1, Shelia Drover1,Michael Grant1, Ken Hirasawa1,RodneyRussell11MemorialUniversity

Pyroptosis, one of several described inflammatory types of programmed cell death, is induced after activation of theNLRP3 inflammasome, which ultimately results in pore formation and cell lysis. One of the main factors in thedevelopmentofthelivercirrhosisassociatedwithHepatitisCvirus(HCV)infectioniscaspase-3-mediatedapoptosis(non-inflammatory).However,recentlyitwasshownthatbothapoptosisandpyroptosisoccurinculturedhumanhepatocyte-likecellsinfectedwithHCVaswellasinuninfectedbystandercells.Itremainsunclearwhetherpyroptosisisbeneficialtothe virus or is an innate antiviral response potentially benefitting the host. The current research has aimed to detectuniversalinflammasomecomponents(cleavedcaspase-1)inHCV-infectedcellsandtostudytheeffectoftheinhibitionofthese components on virus titer. Optimization of a protocol using FAM-FLICA probes has been used effectively tovisualizecaspase-1activation inHCV-infectedcells.Higher levelsofcaspase-1activationwereconsistentlyobserved inHCV-infected cells compared to uninfected cells, indicating more inflammasome activation occurs in infected cells.Interestingly,levelsofNLRP3activationwerehigherinHCV-infectedcellsthanobservedwiththeLPS/Nigericinprimingtypicallyusedasapositivecontrol.TofurtherconfirmtheroleoftheNLRP3inflammasomeinHCV-inducedpyroptosis,weinterferedwithinflammasomeactivationusingaNLRP3inhibitoraswellasaNLRP3knockoutcellline.WefoundthatvirustiterwassignificantlydecreasedwhenNLRP3-mediatedpyroptosiswas inhibitedbyeithermethod. Inconclusion,thesedataconfirmthepresenceofpyroptosisinHCVinfectedcellsandspecificallydemonstratetheinvolvementoftheNLRP3inflammasome.TheseresultssuggestthatpyroptosisisamechanismusedbyHCVtocausepathogenesis.


29-PhagemoronJBD26-15disruptsPseudomonasaeruginosaflagellabiosynthesisZafrinIslam1,VéroniqueL.Taylor1,Yu-FanTsao1,KarenMaxwell11DepartmentofBiochemistry,UniversityofToronto

Bacteriophages(phages),virusesthatinfectbacteria,canintegratetheirgenomesintobacterialchromosomesandexistasprophages. In this state,mostphagegenesaresilenced;however,a smallnumberofgenesknownas ‘morons’areactively expressed from theprophage. Thesegenesarenot required for the life cycleof thephage itself, butprovidesome selective advantage to thephages that carry them. Someof thesephagemoronshavebeen shown to increasebacterial virulence by mediating toxin production, increasing antibiotic resistance, and immune evasion. Our lab isstudyingmoronsisolatedfromphagesthatinfecttheopportunisticpathogenPseudomonasaeruginosa.WediscoveredthattheexpressionofmoronJBD26-15inP.aeruginosastrainPAO1abrogatesswimmingandtwitchingmotility,twokeyformsofbacterialmotility.Whiletwitchingmotilityisabrogated,thebacteriaremainsusceptibletophagesthatrequirethe type IV pilus for infection. This lack of phage resistance suggested the pili responsible for twitching motility arepresentonthesurfaceofthecellsbutaredefective,renderingthebacterianonmotile.Asswimmingmotilityisdrivenbyasinglepolarflagelluminliquidenvironments,andtwitchingmotilityismediatedbytypeIVpiliuponadherencetosolidsurfaces,we examined the production of these bacterial structures in the presence of JBD26-15 expression. Bacterialsurface protein-shearing assays showed that expression of JBD26-15 downregulates localization of FliC, an essentialflagellarcomponent,tothesurfaceofthecell.Inaddition,weobservedanincreaseinPilA,thetypeIVpilusmonomer,relative towild typePAO1, indicatingahyperpilliatedphenotype.Ahyperpiliatedphenotypeexplainsboth the lossoftwitchingmotilityandtheretainedsensitivitytophageinfectionandisconsistentwithafliCknockoutstrainofPAO1.Toourknowledgethisisthefirstinstanceofphage-mediateddisruptionofflagellarbiosynthesisinP.aeruginosa.Additionalexperiments todetermine themechanismbywhich JBD26-15 impairs swimmingand twitchingmotilityareunderway.JBD26-15 is a nonessential phage gene, but its conservation amongmany prophages in P. aeruginosa clinical isolatessuggestsitconfersafitnessadvantage.Thisfitnessadvantageislikelyincreasedresistancetophages-thesinglebiggestdanger toPseudomonas survival.However, since flagella arehighly immunogenic,webelieve that JBD26-15may alsoplayanimportantroleinhumandiseasebydecreasingtheimmunereactivityofthestrainsexpressingthemoron,whileallowing for persistence within the human host. Understanding the role of JBD26-15 in PAO1 will further ourunderstandingofphage-hostinteractionsandultimatelyhowtheseinteractionsaffectthecourseofbacterialinfections.30-RegulationofIRF1antiviralfunctionsbyitsSUMOylationYsabelMeneses1,YumikoKomatsu1,MariaLicursi1,DanyangXu1,SuzetteRutihinda1,KensukeHirasawa11MemorialUniversityofNewfoundland

Interferon regulatory factor 1 (IRF1) is a transcriptional factor that regulates expression of antiviral, antitumor andimmunoregulatorygenes.WerecentlydemonstratedthatoncogenicRas/MEKactivationsuppressesantiviralfunctionsofIRF1,whichisoneofthemechanismsunderlyingRas-dependentviraloncolysis1.Inthisstudy,wesoughttoclarifyhowRas/MEK activation downregulates antiviral functions of IRF1 in human cancer cells. First we determined whetherRas/MEK activation regulates IRF1 post-translationalmodifications such as acetylation, phosphorylation, SUMOylationandubiquitination. SUMOylation,butnot theotherpost-translationalmodifications,was suppressed inhumancancercellswhentheRas/MEKpathwaywasinhibited.TodeterminewhetherIRF1SUMOylationregulatesitsantiviralfunctions,we prepared SUMO-conjugated IRF1 constructs. Human cancer cells transfected with wild type IRF1 construct wasprotectedfrominfectionofoncolyticvesicularstomatitisvirus(VSV)whilethosetransfectedwithSUMO-conjugatedIRF1constructswerenot.SUMO-conjugatedIRF1wasnotcapableofactivatingtranscriptionofantiviralgenes.ToidentifyanIRF1SUMOylationsite(s)targetedbyRas/MEK,weidentifiedpredictedIRF1SUMOylationsitesby insilicoanalysisandgenerated SUMO-resistant IRF1 mutants by mutating the predicted SUMOylation sites (78K, 254K, 275K, and299K). HumancancercellstransfectedwiththewildtypeIRF1oraSUMO-resistant IRF1mutantwerechallengedwithoncolyticVSV.Thecellstransfectedwiththe275Kor299KIRF1mutantsweremoreresistanttotheinfectionthanthosetransfectedwiththewildtypeIRF1,suggestingthat275Kand299KaretheSUMOylationsitesofIRF1thatistargetedbyRas/MEKactivation.1.Komatsu,Y.etal.OncogenicRasinhibitsIRF1topromoteviraloncolysis.Oncogene34,3985–3993(2015).


32-T-cellexhaustionisahallmarkofimmunologicalfailuretocontrolchronicviralinfectionandcancer.MohamedS.Abdel-Hakeem1,Jean-ChristopheBeltra1,ZeyuChen1,SasikanthManne1,E.JohnWherry11PennInstituteforImmunology,PerelmanSchoolofMedicine,UniversityofPennsylvania

T-cellexhaustion isahallmarkof immunological failure tocontrol chronicviral infectionandcancer.Blocking immuneinhibitoryreceptorssuchasprogrammeddeath-1(PD-1)canre-invigorateexhaustedTcells(TEX),butmanypatientsfailtoachievedurablediseasecontrol.Thus,adeeperunderstandingofothermolecularpathwaysunderlyingreversalofT-cell exhaustion is needed. Little is known about “reprogramming” of TEX into recovered T cells (TRECOV) with betterfunctional andmemory profiles following non-immunological cure of chronic disease.Here,we aim to determine thecellular profiles of TRECOV, andmolecularmechanismsunderlying recovery.For this,weutilized thewell-defined LCMVmouse model, that was initially used to define immune exhaustion and re-invigoration using checkpoint blockade.Experimentally, we adoptively transferred TEX frommice chronically infectedwith LCMV-clone 13 into LCMV-immunemice to examine changes in TEX following antigen elimination.We also investigated the recall capacity of TRECOVuponantigenre-exposurecomparedtoTEXandmemoryTcells (TMEM).Ourdata indicate thatuponcureofchronic infectionsomemarkers of exhaustion are downregulated on TRECOV, while somemarkers of TMEM are recovered. For example,previously-exhausted T cells gradually downregulated the expression of the exhaustion marker PD-1 and increasedexpressionofthememorymarkerCD127.Nevertheless,PD-1expressionwasstillhigherandthepercentageofCD127+cells were lower on those recovering T cells when compared to bona fidememory T cells. These changes,were alsoaccompanied by partial recovery from dysfunction,where TRECOV partially restored the capacity of dual production ofeffector cytokines. However, not all aspects of TEXbiology are corrected simply by eliminating chronic infection.Transcriptionalprofilingusingsingle-cellRNAsequencing(scRNA-seq)confirmedourcellularprofiling,asitsuggeststhatthegene-expressionprofileofTRECOVresemblesTMEMinsomerespects,whileotherfeaturesarestillremnantsofTEXcells.TRECOVareheterogeneousandfallinto2mainclusters;onethatismemory-likeandtheotherthatismoreeffector-like.Anothermain features ofmemory cells is their ability to generate an effective recall response upon re-encounteringantigen. Results from rechallenge studies show that upon re-encountering antigen TRECOV had an enhanced ability toexpand compared to TEX.However, these cellswere still inferior on a per cell basis compared to true TMEM. Together,these results suggest that following elimination of persistent antigenic stimulation and inflammation previously-exhaustedTcellsareabletorecoversomepropertiesofTMEM,whileotheraspectsremained“scarred”fromtheirhistoryof exhaustion.We are currently performing deeper analysis of the scRNA-seq data to unravel mechanistic pathwaysunderlyingtheremainingdefects inTRECOV.Wearealsoexaminingtheepigenetic landscapeofTRECOVtoverifywhetherthe cellular and transcriptional defects we observed are imprinted epigenetically. These studies will enhance ourunderstanding of immunological andmolecularmechanisms of TEXrecovery, and have the potential to identify noveltherapeutic strategies for recovery of more functional T cells. This will have major implications on the treatment ofcancerorchronicinfection.


38-TRIM5αsensingofHIV-1counteractstranscriptionallatencyNatachaMerindol1,LaurenceBlondin-Ladrie1,MohamedEl-Far2,CécileTremblay2,LionelBerthoux11DeptofMedicalBiology,UniversitéduQuébecàTrois-Rivières,Trois-Rivières,Canada,2CR-CHUM,UniversitédeMontréal,Montréal,Canada

Elitecontrollers(ECs)areararesubsetofHIV-1infectedindividualsthatmaintainundetectableviremiaandnormalCD4+T cell counts without antiretroviral therapy. They also display a relatively small reservoir of latent proviruses. In aprevious study,we showed thatHIV-1 capsid cores isolated fromECswere efficiently detectedby humanTRIM5α. Inadditiontoblocking their replication,TRIM5αsensingofHIV-1 fromECsactivatedTAK1aswellas theNF-κBandAP-1pro-inflammatorypathways, leadingtoanantiviralstate.BothNF-κBandAP-1aretranscriptionfactorsthatbindwell-characterizedDNAmotifsintheHIV-1promoterandactivatetranscriptionoftheprovirus.Here,weaskedwhetherpro-inflammatory signals triggeredbyTRIM5αcould1)prevent latencyestablishment, 2) reactivate transcriptionof latentproviruses.Toaddressthefirstquestion,weinfectedTRIM5α-knockout(KO)andcontrolTHP-1cellswithGFP-expressingchimericHIV-1vectors constructedwithcapsidsderived fromECsor fromnormalprogressors (NPs).Threedays later,latent proviruses were reactivated by PMA (Phorbol 12-myristate 13-acetate) or SAHA (suberanilohydroxamic acid)treatments,andlevelsoflatencyestablishmentwerethenanalyzedbyFACS.WeobservedthatTRIM5α-sensitivevectorsderived fromECsweresignificantly lesspronetobecome latentproviruses incells thatexpressTRIM5α.Furthermore,latency levelswere independent of TRIM5α forHIV-1 vectors inwhichGFPwas expressed from a promoter differentfromtheviralLTR.Also,preventionoflatencyestablishmentbyTRIM5αwascounteractedbypre-treatingthecellswithNF-κBorTAK-1pharmacological inhibitors.Foroursecondobjective,weusedtheJurkat-derivativeJLat6.3cells,whichcontainalatentGFP-expressingHIV-1provirusintheirgenome.WeinfectedJLat6.3cellsthatwereTRIM5α-knockoutornotwithDsRed-expressingHIV-1vectorsinwhichthecapsidwasderivedfromECsorNPs.LatentGFP+provirusesweresignificantly more reactivated by the EC-derived vectors and only in cells that express TRIM5α. Similar to what wasobservedinthefirstexperimentalsetting,latentprovirusreactivationwasdependentonNF-κBorTAK-1.Inconclusion,HIV-1 recognition by TRIM5αboth prevents latency and reverts the transcriptional silencing of latent proviruses. ThisstudyproposesanewrolefortherestrictionfactorTRIM5αandprovidesanovelrationaleforthesmalllatentreservoirinelitecontrollers.39-InvestigatingtheresponseofactivatedmacrophagestoLCMVinfectionAndraBanete1,SamehBasta11DepartmentofBiomedicalandMolecularSciences,Queen’sUniversity,KingstonON,Canada

Macrophages (Mφ) are innate immune tissue sentinels with a variety of functional phenotypes depending on thecytokinemicroenvironment.Parallel toThelper cell polarization intoTh1andTh2 cells,Mφexhibitdistinct activationpatternsacrossacontinuumfrompro-inflammatory(M1),associatedwiththeTh1cytokine IFNγ,toanti-inflammatory(M2a),associatedwiththeTh2cytokineIL-4.GiventhecriticalroleofMφinregulatinginnateandadaptiveimmunity,itisnotsurprisingthatMφactivationstatusdirectlyinfluencestheoutcomeofviralinfections.Generally,M1polarizationrestricts viral replication. However, viruses can exploit inflammation to bypass host barriers and support replication.Additionally,virusessuchaslymphocyticchoriomeningitisvirus(LCMV)andLassaVirusdirectlysuppressMφactivationleadingtoviralchronicityandseveredisease.StudiesusingHIVshowedthatcertainactivationstatesareabletopreventviral infectionofcells,whileothersareabletostopviralreplication insidethecells.However, themechanismthroughwhich polarized macrophages are able to induce this resistance is unknown. Here, we investigate the response ofactivated spleen Mφ (SpM) to LCMV infection to understand how polarization influences the initiation of cellularmechanismsthatregulateanadequateimmuneresponse.Followinginfection,wediscoveredthatpolarizedMφshowanaltered resistance to LCMV infection,whichwas not due tomodifications in viral entry. Protein expression of innaterecognitionmoleculesinvolvedintheRLRpathwaywereevaluatedintheactivatedMφ.ResearchfundedbyNSERCandCIHR.


40-CharacterizationofthenoveltemperateStenotrophomonasmaltophiliabacteriophageDLP5DaniellePeters2,JonathanJDennis21BiologicalSciences,UniversityofAlberta,2CW405BiologicalSciencesBuilding,DepartmentofBiologicalSciences,UniversityofAlberta,11455SaskatchewanDr.NW,Edmonton,AB,Canada,T6G2E9.

Stenotrophomonas maltophilia is an aerobic, opportunistic Gram-negative bacterium ubiquitous in aqueousenvironments,soils,plantrhizospheresandhospitalsettings1.S.maltophiliaiscapableofcausingavarietyofinfectionsand limited treatment options exist due to S. maltophilia’s exquisite innatemultidrug resistance to a broad array ofantibiotics1–3.Alternative treatmentoptions, suchas thetherapeuticuseofbacteriophages,arebeingexaminedto tryandstemantibioticresistancerelateddeaths.Inordertousephagestherapeutically,initialresearchmustbeginwiththecharacterizationofphagestoensuretheydonotcontainharmfulmorongenes.ThenovelS.maltophiliabacteriophageDLP5 (vB_SmaS_DLP_5) was isolated from garden soil collected in Edmonton, Alberta, Canada using the clinical S.maltophiliastrainD1614.TransmissionelectronmicrographsidentifyDLP5asaB1morphotypeSiphoviridaephagedueto its icosahedral head and long noncontractile tail. DLP5 possesses a relatively narrow tropism, infecting only 5/27clinical S. maltophilia isolates tested. Successful infection with DLP5 results in clear plaques with defined boardersaveraging 0.5±0.2 mm and an average burst size of 36. Restriction fragment length polymorphism analysis of DLP5genomicDNA(gDNA)suggestsitishighlymodified,withonly4/36endonucleasestestedcapableofdigestingthegDNA.AdenovoSPAdesassemblyofpaired-endreadsproduceda96,542bpcontig(295-foldcoverage)withaGCcontentof58.4%.Thecontigispredictedtoencode149ORFsincludingfourtRNAs(Sup-CTA,Glu-TTC,Gly-TCCandSer-GCT).Only39ORFscouldbeassignedaputativefunctionbasedonBLASTpandCD-searchanalysis.Surprisingly,sixoftheseORFsarepredictedtobevirulencefactors:serineproteaseXkdF,SAMmethyltransferase,rhomboidmembraneprotein,PIG-Lfamilydeacetylase,WecE,andanSPFHdomain-containingprotein.Oftheremaining33ORFsassignedputativefunctions,phage partitioning proteins were identified which suggested DLP5 is capable of lysogenizing its host as a phagemid.Experimental evidence confirms DLP5 forms as a stable phagemid during its lysogenic cycle. Studying the growthcharacteristicsofthewildtypeandlysogenicD1614strainsrevealeddifferencesinthelagphaseofgrowth,withwildtypeD1614exhibitingaslowergrowthratewhencomparedtoitslysogeniccounterpart.ThisisthefirstS.maltophliaphageidentifiedtodatecapableoflysogenizingitshostasaphagemid.FuturestudieswithDLP5willattempttoinvestigatethepotential role eachmoron gene plays in the virulencemodulation of strain D1614. Although DLP5 is not suitable fortherapeuticuseduetoitstemperatelifestyleandthepresenceofmorongenes,theseresultshighlighttheneedformoreresearchonStenotrophomonasmaltophiliatemperatephagesandtheirpotentialinvolvementinvirulencemodulationintheirhosts.


42-UsingphylogeneticstoreconstructtheintegrationdatesofindividuallatenthumanimmunodeficiencyvirusreservoirsequencesBradleyJones1,2,NatalieKinloch3,JoshuaHoracsek1,BruceGanase1,MarianneHarris1,PRichardHarrigan2,RBradJones4,MarkBrockman1,3,JeffreyJoy1,2,ArtPoon5,ZabrinaBrumme1,31BCCentreforExcellenceinHIV/AIDS,2UniversityofBritishColumbia,3SimonFraserUniversity,4GeorgeWashingtonUniversity,5UniversityofWesternOntario

Background: Human immunodeficiency virus (HIV) still contributes to a global pandemic. Though combinationantiretroviral therapies (cART) can reduceHIVviremia in theblooddown toundetectable levels, limiting transmissionanddiseaseprogression,thereisstillnocure.AmajorbarriertocuredevelopmentisthefactthatHIVintegratesintothehost’s DNA. These integrated proviruses can remain in host cells for years in the so-called latent reservoir, only tospontaneouslyreactivateandproducevirionsata laterdate.However little isknownaboutthedynamicsofthelatentHIV reservoir including how often new latent reservoirs are seeded, and how long latently-infected cells persist. Toaddress this, we developed a phylogenetic method to estimate the integration dates of HIV sequences in the latentreservoir.Methods: The framework first involves inference, and optimal rooting, of amaximum-likelihood phylogenyrelatinglongitudinalwithin-hostplasmaHIVRNAandputativelatentsequences.PhylogenieswereinferredwithRAxMLandoptimallyrootedviaroot-to-tipregressionusingtheRpackageape.Alinearmodelrelatingtheroot-to-tipdistancesof plasma HIV RNA sequences to their sampling dates was used to predict the establishment (integration) dates ofputativelatentlineagesfromtheirroot-to-tipdistances.Threevalidationswereperformed.Inthefirst,weattemptedtoreconstruct thecollectiondatesof theplasmaHIVRNAsequencesby removing theputative reservoir sequences fromthedatasetofoneindividualandthendesignatingsequencesfromselecttimepointsascensoredsequences.Wethenused the used the remaining sequences to train the linearmodel and estimate the collection dates of the censoredsequences.Inthesecondvalidation,weremovedwholetimepointsofplasmaHIVRNAsequencesfromthedatasetofoneindividualandattemptedtofitthelinearmodelinordertoestimatetheintegrationdatesoftheputativereservoirsequences.Weperformedbothof thesevalidationson1096different sets. Forour final validation,wecomparedourrootingmethod(root-to-tipregression)tousingoutgrouprootingwithHX-B2astheoutgroupinourframeworkinstead.Results:ModelperformancewasfirstvalidatedonsimulatedandpublishedlongitudinalHIVenvsequences.Themodelwassubsequentlyapplied to single-genome-amplifiedHIVnef sequences sampled in-depthovera~20year timeframe(including ~10 years on suppressive cART) in two individuals with long-term viremia suppression. Results revealed ageneticallyheterogeneous reservoir that recapitulatedHIV'swithin-hostevolutionaryhistory,whereputative reservoirsequencesinterspersedthroughoutmultiplewithin-host lineageswiththeoldestdatingto>20yearspriortosampling.Notably,analysisofplasmaHIVRNAsequencesisolatedfromaviremiabliponsuppressivecARTrevealedageneticallydiverseviralpoolspanninga20-yearagerange,consistentwithspontaneouswithin-hostHIVreactivationfroma largepoolof latently-infectedcells.Thevalidations showed that linearmodels couldbecalibrated fromas fewas two timepointsandthatmodelpredictionswererobusttorootingmethod.Conclusion:Ourreservoirdatingmethodprovidesanovelandpowerful tool that can revealkey insights into latentHIV reservoirestablishmentand longevity.Our resultsshow that the latent reservoir is comprised of a genetically heterogeneous population with long persistence. Thisheterogeneityand longevityof the latentHIVreservoirposesachallengeto immune-basedHIVeliminationstrategies,whichaimtoreactivethelatentreservoirandassistthehost’simmunesystemtocleartheinfection.


44-CellularproteinkinaseDmodulatorsplayaroleupstreamanddownstreamoftheTGNduringherpessimplexvirustype1egressÉlisabethRoussel1,DianeGingras1,RogerLippé11UniversitédeMontréal

Theassemblyofnovelherpessimplexvirustype1particlestakesplaceinthenucleus.Theseparticlesthentravelacrossthetwonuclearmembranesandacquireafinalenvelopefromacellularcompartment,likelytheTGNbasedonstudiesfromseverallaboratories.Themolecularactorsinvolvedinthereleaseofthevirusfromthiscompartmentarehoweverlittleknown.WepreviouslydemonstratedthatthehostproteinkinaseDplaysacrucialroleinreleasingvirusfromtheTGNtotheplasmamembraneandthatdiacylglycerol,alipidthatfacilitatestherecruitmentofthekinasetotheTGN,isalsoimplicated.Giventheroleofthesemoleculesintheherpessimplexvirustype1lifecycle,weaimedtodeterminetowhatextentthisvirusutilizestheproteinkinaseDpathway.Thus,bothupstreamanddownstreammolecularplayersofthis pathway were targeted by RNA interference and viral production quantified. Surprisingly, several of thesemodulatorsnegativelyimpactedtheextracellularreleaseofthevirus.TheuseofmutantandpharmacologicalreagentstargetingCERT,alipidtransferprotein,suggestedthattheseeffectswereindependentoftotalintracellulardiacylglycerollevels.Electronmicroscopy revealed that thehostproteinsNir2andproteinkinaseDmayalsomodulateviralnuclearegress.Altogether, this suggested a complex scenario whereby upstream or downstream modulators of the proteinkinaseDpathwaycanimpactthevirusatdifferentstagesofitsegress.47-AGenome-wideScreenofEpstein-BarrVirusProteinsthatModulateHostSUMOylationIdentifiesaSUMOE3LigaseconservedinHerpesvirusesCarlosDeLaCruzHerrera1,KathyShire1,LoriFrappier11UniversityofToronto

Epstein-Barr virus (EBV) infects most people worldwide and is a causative agent in several kinds of lymphoma,nasopharyngeal carcinoma and 10% of gastric carcinoma. EBV encodes ~80 proteins many of which are poorly orcompletely uncharacterized, but are thought to modulate cellular processes. Modification by the small ubiquitin-likemodifier(SUMO)controlstheactivityofmanyproteinscentraltoantiviralresponsesandoncogenesis,andthereforetheSUMO pathway is a likely target of EBV proteins. We generated an expression library of most of the encoded EBVproteins and screened them for the ability to globally modulate SUMOylation.We screened for cellular SUMO1 andSUMO2 modifications in three different cellular systems, including nasopharyngeal carcinoma cells relevant for EBVinfection.OneEBVprotein (BRLF1;Rta)globally inducedthe lossofSUMOylatedproteins, inaproteasome-dependentmanner,aswellasthelossofpromeylocyticleukemianuclearbodies,suggestingthat,likeitshomologue(Rta)inKaposi’ssarcomaassociatedherpesvirus, it isaSUMO-targetedubiquitin ligase.FourEBVproteinsconsistentlyupregulatedthelevelsofSUMO-conjugatedproteins, includingtheEBVSM(EB2)protein.SMhomologuesexist inherpessimplexvirus(UL54 or ICP27) and cytomegalovirus (UL69), prompting us to examine the effects of these proteins on cellularSUMOylation.LikeSM,HSV-1UL54andCMVUL69 inducedglobalSUMOylation.All threeviralproteinswerepurifiedandtestedinaninvitroassayforSUMOylationusingp53asasubstrate,revealingthatallthreeproteinshadtitratableSUMOE3ligaseactivity.Consistentwiththisactivity,allthreeviralhomologuesboundSUMOandtheSUMOE2,Ubc9.These are the first SUMOE3 ligases discovered for EBV,HSV-1 andCMV. Interestingly the homologues haddifferentspecificities for SUMO1 and SUMO2, with SM and UL69 preferentially binding SUMO1 and inducing SUMO1modifications, and UL54 preferentially binding SUMO2 and inducing SUMO2 modifications. The results provide newinsights into the function of this family of conserved herpesvirus proteins. The conservation of this SUMO E3 ligaseactivityacrossdiverseherpesvirusessuggeststheimportanceofthisactivityforherpesvirusinfections.


48-IsolationandcharacterizationoftwonovelcyanophagesfromasubarcticCanadianlakeAliceLévesque1,AntonyT.Vincent1,SimonJ.Labrie1,SylvainMoineau1,WarwickF.Vincent1,AlexanderI.Culley11UniversitéLaval

Cyanobacteriaarekeycomponentsofarcticaquaticecosystems,asbothessentialprimaryproducers,andassignificantcontributors to the biogeochemical cycling of essential nutrients such as carbon and nitrogen. The two primarymechanismsoftop-downcontrolofcyanobacterialpopulationsaregrazingandviral lysis.VirusesarethemostdiverseandabundantbiologicalentitiesonEarthwithestimatesof1031viralparticlesgloballyand104virotypespermLofwater.It is now widely recognized that viruses are important drivers of host evolution and influence nutrients cycles andmicrobialdynamicsthroughvirallysis.Cyanophages,virusesthatinfectcyanobacteria,infectkeystoneprimaryproducersinmarineandfreshwaterenvironments,andhavebeenshowntoharbourhost-likegenesthatareinvolvedinessentialhostmetabolicprocesses.Inordertobetterunderstandcyanophagesdynamics,genomicsandecology,wefocusedourresearchon the isolationandcharacterizationofcyanophages that infectpolarcyanobacteria.Water fromaCanadiansubarcticlakelocatedinthevicinityofWhapmagoostui-Kuujjuarapik(Nunavik,Canada)wassampledinAugust2015andwas filtered through 0.45µm and 0.22µm pore size filters to remove large particles (cells, clay, etc.). The filtratecontaining viruses was used to inoculate different cyanobacterial cultures for viral amplification. Cultures presentingsigns of lysis relative to an untreated control were serially diluted to isolate a single virus. Clonal isolates weresubsequently sequenced on a NGS sequencing platform (MiSeq, Illumina). We succeeded in isolating two novelcyanophages (B3 and B23). Electronmicrographs revealed that both isolates possessed icosahedral capsid and a longcontractiletail,typicalfeaturesofvirusesintheorderCaudovirales,familyMyoviridae.B3andB23genomelengthswere244,930 bp and 243,633bp respectively. Genomic analysis and ORFs annotation indicated the presence of two host-relatedmetabolicgenes(hliandphoH) inbothgenomes.However, thefunctionofonly25%oftheputativeORFswasidentified using RAST based on homology with sequences in the SEED database. Phylogenetic analysis based onalignmentsoftheB3andB23primasegeneandthosefromphagesfromarepresentativenumberoftaxashowedthatboth viruses clustered with other cyanophages. These results suggest that B3 and B23 are highly divergent frompreviouslydescribedcyanophagesandthatthisstudywillresult innewinsightintotheworldofarcticcyanophages,ofwhichlittleisknown.49-PersistenceofentericvirussurrogatesinstaticbacterialbiofilmsJulieBrassard1,Marie-JoséeGagné11AgricultureandAgri-FoodCanada

Background:Norovirusandotherentericvirusesarerecognizedasleadingcauseoffoodborneinfections.Freshproduce,meats and ready to eat foods provide an ideal route of transmission for those viruses. Unlike most microbiologicalagents,virusescannotreplicateonfood,thereforecontaminationlevelsdonotincreaseduringprocessingorstorageandveryfewviralinfectiousparticlesarenecessarytoinducedisease.Entericvirusesareknownfortheirstabilityinvariousenvironmentsandonsurfaces.Also,foodprocessingenvironmentsareprimelocationsforthedevelopmentofsurface-associated microbial communities known as biofilms. It is well known that biofilms contribute to contamination,persistence and hygiene failure in the food industry. Still, relatively little is known about the behavior of foodborneviruses dwelling within these complex communities. The aim of this study was to evaluate the association betweenenteric virusesandbiofilms.Methods:MurinenorovirusMNV-1and rotavirusWawereusedas surrogateandbiofilmformation(48h)ofmono-cultureandmix-cultures(L.plantarum;L.rhamnosus;L.pseudomesenteroides;P.fluoresences)werepreparedon3surfaces:glass,stainlesssteelandpolystyrenecoupons.Viruseswereaddedatfinalconcentrationof105pfu/mlwitheachformedbiofilmandincubatedfor15,90minand24hrs.Infectiousviralparticlesweredetectedinbiofilmsattachedat surfacesbyplaqueassays.Results:Anaveragepersistenceof2.4 log/mlof infectiousparticlesofMNV on the 3 surfaces tested without biofilm at all incubation times was observed. No significant association orpersistencehasbeennotedbetweenMNVandthemixedbiofilm.However,asignificantincreaseofitspersistencewasdetected in presence of simple formed biofilms from the two Lactobacillus used in this study. For rotavirus, theassociationbetweenthevirusandthesurfaceswithoutbiofilmat15min,90min,24hwereshownwitharecoveryof2.5,2.4,and0.1log/ml,respectively.However,significantamountsofinfectiousparticleswererecoveredafter24hourswithsomesimpleandmixedbiofilms.Theseresultsshowedthatbiofilmscaninfluencethepersistenceoftherotavirusovertimeontheglass,stainlesssteelandpolystyrenesurfaces.Conclusion:Biofilmsonsurfacesusedinthefoodindustrycaninfluencethepresenceandpersistenceofentericvirusesintheenvironment.However,variousfactorssurroundingthisassociationareunknownandappeartovarydependingonvirusesandbiofilmcomposition.


50-AdaptivemutationsininfluenzaA/California/07/2009enhancepolymeraseactivityandinfectiousvirionproductionPatrickD.Slaine1,CaraMacRae2,MarielKleer1,EmilyLamoureux3,SarahMcAlpine4,MichelleWarhuus4,AndréComeau3,CraigMcCormi1,ToddHatchette4,DenysKhaperskyy11DepartmentofMicrobiologyandImmunology,DalhousieUniversity,5850CollegeStreet,HalifaxNS,CanadaB3H4R2,2TheHospitalforSickChildren,UniversityHealthNetwork,Toronto,ON,Canada,3CGEB-IntegratedMicrobiomeResource(IMR)andDepartmentofPharmacology,DalhousieUniversity,5850CollegeStreet,HalifaxNS,CanadaB3H4R2,4DivisionofMicrobiology,DepartmentofPathologyandLaboratoryMedicine,NovaScotiaHealthAuthority(NSHA),Halifax,NS,Canada

Mice are not natural hosts for influenza A viruses (IAVs), but they are useful models for studying antiviral immuneresponsesandpathogenesis.Serialpassageof IAV inmice invariablycauses theemergenceofadaptivemutationsandincreasedvirulence.Typically,mouse-adaptationstudiesareconductedininbredlaboratorystrainsBALB/candC57BL/6,which have defects in the antiviralMx1 gene that results in increased susceptibility to infection and disease severity.Here, we report the adaptation of IAV reference strain A/California/07/2009(H1N1) (a.k.a. CA/07) in outbred SwissWebstermice. Serial passage led to increased virulence and lung titers, and dissemination of the virus to brains.Weadaptedadeep-sequencingprotocoltoidentifyandenumerateadaptivemutationsacrossallgenomesegments.Amongmutations that emerged during mouse-adaptation, we focused on amino acid substitutions in polymerase subunits:polymerasebasic-1 (PB1)T156AandF740L,andpolymeraseacidic (PA)E349G.Thesemutationswereevaluatedsinglyand in combination in minigenome replicon assays, which revealed that PA E349G increased polymerase activity. ByselectivelyengineeringthesethreeadaptivePB1andPAmutationsintotheparentalCA/07strain,wedemonstratedthatadaptivemutationsinpolymerasesubunitsdecreasedtheproductionofdefectiveviralgenomesegmentswithinternaldeletions,anddramaticallyincreasedthereleaseofinfectiousvirionsfrommousecells.Together,thesefindingsincreaseourunderstandingofthecontributionofpolymerasesubunitstosuccessfulhostadaptation.51-ConstructionofaSyntheticKSHVGenomebyTransformation-AssociatedRecombinationThornburyM1,DugayB1,RohdeJ1,CraigMcCormick11DalhousieUniversity

Kaposi’s sarcoma-associated herpesvirus (KSHV) is the infectious cause of Kaposi’s sarcoma (KS), primary effusionlymphoma(PEL)andmulticentricCastleman’sdisease(MCD).ThereisnoKSHVvaccine.Rationalattenuationofvirusesforvaccinedevelopmentrequiresintimateunderstandingofviralantigensandviralgenefunction,andefficientmethodsfor viral genome engineering. Despite recent advances in en passant mutagenesis and CRISPR/Cas9-based genomeediting,geneticmanipulationofKSHVremainscumbersome.Developmentsinsyntheticbiologyhasmadeitfeasibletosynthesize,assemble,and re-code largeviral genomes.Here,wedescribeourapproach toassemblea syntheticKSHVgenomeinyeastviatransformation-associatedrecombination(TAR).Thewidely-usedKSHVstrainJSC1(~165kbp)isthereferencegenome for this study.TheTARcloningvector containsbacmidandyeast centromerecassettes to facilitategrowth inE.coliandS.cerevisiae, respectively.TheKSHVgenomewillbeshearedandoligonucleotideprimerswillbeusedtoclone~14kbpfragmentsintotheTARVectorpCC1-BAC.GenomefragmentswillbecleavedfromtheTARvectorand co-transformed into yeast; 40 base pairs of conserved overlapping sequence at the end of each fragment (“TARhooks”)willfacilitatehomologousrecombinationinyeast.This‘parental’recombinantKSHVgenomewillbesequencedvia IlluminaMiSeq to ensure faithful assembly. In cell culture, KSHV establishes a non-productive latent infection. Torecover infectiousvirus,theKSHV-TARgenomewillbetransfected intoawidelyusedepithelialcell lineexpressingtheKSHVlyticswitchproteinRTAonatetracycline-regulatedpromoter,allowinginduciblereplicationofvirus.Virusstockswillbeamplifiedandplaque-purifiedonacell linethatconstitutivelyexpresseslowlevelsofRTAtosupportKSHVlyticreplication.EstablishmentofTARmethodsforKSHVwillfacilitaterapidmutagenesisinyeast,allowingthecreationofre-codedvirusesthat lackimmuneevasiongenes,orhaveotherdefects leadingtoattenuation.Thistechnologywill likelyadvance fundamental studies of virus gene function, while accelerating discovery and development of candidateattenuatedvaccineswithdesirableproperties.


52-ModulationandrecruitmentofTRF2atviraltelomeresduringHHV-6A/BinfectionsShellaGilbert-Girard1,AnnieGravel1,BenediktB.Kaufer2,LouisFlamand11UniversitéLaval,2FreieUniversity,Berlin,Germany

Human herpesvirus-6A (HHV-6A) and HHV-6B are two distinct beta herpesviruses with different epidemiological andbiologicalcharacteristics.HHV-6Bisaubiquitousvirusthatinfectsnearly100%ofworldpopulationandistheetiologicalagentofroseolainfantum,aninfantilefebrileillnesscharacterizedbyhighfeverwithoccasionalskinrash.HHV-6Bisalsoa concern in hematopoietic stem cell and solid organ transplant recipients with frequent reactivation and medicalcomplications. Pathological and epidemiological data on HHV-6A remain scarce. The viral genomes of HHV-6A/B arecomposedofauniquesegmentofapproximately143kbpflankedatbothextremitieswithidenticalanddirectlyrepeated(DR)terminiofapproximately9kbpeach.EachDRcontainstworegionswithrepeatedTTAGGGtelomericsequencethatplay a role in the ability of these viruses to integrate their genomes into human chromosomes. Although HHV-6integrationcanoccurinseveraldistinctchromosomes,itinvariablytakesplaceinthetelomeric/sub-telomericregionsofchromosomes. To prevent activation of the damage recognition pathways at inappropriate times, telomeres areprotectedbytheshelterincomplex.TheshelterincomplexfoldsthetelomereinastructurecalledtheT-Loop,preventingtherecognitionofthetelomereextremityasadouble-strandbreak(DSB).Theshelterincomplexismadeofsixproteins:TRF1,TRF2,TPP1,RAP1,TPP2andPOT1.TRF1andTRF2bothformhomodimersthatbinddirectlytothedouble-strandTTAGGGrepeatsinasequence-specificmanner.TRF2repressestheATMpathwayandcellcyclearrestandplaysaroleinthe repression of telomere repair by non-homologous end-joining (NHEJ) and homologous recombination (HR). POT1bindstothesingle-strandsectionofthetelomeresandprotectsthetelomeresagainstrecognitionbytheATRpathway.During infectionbyDNAvirusessuchasherpesviruses, thedsDNAviralgenomesare likely toberecognizedasbrokenDNAmolecules and trigger a DNA-damage response. In the present study, we analyzed whether sheltherin proteinswouldassociatewithviraltelomericsequencesduringinfectionandifthesecouldpreventaDNAdamageresponse.HHV-6AinfectionofHSB-2cellsledtoincreasedexpressionofTRF1,TRF2,RAP1andTPP1mRNAs.Similarly,HHV-6Binfectionof MOLT-3 cells caused a significant increase in TRF2 and TPP1 mRNAs levels relative to uninfected controlcells.VariationsinPOT1,TIN2andTERTmRNAlevelswereminimal.IncreaseinTRF2mRNAlevelswerevalidatedattheproteinlevel.OurresultsalsoindicatethatHHV-6AIE2colocalizeswithsheltherincomplexproteinsattelomeresduringinfection. We could also document that TRF2 colocalizes with viral replication compartment (VRC) during infection,suggesting that telomeric sequenceswithinHHV-6 genome are likely recognized and boundby TRF2during infection.SuchhypothesiswasconfirmedbyELISAandChIP.IncellstransientlyknockdownforTRF2,arobustDDRwasobservedatVCRduringinfection.Insummary,ourresultsindicatethatHHV-6A/Binfectionmodulatesheltherinproteinexpressionlevels.TRF2isrecruitedandbindstoviralDNAduringinfectionandprotectsagainstthedevelopmentofDDRatVRC.54-ViralmanipulationofhostcholesterolhomeostasisisrequiredforviralpackagingandegressCarolyn-AnnRobinson1,EricS.Pringle1,AndreaMonjo1,CraigMcCormick1,21DepartmentofMicrobiologyandImmunology,DalhousieUniversity,2BeatriceHunterCancerResearchInstitute,Halifax,NovaScotia

Kaposi’s sarcoma-associatedherpesvirus (KSHV) is the etiological agent of Kaposi’s sarcoma (KS) andprimary effusionlymphoma (PEL). Like all herpesviruses, newly-assembled KSHV particles acquire a host-derived lipid envelope duringegress. To acquire this envelope, KSHV must reorganize host cellular compartments and manipulate cholesterolhomeostasis.TheliverXreceptorα(LXRα)transcriptionfactorisacentralregulatoroflipidhomeostasis.LXRαactivationbyoxysterols stimulates transcriptionofcholesteroleffluxand fattyacidsynthesisandstoragegenes, therebyalteringthe bioavailability of host-derived cholesterol. We employed a specific endogenous LXRα agonist, 22R-hydroxycholesterol (22OH) to investigate the impact of lipid homeostasis perturbations on KSHV replication. KSHVinfectionwasmonitorediniSLK.219cellsthatarelatently infectedwithaGFP-expressingrecombinantKSHVwhichcanbe reactivated from latency by addition of doxycycline. These cells were also monitored for cholesterol distribution,storage, and efflux during latency and throughout lytic replication. Treatment of iSLK.219 cells with 22OH causedincreased reactivation from latency compared to controls. Strikingly, LXRα activation significantly decreased theproduction of infectious virions from these cells by impeding the egress of viral capsids from the host cell nucleus.Furthermore,22OHtreatmentdepletedbothfreeandstoredhostcellcholesterol,therebydecreasingitsbioavailabilityfor viral envelopes. Taken together, these findings suggest that KSHV replication requires perturbations in host lipidhomeostasis,andthatLXRαactivationdisruptsKSHV-mediatedlipidchangestoblocktheproductionofinfectiousvirions.


58-ExtensivehostimmuneadaptationintheSaskatchewanHIVepidemicZabrinaBrumme1,2,NatalieKinloch1,StephenSanche3,AlexanderWong4,EricMartin1,KyleCobarrubias1,2,PaulSandstrom5,PaulLevett6,7,P.RichardHarrigan8,JeffreyJoy2,81SimonFraserUniversity,2BritishColumbiaCentreforExcellenceinHIV/AIDS,3UniversityofSaskatchewanCollegeofMedicine,4DepartmentofMedicine,UniversityofSaskatchewan,5NationalHIVRetrovirologyLaboratories,PublicHealthAgencyofCanada,6SaskatchewanDiseaseControlLaboratory,7DepartmentofBiology,UniversityofRegina,8FacultyofMedicine,UniversityofBritishColumbia

Background: Since2003, a concentratedHIVepidemichasbeenexpanding in theCanadianprovinceof Saskatchewan(SK),where injection drug use represents the primarymode of transmission (60%) andwhere nearly 80%of infectedpersonsself-identifyasIndigenous.WhiletheoverallHIVdiagnosisrateinSK(14.5per100,000in2016)isalreadymorethantwicethenationalaverage,regionalratesareuptofourfoldhigher(e.g.67.8per100,000inPrinceAlbert).TheSKHIVepidemicmayalsohaveuniqueclinicalcharacteristics.AnecdotalreportsofmorerapidthanexpectedprogressiontoHIV-induced immunodeficiency are emerging from the province, and the 2016 SK HIV Prevention and Control reportestimated that fewer than 50% of persons diagnosed since 2007 remained alive in 2016. Furthermore, studies inneighbouring Manitoba have reported accelerated HIV progression among individuals expressing certain HumanLeukocyteAntigen (HLA) class I alleles, including the typicallyprotectiveB*51.This isnotable,asa study investigatingHLAdiversityamongIndigenousSaskatchewanresidentsidentifiedB*51asthemostcommonHLA-Balleleinthisgroup.GiventhatacquisitionofHIVpre-adaptedtohostHLAcansignificantlyaccelerateclinicalprogression,wehypothesizedthat elevated HIV adaptation to host HLA may explain the reports emerging from the region. Specifically, given theestablished linkbetweenpopulation-levelspreadof theB*51-associatedRT-I135XescapemutationanderosionofHIVcontrolbythisalleleinotherglobalregions,notabalyJapanwhereB*51prevalenceishigh,wefurtherreasonedthatHIVadaptation to B*51 might be particularly elevated in SK. We investigated our hypotheses using comprehensivelongitudinalHIVPolsequencedatasetscollectedbetween2000-2016fromSaskatchewanandelsewhereinCanadaandtheUSA.Methods:Weanalyzed1,144HIVsubtypeBPolsequencesfromuniqueSaskachewanresidentsspanning2000-2016 for the presence of 70 known HLA-associated Polmutations. These sequenceswere originally collected for thepurposesofHIVdrugresistancegenotypingandrepresent~65%ofcumulativeHIVcasesdiagnosedinSaskatchewantodate.HIVPolsequences fromBritishColumbia (N=6,525)andelsewhere inCanada/USA(N=6,517), spanningthesamedateranges,wereusedforcomparison.OverallHIVadaptationlevelsto34HLAalleleswerealsoquantified.PutativeHIVtransmissionclusters,andthedistributionofHLA-associatedadaptationswithinandexternaltotheseclusters,wasalsoinvestigated. Results: The molecular epidemiology of HIV in Saskatchewan is highly unique, with 78% of circulatingsequencesresidinginlargephylogeneticclusters(comparedto48%inthedensely-sampledBCepidemicandonly15%intheNorthAmericadataset).AnalysesconfirmedsignificantlyelevatedlevelsofHIVadaptationtonumerousHLAallelescommonly expressed in Indigenous populations in Saskatchewan, in particular B*51. The prevalence ofmost of theseHLA-adapted viral polymorphisms was also increasing over time. Notably, HIV strains harboring HLA-adaptedpolymorphismswere significantly overrepresentedwithin large phylogenetic clusters in SK, indicating the preferentialandwidespread transmission of these polymorphismswithin the province. Conclusions: High levels of circulating HIVadaptation to HLA in Saskatchewan provide a plausible explanation for reports of accelerated progression in theprovince,andidentifySaskatchewanasthefirstexampleofaNorthAmericanHIVepidemiccharacterizedbysignificantcirculatingviraladaptationtohost immunity.ResultshighlighttheutilityofPolsequences,routinelycollectedfordrugresistancemonitoring, for surveillanceofHIV-hostadaptation,andunderscore theurgentneed toexpandHIV testing,treatmentandharmreductionprogrammesintheregion.


59-TypeIInterferon-AssociatedImpairmentoftheHumoralImmuneResponseagainstLCMVMatthieuDaugan2,ArmstrongMurira2,AlainLamarre21InstitutArmandFrappier,2INRS-IAF

Type I interferon (IFN-I) has been characterized to enhance cell-mediated immune responses against acute viralinfectionswhilst impair immuneactivation in chronic viral settings aswouldbe in the caseofHCVandHIV.Here,weshow that inaddition to itseffectonT cells, IFN-Idrives impairmentofeffectivehumoral immune responses throughdirect interaction with B cells upon chronic viral infection. Using the classic LCMV murine infection model, we co-administered4-hydroxy-3-nitrophenyl(NP)atthetimeofinfectionwherebyflowcytometryanalysisofBcellproportionsandELISPOTdata revealed thatcompared toanormalhumoral immuneresponse in theVSV (acute) infection, LCMV-infected mice developed non-specific hypergammaglobulinemia along with diminished NP-specific responses shortlyafterinfection.Notably,duringpersistentviralinfections,infectedhostsalsoexhibitaberranciestothehumoralresponsesuch as polyclonal activation and hypergammaglobulinemia. Accompanying this dysregulation, the emergence ofneutralizingantibodies(nAbs)isdelayedandbearsnegligibleimpactontheprogressionofthediseasebythetimetheyare successfully elicited. Altogether, these perturbations result in a diminished antigen-specific Ab response and anenhancednon-specificpolyclonalresponse.Thesearehallmarksofdisruptioninthehumoralimmuneresponseduringachronicinfection.OurresultsalsodemonstratedthatthisimpairmentwaslimitedtotheT-celldependentB-cellresponseandfunctionwasrestoredbyablationofIFNsignalingthroughantibody-dependentIFNreceptorblockadeaswellasB-cellspecificIFNreceptorknockouts.Inaddition,disruptedlymphoidarchitectureobservedfollowingimmunofluorescentmicroscopywasalsorestoreduponeliminationofB-cellspecificIFNsignaling.Importantly,restorationofeffectiveB-cellresponses intransgenicmicealsofeatured increasedneutralizingantibodytiters inELISAassays,whichwereabsent inthe wildtype model with functional IFN signaling. Our findings illustrate the role played by IFN in limiting effectiveantibodyresponsesbyactiononB-cells.WhereascompleteblockadeofIFNsignalingwouldbedeleterious,targetedB-cell specific restriction could improve humoral responses towards effective therapeutic and prophylactic measuresagainstchronicinfectionssuchasHCV.62-Oncolyticvirus-inducedexpressionofanalternativeleaderdrivestranslationofanmRNAencodinganinositolphosphatasethatlimitsinfection.TysonGraber1,Huy-DungHoang1,Jian-JunJia1,BrunoFonseca1,ChristosGkogkas3,MehdiJafarnejad2,TommyAlain11Children'sHospitalofEasternOntarioResearchInstituteandUniversityofOttawa,2McGillUniversity,3DepartmentofIntegrativePhysiology,PatrickWildCentre,UniversityofEdinburgh,Edinburgh,UK

Innatecellularimmunityprotectsfrominvadingpathogensthroughanetworkofmolecularsensorsthatactivateantiviraleffectorsanddampenpro-viralcellularactivities.Theproteinsynthesismachineryconstitutesavaluablearmof innateimmunityyetbecomesderegulated inthecontextsofviral infectionandcancer.The inabilitytomountanappropriateinnate immune response servedas theoriginalbasis for the cancer cell tropismofoncolytic viral therapies.However,residual innate immunity represents a significant barrier to therapeutic efficacy. We sought to identify new innateimmuneeffectorsthataremodulatedatthelevelofmRNAtranslationduringinfectionofmurinebreastcancercellswiththree different oncolytic viruses (HSV-1, Reovirus, Vaccinia). Using ribosome profiling,we found that the response ofcancer cells to all three viruses at the level of mRNA translation was distinct from the transcriptional response.Weidentifiedatranslationde-repressionphenotypeassociatedwiththeupregulatedmRNAsetcommontoallthreevirusesandfoundthatacandidatemRNAencodinganinositolphosphatase(Inpp5e)harboursanalternativelyspliced5’leader,with the shorter isoform favourably expressed during infection. Critically, this shorter 5’ leader leads to a highertranslationalefficiencyof the Inpp5eORF.Moreover, interruptionof INPP5EexpressionbyRNAiorCRISPR-Cas9 ledtoenhanced infectionbyvariousoncolytic virusesandappears tobemediatedbypleiotropicmechanisms.Thus,usingaribosome profiling approach, we have uncovered an innate immunity effector that could be exploited to engineeroncolyticviruseswithincreasedclinicalefficacy.


63-PoxvirusgenomeeditinginthedevelopmentofasafeandefficientoncolyticplatformAdrianPelin1,FabriceLeBoeuf1,MikeHuh1,MatthewTang1,John.CBell11OttawaHospitalResearchInstitute,CenterforCancerTherapeutics,Ottawa,Ontario,Canada

Vacciniavirushasalargeandstill incompletelyunderstoodgenomealthoughseveralstrainsofthisvirusarealreadyinclinicaldevelopment. For themostpart, clinical candidateshavebeenattenuated from theirwild typevaccine strainsthrough deletion of metabolic genes like the viral thymidine kinase gene.We decided to carry out a more in depthunderstanding of the genetic elements of vaccinia which could be modulated to improve the oncolytic/therapeuticcharacteristicsofthevirus.Usingavarietyofcancercelllinesandprimarytumorexplants,weperformedafitnessassaythat compares head to head five wild-type Vaccinia strains to identify the genetic elements that together create anoptimal “oncolytic engine”. Using a transposon insertion strategy and deep sequencing of viral populations wesystematicallyexaminedvacciniagenesthatdoordonotplayaroleinthetherapeuticactivityofthevirus.Ourstudiesallowedustoidentify largeareasofthevacciniagenomethatwhendeleted,augmenttheoncolyticactivityofanewlycreatedrecombinantvirus(35genesdeleted).Thisnovelviruswascomparedtofivevacciniastrainscurrentlyintheclinicand in a variety of assays, the deleted virus displayed superior therapeutic activity and an enhanced safetyprofile. Studies in a variety of in vitro and in vivomodelswill be presented illustrating the strategywehaveused tocreatethisoptimizedoncolyticvirusplatform.Wearecurrentlycarryingoutfurtherpre-clinicalstudiestoacceleratethetranslationofthisnewvirusintotheclinic.64-InfluenzaAvirushostshutoffnucleasePA-X:targetspecificityandprotein-proteininteractionsDenysKhaperskyy1,BrittanyPorter1,LeaGaucherand3,MartaGaglia3,CraigMcCormick11DalhousieUniversity,2DalhousieUniversity,3TuftsUniversity

InfluenzaAviruses (IAVs) inhibithostgeneexpressionbyaprocessknownashost shutoff.Thehost shutoff facilitatesvirus replication primarily by blocking induction of antiviral factors. Several IAV proteins participate in host shutoff,includingtheendoribonucleasePA-X.Ininvivomodelsofinfection,PA-Xdeficiencyexacerbateslunginflammationandincreasesmorbidity andmortality. However, amechanistic understanding of PA-X effects on the cell transcriptome islacking.WepreviouslydemonstratedthatPA-XspecificallydegradestranscriptsproducedbyhostRNApolymeraseII,yethow PA-X distinguishes between different host transcripts as well as between viral and host mRNAs is currentlyunknown. Our recent findings suggest that the target discrimination is linked to nuclearmRNA biogenesis pathways.Indeed,uponectopicexpressionPA-XlocalizedtothecellnucleusandwehypothesizethatPA-Xisrecruitedtoitstargettranscripts through interaction with mRNA processing factors, and that the differential sensitivity of RNAs reflectsdifferential engagementof thismachinery.Weemployed two complementary approaches to test this hypothesis: thetranscriptome-wideprofiling of host PA-X targets and the proteomic analysis of PA-X-interacting proteins usingBioID.Usingtheseapproaches,wediscoveredthattheRNAsplicingisadeterminantofsusceptibilitytoPA-XandthatthePA-XC-terminaldomain interactswithanumberofnuclear,nucleolar,andnucleocytoplasmic shuttlingproteins involved inRNA processing. Taken together, our data support a model in which PA-X interacts with cellular mRNA processingpathwaystotargetsubsetsofhostRNApolymeraseIItranscripts.ThistargetdiscriminationmechanismsetsPA-Xapartfromotherviralhostshutoffnucleases,whichpreferentiallytargetactivelytranslatedmessengerribonucleoproteins inthecytoplasm.


65-CharacterizationofaNovelHost-TargetedAntiviralMoleculeMarielKleer1,PatrickD.Slaine1,DenysA.Khaperskyy1,2,CraigMcCormick1,21DepartmentofMicrobiologyandImmunology,DalhousieUniversity,2CanadianCenterforVaccinology

Introduction: Current front-line influenza antivirals rely on targeting specific viral proteins to limit replication. Thesedrugsareknownasdirect-actingantivirals(DAAs).However,rapidviralevolutionallowsfortheemergenceofmutationsthat confer resistance to these typesof antivirals. Because influenza viruses dependonmanyhost processes for viralreplication, host-targeted antivirals (HTAs), provide an attractive alternative to limit the emergence of drug-resistantviruses.Here,wecharacterizeanovelhost-directedantiviral,SG3,thateffectivelylimitsinfluenzaAvirus(IAV)replicationin vitro.Methods:A549 lungepithelial cellswere infectedwith IAVand treatedwith SG3at4hpi.Releaseofprogenyvirionswas analyzed by plaque assays and protein expressionwas assessed via immunoblotting. Results: SG3-treatedA549cellsexhibiteda2-logreductionininfectiousvirionscomparedtocontrolcells.SG3treatmentcausedactivationofboththePERKandIRE1αarmsoftheunfoldedproteinresponse(UPR),andselectivelyreducedaccumulationofviralHAandNAglycoproteinswhilehavingminimaleffectsonNPandM1expression.Conclusions:SG3treatmentreduces IAVreplication and accumulation of viral glycoproteins HA and NA, while activating the unfolded protein response. Ourfindings demonstrate that SG3 is a promising candidate HTA for influenza. These in vitro findings will have to becorroboratedinanappropriatepre-clinicalinvivomodel.66-PhotodynamicInactivationofHerpesSimplexVirusesAndreaMonjo1,EricPringle1,MackenzieThornbury1,SherriMcFarland3,4,CraigMcCormick11DalhousieUniversity,2DalhousieUniversity,3DepartmentofChemistryandBiochemistry,UniversityofNorthCarolinaGreensboro,4PhotoDynamic,Inc.

Introduction:Photodynamicinactivation(PDI)employsaphotosensitizer,light,andoxygentoinactivatemicroorganismsthroughtheproductionofreactiveoxygenspecies.PDI’smechanismofactionisnonspecificbutitseffect is immediateandconfinedtoregionswhereaphotosensitizer, light,andoxygenoverlapinspaceandtime.OrthoquinTM,abotanicalplant extract developed by PhotoDynamic Inc., is a powerful photosensitizer with antimicrobial properties that areamplified by exposure to visible light. OrthoquinTM-mediated PDI has been shown to kill bacteria and destroy biofilmwithoutcausinginflammation,makingitanattractivecandidatefortreatingoralbacterialdiseases.Herpessimplexvirusinfections are commonglobally (affecting67%of people under age50) and symptoms includepainful oral (HSV-1) orgenital/anal (HSV-2) lesions. OrthoquinTM-mediated PDI was investigated as a potential treatment for symptomsassociated with these two viral infections.Methods: OrthoquinTM-mediated PDI was tested against HSV-1 and HSV-2infectionofHeLacellsusingplaquereductionassaysandsinglecycle infectionassays(viaflowcytometry),wherevirusinoculumwas incubatedwith different concentrations of OrthoquinTM and exposed to visible light before inoculatingcells. Results: Upon photoactivation, OrthoquinTM-mediated PDI inhibited HSV-1 and HSV-2 infections in a dose-dependentmanner, thoughwith different efficacies against each virus. Conclusions: PDI is a promising alternative totraditional antibiotics and antivirals due to its immediate and selective mechanism of action. We showed thatOrthoquinTMinhibitsherpessimplexvirusinfection.


68-KSHVhijackstheunfoldedproteinresponsetopromotelyticreplicationBenJohnston1,CraigMcCormick11DalhousieUniversity

Kaposi’s sarcoma-associated herpesvirus (KSHV) is the infectious cause of the complex endothelial neoplasm Kaposi’ssarcoma, and two rare lymphoproliferative disorders, primary effusion lymphoma (PEL) andmulticentric Castleman’sdisease. KSHVactivatesmultiple cellular stress responsesduring infection, but our understandingof their role in viralreplicationandtumorigenesisislacking.Onesuchstressmanagementpathwayistheunfoldedproteinresponse(UPR),which is activatedby endoplasmic reticulum (ER) stress. The three sensorsof ER stress that initiateUPR signaling arePERK, IRE1, and ATF6, which induce a translational and transcriptional response to restore homeostasis. Failure torestorehomeostasisresultsintheUPRshiftingfromanadaptiveresponsetopro-apoptosis.IRE1playsakeyroleinthislife-or-deathdecision.DuringERstressIRE1splicesouta26-nucleotideregionofxbp1(X-boxbindingprotein1)mRNA,shiftingthereadingframetotranslatetheactivetranscriptionfactorXBP1s.Inlatentlyinfectedcells,chemicalinductionofERstressinitiateslyticreactivationthroughXBP1-mediatedtranscriptionoftheKSHVlatent-lyticswitchgene,K-RTA.Thus,itappearsthatKSHVhasevolvedamechanismtorespondtoERstress.IRE1alsocleavesanddegradesasubsetofER-boundmRNAsinaprocessknownasregulatedIRE1-dependentdecay(RIDD),whichreducestheproteinloadwithintheER, and is also thought tobe important for inducingapoptosis. In this study,we investigated the roleof theUPRduring lytic replication. We found that lytic replication activates all three sensors of the UPR but the downstreamsignaling events, except for RIDD, are inhibited. Knockdownor chemical inhibition of any of theUPR sensor inhibitedvirusreplication.Surprisingly,ectopicexpressionofXBP1salsoinhibitedviralproduction.Therefore,whileXBP1splaysanimportantrole inreactivationfromlatency, it inhibits latersteps in lyticreplication.Thesedataalso indicatethatRIDDmay be important for KSHV replication. Overall our findings suggest that KSHV hijacks each of the UPR sensors topromotevirusproductioninsteadofresolvingERstress.ThesefindingsidentifyakeysignalingnodeinKSHVreplication,whichmayleadtothedevelopmentofnewtherapeuticstrategiestoinhibitKSHVreplicationandtumorigenesis.69-UnderstandingviraldsRNA-mediatedinnateimmunesensingandcellularresponsesinrainbowtroutcellsSarahJPoynter1,StephanieJDeWitte-Orr21UniversityofWaterloo,2WilfridLaurierUniversity

VirusesacrossgenometypesproducelongdsRNA(viral(v-)dsRNA)moleculesduringtheirreplicativecycle.ThesedsRNAmolecules are potent immunostimulatory molecules and induce type I interferon (IFN), that in turn induces theproductionofaseriesofgenesknownasinterferon-stimulatedgenes(ISGs),theaccumulationofISGswithinacellcanresult in a protective antiviral state. Current research on dsRNA-induced immune responses often uses polyinosinic:polycytidylicacid (poly I:C),acommerciallyavailableproductwith limitedbiological relevancy.AnalternativesyntheticdsRNAcanbeproducedbyinvitrotranscription,(ivt-)dsRNA,withadefinedsequenceandlength.Wehypothesizedthativt-dsRNA,containingaviralsequenceandlength,wouldbeabetterproxyforv-dsRNA,comparedtopolyI:C.Thisisthefirststudyto investigatetheeffectsofv-dsRNAonthe innateantiviralresponseandtocomparev-dsRNAto ivt-dsRNAinducedresponsesinrainbowtroutcells.InrainbowtroutgutcellsclassAscavengerreceptors(SR-As)werefoundtobeasurfacereceptorforpolyI:C.Inthecurrentstudy,ivt-dsRNAbindingwasblockedbypolyI:Candv-dsRNA,aswellasbypoly I,aSR-Acompetitive ligand.Thesedatasuggestall threedsRNAmoleculesarerecognisedbythesamereceptor,likelyanSR-A.TodeterminetheabilityfordsRNAmoleculestostimulatetheinnateimmuneresponse,IFN1andtheISG,vig-4, transcript levelsweremeasuredusingqRT-PCR, and a protective antiviral state against twoaquatic viruseswasobserved inrainbowtroutgonadalcells.V-dsRNAwasableto induce IFNandISGtranscriptproductionat3hand24h,anditseffectsincreasedinalength-dependentmanner,ashasbeenpreviouslyseeninrainbowtroutcellswithivt-dsRNAandpolyI:C.V-dsRNAandivt-dsRNAmoleculesthatwerelengthandsequencematchedbothinducedsimilarIFNandISGtranscript levels, and subsequently similar antiviral states. Two novel putative dsRNA-receptors were also identified,DHX9andDDX3.Thesesensorshavesimilardomainstotheirmammaliancounterpartsandbothwerefoundtobindtoivt-dsRNA.Thisstudyisthefirstofitskindtolookattheeffectsv-dsRNAinfishcells;andthefirsttocomparematchedivt-dsRNA tov-dsRNAmolecules. Taken together these results suggest that ivt-dsRNAmaybeagoodsurrogate forv-dsRNA in the study of dsRNA-induced responses and may be a candidate molecule for dsRNA-based antiviraltherapies.


72-ViralamyloidproteinPsylviaLéger1,KarstenRichter2,MeganStanifer1,2,SteeveBoulant1,2,MichèleBouloy3,CarmenNussbaum1,Pierre-YvesLozach11UniversityHospitalHeidelberg,2DKFZHeidelberg,3InstitutPasteurParis

Theaberrantassemblyofhostcellproteinsintoamyloidfibrilscausesmanyfatalbraindisorders,e.g.Creutzfeldt-Jacob,Alzheimer’s, and Parkinson’s disease. These conditions can arise spontaneously, be inherited, or in the case of priondiseases, occur through infection. Investigating the mosquito-borne Rift Valley fever virus, a neurotropic humanpathogen,wefoundthatthevirus-encodedNSsproteinformsmassivebundlesoftwistedfibrillarytangles,reminiscentof the twists inamyloidogenic fibrils.Wealso identifiedNSs structures resemblingamyloidoligomersandprotofibrils.Accordingly,alargefractionofNSsmultimersremainedresistanttoSDSand,theamyloid-markerthioflavin-TboundtoNSs.Fibrillaraggregatesgrewinmammalianhostcellsuntiltheydie.Filamentousstructuresalsogrewinmosquitocells.Inastarkingcontrasttothedynamicsinmammaliancells,arthropodcellslostNSsfiber-bundlesthereafterinaprocessinvolvingRNAinterferenceandphosphorylationbutnotproteasomalactivityand,cellssurvivedinfection.Togetherourresults confirmed experimentally the concept that viruses can encode amyloid proteins.Our study also highlights theexistenceofendogenouscellularmechanismscontrollingamyloidfibrilsandpavethewayforfutureinvestigationsintoanarrayofneurodegenerativediseases.


Posters73–127:posterSession#2

73-EvaluationoftheinfluenzaAvirusquasispeciespopulationsshedbydifferentavianreservoirhostsJessicaBenkaroun1,HughG.Whitney2,AndrewS.Lang31MemorialUniversityofNewfoundland,2NewfoundlandandLabradorForestryandAgrifoodsAgency,3MemorialUniversityofNewfoundland

InfluenzaAviruses(IAVs)evolvequicklythroughmutation,duetoanerror-proneviralreplicaseenzyme,andthroughtheprocessof segment reassortmentduring co-infections.Mutations generatenewclosely related virus variants that canhavedifferentcapacitiesofinfectionandreplication,andthepoolofcloselyrelatedvariantsthatareproducedduringaninfectionisknownasaquasispecies.WildbirdsarethenaturalreservoirofIAVsandareknowntocarrythemajorityofIAVsubtypesandoverallgeneticdiversity.Mostavian IAVshave lowpathogenicity in theirnaturalhostsandcirculatewithoutcausingovertsymptoms,buttheyarestillofbroaderimportanceduetotheirinvolvementinthegenerationofhighly pathogenic viruses in domestic poultry and viruses infecting humans. To better understand the evolutionarydynamicsof IAVs in theirnaturalhosts,weperformedacomparativeanalysisof thequasispeciespopulationsshedbywild birds. Thiswas done by performing high-throughput sequencing of IAVs present in paired oropharyngeal/cloacalswab samples collected from twoof each of three different groups ofwild bird hosts: seabirds, gulls and ducks.OneH2N3andoneH3N2viruswereanalyzed fromAmericanBlackDucks, twoH1N2viruseswereanalyzed fromCommonMurres, and twoH13N6 viruseswere analyzed fromAmericanHerringGulls.Our analysis shows that thenumbers ofoverallvariantsweresimilarwithinthedifferentbirdhostsbutvariedamonghosts.Theamountofsequencediversity,asquantifiedbyShannonentropy,variedamongthegenesegmentsbutshowedsimilarpatternsforthesamesegmentindifferentvirusesfromthesamehost.Theproportionofsharedvariantsforanypairofsegmentsfortwovirusesfromthesamehostwascorrelatedwiththephylogeneticrelationshipof thesegmentssuchthathigherproportionsof identicalvariantsweredetectedinmorecloselyrelatedsegments.Closeto100%ofthevariantsobservedinthePA,HA,NA,NPandNSsegmentswerefoundinbothofthemurreviruses,andthesesegmentsareallphylogeneticallyverycloseforthetwoviruses.ThemurrevirusPB1andPB2segmentswerephylogeneticallymoredistantandsharedapproximately50%of variants between the two viruses. TheM segments of thesemurre viruses have different phylogeographic origins(Eurasia versus North America) and shared few variants. The two gull viruses are phylogenetically close for all eightsegmentsandahighproportionofidenticalvariantswereobservedforallsegments.Similartothepatternsfortheotherhosts,theduckvirusesshowedvariantconservation levelsthatcorrespondedtothephylogeneticrelatednessforeachsegment.Finally,ahighproportionofnon-synonymoussubstitutionswasobserved for theNSsegment relative to theothersegmentsforallvirusesanalyzed.74-ConservedmicroRNACluster’sRoleinModulatingtheInnateImmuneResponsetoViralInfectionNadineAhmed1,Ragunathsingaravelu1,JohnP.Pezacki11UniversityofOttawa

Successfulrecognitionofviralinfectionsandconsequentialtriggeringofantiviralinnateimmuneresponsesarecrucialforhost persistence and survival. Traditionally, to learn more about these interactions, regulatory networks at thetranscriptionalandtranslational levelshavebeenextensivelystudied.Recently,withthediscoveryofmicroRNAs,therehasbeenashiftinthisdogma.microRNAs(miRNAs)areaclassofhighlyconservedandabundant,smallnon-codingRNAsinvolved in post-transcriptional regulation of gene expression. miRNAs perform their function by binding to the 3ʹ-untranslatedregionoftargetmRNAstoinducedegradationandsuppresstranslation.Hereinwedescribeaninterferon-regulatedmiRNAclusterthatmodulateinterferonproductionandsignalingduringviral infection.TheidentifiedmiRNAclusterappearstoregulatekeyphosphorylationeventswhicharecrucialtotheantiviralsignalingcascade.Wefoundthattheclusterenhancesthe innateantiviral responsebyaugmentingthephosphorylationof transcriptionfactors,suchasinterferonregulatoryfactors(IRF)andsignaltransducerandactivatoroftranscription(STAT),duringvesicularstomatitisviralinfections.WedemonstratethattheclusterelicitsitsantiviralresponsebytargetingnegativeregulatorsoftheIFNsignaling cascade and JAK-STAT signaling pathway.Overall, ourwork reveals a novelmodeof cooperation between amiRNAcluster intheregulationofantiviralcellularresponsesandaids inuncoveringfundamentalrolesfornon-codingRNAsininfectionandimmunity.


75-KickingtheKapoutofKaposi’sSarcoma:ToolsforStudyingaHerpesvirusProteinGrantMacNeil1,EricS.Pringle1,JulieRyu1,2,CraigMcCormick1,JenniferA.Corcoran1,31DalhousieUniversity,2ElancoCanadaLimited,3BeatriceHunterCancerResearchInstitute

Kaposi's sarcoma-associatedherpesvirus (KSHV) is thecauseof theendothelial cell (EC) cancer,Kaposi's sarcoma (KS).KSHVlatentinfectionofECspredominatesinKS,anddrivesviraltumorigenesis.Inlatentinfection,asubsetofviralgenesreprogram normal ECs and induce aberrant proliferation, altered morphology and inflammatory gene expression.Lytically infected cells are far less abundant inKS lesions; however, they serve as a sourceof newvirus andpromotetumorigenesis in a paracrinemanner. KapB is expressed fromahighly repetitive,GC-rich, polycistronic transcript thatalsoencodesKapAandKapC.KapBhasbeenshowntorecapitulatelatentphenotypeswhenectopicallyexpressedinECs.WerecentlyconductedshRNA-mediatedknockdownofthekaposintranscriptinthecontextofKSHVlatentinfectiontostudythecontributionofthisgeneto latencyphenotypes.However, thetoolsto investigateKapB-specificrolesduringeither stateofKSHV infectionhavebeen lacking in the field. Toaddress this,weutilized theBAC16bacterial artificialchromosomecloneofKSHVtoeliminatetheexpressionofKapB.BecausetheKapBandKapCopenreadingframes(ORF)overlap,wesynthesizedageneblockwitharecodedversionofKapC,designedtoeliminatetheKapBORFexpressionbyinsertionofseveralnonsensecodonsintotheKapBreadingframe.Usinghomologousrecombination,thisrecodedKapCwasinsertedintoadelKapBdelKapCbackbone,generatingaKapBknockoutsequence.ThesesequencesarebeingusedtoproducerecombinantmutantvirusesthatwillallowustodetermineKapB-specificeffectsonnormalcellfunctionduringKSHV infectionthatcannowbedifferentiatedfromKapAandKapCphenotypiceffects.Withthesetools,we intendtoreveal,withgreaterprecision,functionsofKapBinbothlatentandlyticinfectioncycles.76-Expressionofthep14FusionAssociatedSmallTransmembraneProteininanOncolyticAdenovirusImprovesVectorEfficiencyJDelPapa1,2,JPetryk3,JCBell2,3,RJParks1,21RegenerativeMedicine,OttawaHospitalResearchInstitute,2Biochemistry,MicrobiologyandImmunology,UniversityofOttawa,3CancerTherapeutics,OttawaHospitalResearchInstitute

Theuseof fusogenicproteins toarmAdenovirus (Ad)hasenormouspotential.Theseproteins functionthroughcell tocell fusionwithinthetumorwhich leadstothegenerationofmultinucleatedsyncytia,withtheconcomitantreleaseofimmunologicallyactivesyncytiosomes.However,thesizeoftypicalviralfusogenicproteinsmakestheirinclusionintoanoncolyticAdgenomedifficultduetoviralDNApackagingconstraints.Thep14FusionAssociatedSmallTransmembrane(p14 FAST) protein is a non-structural viral fusogenic protein originating from reptilian reovirus, which is significantlysmaller than typical fusogenic proteins. Our objective is to assess the anti-cancer potential of an Ad that undergoescancerspecificconditionalreplication(CRAd)incancercells,andexpressesthep14FASTfusogenicprotein(CRAdFAST).Inanimmune-competentmousemodelofcancer,CRAdFASTprovidedsomeanti-cancereffect,limitedbytheabilityofAdtoreplicateinmousecells.InanA549humanlungadenocarcinomaxenograftmodelofcancer,p14FASTexpressionsignificantlyimprovedtheoncolyticabilityofCRAdinvitroandinvivo.Wehavealsoshownthatp14FASTexpressionandassociatedcell fusion induceadramatic increase in theproductionofextracellular vesicles,whichmayhave immuno-stimulatory properties. We are currently exploring the characteristics of fusion-derived extracellular vesicles todeterminetheextentoftheirabilitytoinitiateadaptiveimmunetargetingoftumorassociatedantigens.Takentogether,ourresultssuggestimmunologicallyactivecancersyncytiosomesreleasedduringCRAdFASTinfectionmaypossesspotentanti-canceractivityinitiatedbythedevelopmentofananti-tumoradaptiveimmuneresponse.


78-ClinicalroleofentericvirusesinsucklingpigletsNicolasNantel-Fortier1,VirginieLachapelle1,ElysePoitras2,AnnLetellier1,Yvanl'Homme1,3,JulieBrassard1,21UniversityofMontreal,2AgricultureetAgroalimentaireCanada,3CEGEPGarneau

Neonataldiarrhea isamajorconcern for swineproducersandcauses significanteconomic losses to the industryeachyearduetothedeathofthousandsofanimalsworldwide,orincreasedproductioncostsassociatedwithtreatmentsandextendedperiodsofgrowth.Somevirusesaresuspected,andprobablyunderestimated,fortheirimportanceinneonataldiarrhea in animals. Indeed, enteric viruses such as caliciviruses (including norovirus and sapovirus), rotaviruses,astrovirus,kobuvirusandtorquetenosusvirushavebeenassociatedwithepisodesofdiarrheainpiglets.However,viralexcretion and diarrhea are not always concomitant. In order to monitor the excretion of enteric viruses in sucklingpiglets,a samplingschemeusingdiarrheicpiglets faeces (yellow-brownsoft to liquid)andnormal faeces fromhealthypigletswascarriedout.Atotalof203faecalsamples,70ofwhichwerefromdiarrheicpiglets,werecollectedfrom11farrowing farms in a farrow-to-finish integrated swine production network in the province of Quebec. Samples weretransportedoniceandstoredat-80°C.FaecalsamplesweresuspendedinPBS20%(w/v)andcentrifuged16000xgfor5minutes.Totalnucleicacids (DNA/RNA)wereextractedwithaQIAampViralRNAMiniKit (Qiagen).RotavirusAandC,calicivirusesandkobuvirusweredetectedusinga targetedRT-PCR,astrovirusgenotypes1 to5weredetectedusingamultiplexedRT-qPCRandtorquetenosusviruswasdetectedusingaqPCR.Positivebandsofcaliciviruseswereexcisedfroma2%agarose gel, purified and clonedusing ap-Gem-T vector systemand JM-101 competent cells (Promega).Atotal of 4 colonies per samples were sequenced and consensus sequences were obtained with the BioEdit softwareversion 7.2.6. Astroviruses (genotypes 1 to 5) were by far the most frequently detected viruses with 85% positivesamples, followed by rotavirus C (42%), kobuvirus (41%), torque teno sus virus (25%), rotavirus A (20%) and thecaliciviruses(16%).However,onlytherotavirusC,theastrovirusgenotype3andthecaliciviruseswereassociatedwithsymptomsofdiarrhea,withchi-squarepvaluesof0,001,0,005and0,02respectively.All sequencedcaliciviruseswereclassifiedassapoviruses.Thisstudyre-evaluatedthepresenceofselectedentericvirusesinpigletsfromtheprovinceofQuebecanddetectedforthefirsttimethepresenceofkobuvirus inCanadianpigs.Wewerealsoabletodemonstratethatcaliciviruses,astrovirusgenotype3androtavirusCarepossiblecausativeagents inneonataldiarrhea inCanadianswine, whichmay be useful in future assessment of clinical portraits of diarrheic herds. Although present in a largeproportion of samples, rotavirus A, kobuvirus, torque teno sus virus and the other astroviruses genotypes were notassociatedwithdiarrheaasreportedelsewhere.Onlysapoviruswasfoundinthesequencedcalicivirus-positivesamples,asitisusuallythecaseinyoungerpigs.Furtherworkisneededtoassesstheprevalenceofthesevirusesincoinfectionwith other pathogens such as bacteria and other viruses to provide an in-depth insight as to their role in thepathogenesisofpigletdiarrhea.


79-SamplepretreatmentsaffecttheoutcomeofviralmetagenomicsanalysesNicolasNantel-Fortier1,VirginieLachapelle1,MartinGauthier2,AnnLetellier1,YvanL'Homme1,3,JulieBrassard1,21UniversityofMontreal,2AgricultureetAgroalimentaireCanada,3CEGEPGarneau

High-throughput sequencing ismore affordable than ever before and it is now themethod of choicewhen studyingmicrobiomes.Theporcineentericmicrobiomeiscomposedofgeneticmaterialfrombacteria,archaeaandviruses.Oneofthemanychallengeswhenstudyingthevirome(viralcomponentofthemicrobiome)withhigh-throughputsequencingis the isolation of the viruses from the rest of the microbiota. This is usually accomplished using various samplepretreatments, incorporated intotheproceduretoremoveasmanynon-viralcomponentsaspossible.Thesestepsarepresumedto leadto increasedsequencingdepthand, therefore, facilitatedownstreamanalysis.Todetermine if theseupstream steps were useful for downstream analyses, we performed an evaluation of the differences between highthroughputsequencingresults fromsamplesthatunderwentpretreatmentsfromsamplesthatdidnot.Amaximumof8g of feces were collected from pigs at different periods, ranging from <1 week to 22 weeks old. All samples weresubmitted toahomogenizationstepat20% inPBS,andcentrifuged for5minat16000xg. The followingstepswereeitherperformedoromitted:sampleswerepassedthrough0,45µmand0,22µmfiltersfollowedbyRNaseandDNasetreatments.Atotalof32treatedanduntreatedsamples(8samplesreceiving4differentseriesoftreatments)underwentnucleicacidextraction,randomreversetranscriptionandamplificationaswellaspurificationsteps.Then,librarieswerepreparedand sampleswere sequencedusingan IlluminaMiSeq sequencer.Readswere trimmed forquality, adapterswereremovedandwereclassifiedusingtheCentrifugemicrobialclassificationenginewithacustomdatabasebasedonthe “nt” database from NCBI and visualized using Krona. When the samples went through the complete series ofpretreatments,6/8sampleshadtheirhighestviralreadpercentagesofall4seriestested(from10%ina22weeks-oldpigto86% ina1week-oldpiglet,withameanof27%), theother2hadtheirhighest readpercentageswiththe filtrationsteps only (30% in a 1 week-old piglet and 45% in a 4 weeks-old piglet). In all samples (8/8), the lowest viral readpercentageswereobservedwhenthehomogenatewasusedwithoutpretreatment(from0,06%ina22weeks-oldpigto18%ina4weeks-oldpiglet,withameanof7%).Mostofthenon-viralreadswerebacteriain27/32samples(from6%ina4weeks-oldpigletto86%ina22weeks-oldpig,withameanof57%)andeukaryotesintheother5samples(from26%ina22weeks-oldpigto86%ina14weeks-oldpig,withameanof68%).Withallpretreatments,theyoungestpigs(1and4weeks-old)hadthehigherpercentageofviralreads,withanaverageof49%comparedtotheoldestones(14and22weeks-old)withanaverageof13%.Mostnotableviralfamilyreadcountvariationswhenenzymeswereaddedweretheastroviridae,caliciviridaeandpicornaviridae,withreadcountsvaryingdecreasinglyinsomesamples(70496to39,17271to21and41000to22reads,respectively).Usingpigfeces,wehaveshownthatsamplepretreatmentsprofoundlyaffectthe overall relative quantity of reads from each of the virus, bacteria and eukaryote classes in the pigs’microbiome.Pretreatmentsaffectpigs’viromesdependingontheirage:olderpigs’viromesbenefitfromtheenzymetreatments,asthereseemstobealotmorebacteriaandeukaryoteDNAinthemcomparedtotheyoungerones.Also,RNAvirusessuchas the astroviridae, caliciviridae and picornaviridaeappear to bemore affected by the enzyme treatments than otherviruses from the same samples. Using filtration treatment preceding genomic extraction is therefore paramount toimprovedownstreamanalysesofthepig’svirome.However,theuseofenzymesshouldbeevaluatedaccordingtothenatureofthesampletested.


80-Ebolavirusrequiresphosphatidylinositol(3,5)bisphosphateforviralentryShirley Qiu1,2, Anders Leung3, Yuxia Bo1,2, Robert A. Kozak3, Sai Priya Anand1,2, Corina Warkentin1,2, Fabiola D.R.Salambanga1,2,JenniferCui1,2,GaryKobinger3,DarwynKobasa3,4,MarcelineCôté1,21DepartmentofBiochemistry,MicrobiologyandImmunology,UniversityofOttawa,2OttawaInstituteofSystemsBiology,UniversityofOttawa,3SpecialPathogensProgram,NationalMicrobiologyLaboratory,4DepartmentofMedicalMicrobiology,UniversityofManitoba

Ebolavirus(EBOV)isahighlypathogenicenvelopedfilovirusthatcausessporadicoutbreaksofseverehemorrhagicfeverdisease. EBOV entry requires internalization into host cells and trafficking to late endosomes/lysosomes where itsreceptor,NPC1,resides.Howviraltraffickingoccurs,andwhetheritisregulatedbythevirus,remainunclear.Here,weshowthatthePIKfyve-ArPIKfyve-Sac3cellularcomplex,whichisinvolvedinthemetabolismofphosphatidylinositol(3,5)bisphosphate (PtdIns(3,5)P2), is critical forEBOV infection.Although theexpressionofall subunitsof thecomplexwasrequired, PIKfyve kinase activity and PtdIns(3,5)P2 productionwere found to be the critical determinants for efficiententry. Inhibition of PIKfyve reduced delivery of virions to NPC1 and resulted in virus accumulation in early endocyticcompartments.Furthermore,phosphoinositideprobesandlivecellimagingrevealedanincreaseinPtdIns(3,5)P2-positivevesiclesincellsduringEBOVentry,indicatingpotentialupregulationofthislipidbythevirus.Takentogether,ourstudiessuggestthatEBOVrequiresandpotentiallyupregulatesPtdIns(3,5)P2productionincellstopromoteefficientdeliverytoNPC1.81-EvaluatingfidelitymutantsinanalphavirusEdwardPatterson1,SamanthaWalsdorf1,TiffanyKautz1,NaomiForrester11UniversityofTexasMedicalBranch

RNAviruses replicatewith a degreeof plasticity in their genome, creating a swarmof closely related sequences. Thisdiversitymustbemaintainedintheviruspopulationtonavigatebottlenecksandestablish infectioninnewtissuesandhosts.Thereisaprecisemutationrateforoptimalviralfitness,avoidingerrorcatastropheandlackofdiversity.Alteringthe mutation rate of alphaviruses is a promising method to produce a range of commercially available attenuatedvaccines. Venezuelan equine encephalitis virus (VEEV) fidelity mutants targeting the nsP4, which encodes the RNA-dependent RNA polymerase (RdRp), have recently been discovered. However, the tools available to characterize themutationrateofaltered fidelitymutantsareunreliableandhaveprovided inconsistent results.Optimizinganassay toevaluatemutationratewithreproducibleresultswillbeapriority forvaccinedevelopmentof fidelitymutants. Ideally,onlyasingleroundofinfectionshouldbemeasuredtodeterminetheeffectofaspecificmutation.Thiswouldeliminatebiaseddatafromfitnessimpartedbyrandommutationsthatareselectedduringsequentialinfectionofcells.Foreaseofprocessing,virusparticlescontainingRNAshouldalsobereleasedintothemedia.Tocreateamethodtoevaluatefidelitymutants,severalpreviouslycharacterizedresidues,E3Δ56-59,E1F253L,andE1G91D,weremutated in thestructuralproteins of the VEEV vaccine strain, TC-83. Electroporation of the transcribed VEEV genome into Vero cells allowedreplicationofnon-infectiousviruses.RT-PCRwasusedtodeterminethepresenceofviralRNAinthemedia,andgenomescontainingaGFPreportergenewereusedtoconfirmthatresultingviruseswerenon-infectious.Theoptimalstructuralmutationwas chosen for combinationwithVEEVnsP4 fidelitymutants,G14R,A106T,C488Y (previously characterizedfidelitymutantfromchikungunyavirus),G14R+E37G+A106T,andG14R+E37G+A106T+C488Y.Sequencediversitywillbecalculated fromnextgeneration sequencingdataandcompared todiversitymeasured from infectiousvirusgenomes.Usingthesenon-infectiouscloneswillallowcharacterizationofallnon-structuralmutationsandamajorityofstructuralmutations that are suspected to alter fidelity. Implementation of this pipelinewill be vital to accurately characterizemutationsandevaluatetheireffectsonmutationrateineffortstoexpeditevaccinedevelopment.


82-Protein-proteininteractionsofME53fromAutographacalifornicanucleopolyhedrovirusEmineÖzşahin1,ÉvaNagy1,PeterKrell11UnivesityofGuelph

ME53isa53kDaproteinfromthebaculovirusAutographacalifornicamultiplenucleopoly-hedrovirus(AcMNPV)andisconserved inall sequencedalpha- andbetabaculovirusgenomesbut itsprecise function remainsunknown.ME53 is astructural protein incorporated into thenucleocapsidofbothbuddedandocclusion-derived viruses. It is expressedatbothearlyandlatetimesofinfection.WhiledeletionofME53doesnotaffectviralDNAsynthesislackofME53reducesvirusproductionby99%causingarestrictionof thecell tocellspreadof thevirus.ME53 localizestothecytoplasmatearly times post-infection and forms foci on the cytoplasmic membrane with GP64, which is the major envelopeglycoprotein.Despite theabsenceof a recognizablenuclear localization signal,ME53also translocates to thenucleusmainly at the ring zone where occlusion-derived viruses form at late times post-infection. We hypothesize that thenuclear localization is accomplished through a viral or host chaperon protein using theME53 “nuclear translocationsequence” locatedbetweenaminoacids109and137.Therefore,weaimedto identifytheviralandhostproteinsthatbindtoME53inordertobebetterunderstandthemolecularmechanismofME53intracellulartranslocationandME53function. Yeast two-hybrid and mass spectrometry analyses were used to identify protein interacting partners ofME53. AcMNPV-infected or uninfected Sf9 cells were used to construct prey cDNA libraries to identify the potentialprotein-bindingpartnersofME53usingME53proteinasbait.Cellular receptor foractivatedproteinkinasec (RACK1)wasidentifiedasoneputativeME53cellularproteininteractingpartnerfromacDNAlibraryconstructedfromAcMNPVuninfectedcells.TwoAcMNPVproteins,VP80(anucleocapsidproteinwhichinteractswithnuclearactin)andLEF5(lateexpressionfactor5),werealsodeterminedasputativeME53proteinbindingpartnersfromacDNAlibraryconstructedfromAcMNPV-infectedcells.Thesethreegenes(RACK1,VP80,andLEF5)andseveraltargetedAcMNPVORFs includingp6.9,Exon0,VP39,GP64andsegmentsofME53,wereclonedasreciprocalbaitsandpreysandtestedforprotein-proteininteraction in the yeast two-hybrid system.UsingN terminally fusedGFPME53 to pull downME53 -binding partnersfollowedbymassspectrometryanalysisweidentifiedHsp60(heatshockprotein60)asanotherpotentialME53bindingprotein.The interactionbetweenVP80andME53wasconfirmedbybimolecular fluorescencecomplementation.SinceVP80isastructuralproteininteractingwithVP39(majorcapsidprotein),theinteractionbetweenVP80andME53mayenabletheattachmentofnucleocapsidstothecellularmembraneallowingfornucleocapsidbudding.Itmayalsoinvolveocclusion-derivedvirusformationinthenucleusattheringzoneorfacilitatenucleocapsidtransportfromthenucleustothecellularmembrane.


83-DecodingIntraviralandVirus-HostProteinInteractionNetworksofPorcineNidovirusesbyQuantitativeProteomicsLaura Jhoana Sanchez Mendoza1, Camila Andrea Valle Tejada1, Chantale Provost1, Carl A Gagnon1, Francis Beaudry1,LevonAbrahamyan11Facultédemédecinevétérinaire,UniversitédeMontréal

The long-term goal of our research program is to better understandmolecular mechanisms of interactions betweenanimalnidovirusesandtheirhostsinordertodevelopnewstrategiesforeffectivecontrolofviralinfections.Thepresentresearchprojectisfocusedonemergingswinepathogensofunprecedentedeconomicsignificance,suchastheporcinereproductive and respiratory syndrome virus (PRRSV) and the porcine epidemic diarrhea virus (PEDV). The PEDV andPRRSV are responsible for severe economic losses and considered as the primary emerging livestock pathogensworldwide. Insufficientunderstandingofvirus-host interactions impedes thedevelopmentofeffectiveanimalvaccinesagainstPRRSVandPEDV.Host-virusinteractionsarehighlydynamicandmayinvolvemultiproteincomplexes.Earlier,ourgroupemployedbiochemicalandproteomicsapproachestoidentifyvirus-hostmultiproteincomplexes,andshowedthattheir composition is controlled by the virus either by direct recruitment of or by binding to host proteins. Growingevidence indicates thatextracellularmicrovesiclesplayan important role inviralpathogenesisandmodulationofhostimmuneresponsestoinfection.Consequently,thecharacterizationofthecompositionoftheextracellularmicrovesiclesand identificationofthehostproteinsthatarespecificallyencapsidated intoorboundtovirionsare importantforourfurtherunderstandingofvirus-hostinteractions.Toaccomplishthisobjective,weproducedandpurifiedPRRSVandPEDVusingboththesimiancellculturesthatareroutinelyusedforvirusproductionandPRRSV/PEDVnaturaltargetcells(ofporcineorigin).WehypothesizedthatthecompositionofPRRSVandPEDVvirionsandvirus-hostmolecularcomplexeswill reflect changes in environmental conditions (e.g., pH, activities of host proteases, and tissue-specificity).Furthermore,wehypothesizedthat thetight interactionsbetweenhostandviralproteinsdefines the fateof infectionandpathogenesis.Proteomics is thebestmethod todirectly characterize themultimolecular complexes important forvirusentryandpathogenesis.Weexaminedthecompositionofprogenyvirionsinordertoidentifycellularproteinsthatare associated with or encapsidated into viral particles using state-of-the-art mass spectrometry (MS) strategies,including ahigh-resolutionhybridQuadrupole-OrbitrapMS. Thepresent studyhasdemonstrated the incorporationofcellularproteinsinPRRSVandPEDVvirions.WefoundthatthePRRSVandPEDVinfectionsaffectedtheabundancelevelsofnumeroushostproteinsassociatedwithmicrovesicles.Ourproteomicdata showed that theabundanceofproteinsinvolvedinimmuneresponsesandmetabolicprocesseswasdramaticallyaffectedbyPRRSVinfection.TheabundanceofproteinsinvolvedinimmuneresponseswasalsochangedinPEDVinfectedcells.Interestingly,inPEDVinfectedcells,hostproteins involved in cell cycle regulation and cytoskeletal systemwere affected in abundance,which is not surprisingbecause several investigators reported that cytoskeletal proteins are actively participating in moving the viralcomponents to the assembly site, and thatmany virusesmanipulate DNA repair and cell cycle in order to achieve acellularenvironmentfavorablefortheirreplication.Furtherinvestigationsareneededtoevaluatetheroleofindividualcellularproteinsinthenidoviralreplication,assembly,andpathogenesis.


86-DevelopmentofaHumanizedAntibodyforPreventionofEpstein-BarrVirusInfectionandAssociatedCancersJeromeTanner1,JingHu1,CarolinaAlfieri1,21CHUSainte-Justine,2DepartmentofMicrobiology,InfectiologyandImmunology,UniversityofMontréal

Acute Epstein-Barr virus (EBV) infection in immunosuppressed transplant patients can give rise to malignant B-cellproliferation known as post-transplant lymphoproliferative disease (PTLD). The EBVmajor virion surface glycoprotein(gp)350 is aprincipal targetofnaturallyoccurringneutralizingantibodies, and is viewedas thebest target topreventacute infectionandPTLD inat-risktransplantrecipients.Wehaveconstructedahumanized(hu)versionofthemurineanti-gp350neutralizingmonoclonalantibody72a1.Thehu72a1IgG1antibodydisplaysnosignificantanti-mouseactivity,recognizesbothgp350anditssplicevariantgp220,aswellasagp350peptidethatwasshowntoconstitutetheprincipalEBVgp350neutralizingepitopewhen tested in immunoassays. Further,hu72a1antibody canblockEBV infectionofBcells invitroata levelequaltothatofamouse-humanchimeric72a1antibodyconstruct.Thisworkprovidesafurtherstructuralandimmunologicalunderstandingofthe72a1antibodyinteractionwithEBVgp350,aswellasalaunchpointfor future anti-EBV therapeutic antibodies designed to block EBV infection and prevent PTLD while eliminating thedeleteriousantigenicmurinefeaturesoftheoriginal72a1antibody. 87-ViDiT-CACTUS:afast,low-costandsensitivewrapper-methodforvirusdiscoveryandothermicrobiologyapplicationsJoostVerhoeven1,MartaCanuti1,HannahMunro1,SuzanneDufour1,AndrewLang1,21MemorialUniversityofNewfoundland,2MemorialUniversityofNewfoundland

Astheper-basecostsofhigh-throughputsequencing(HTS)technologiesdecreaseitisbecomingincreasinglycommoninmicrobiology. In contrast, library preparation methods often carry a high per-sample cost, possess strict inputconcentration requirements and differentmethods are usually needed for different applications. These aspectsmakeHTSdifficulttoimplementinsomenicheapplicationsandespeciallyforresearchgroupsonabudget.Furthermore,theimmense volume of HTS-generated data often requires bioinformatic expertise and high-end equipment for dataanalyses.Toanswertheseneeds,wedevelopedViDiT,auniversalPCR-basedlibrarypreparationmethodoptimizedforthe Ion Torrent PGM sequencer, that includes 3 steps: DNA amplification with tailed random octamers; librarygeneration,whereadaptorsequencesareintroduced;andlibraryenrichmenttoincreasethelibraryconcentration.Wefurther developed CACTUS, an open-source Python wrapper-script that handles sequence quality checks, filtering,PHRAP-based assembly and alignment of sequences to reference databases using BLAST and DIAMOND. CACTUS isdesignedtooff-loadintensiveworkloadstocloudplatformstogeneratehigh-qualitydatawithouttheneedforhigh-endservers.We validated our method within three applications: virus discovery, performed on 36 oropharyngeal-cloacalswabs collected from avian influenza virus (AIV)- negative wild birds; amplicon-based full-genome sequencing,implementedonproductsoftheinfluenzaviruswholegenomeRT-PCRcompletedon3AIV-positivecloacalswabs;andmicrobiomecharacterizationofadeep-seacarnivorous sponge.ViDiT-CACTUSwas successfullyusedasvirusdiscoverytool as it allowed the identification of fragments of putative viruses from 25 viral families, including 10 vertebrate-infectingviruses,evenfromsampleswithundetectablenucleicacid(NA)concentrations.Mostofthe identifiedvirusesshared low identitywith reference sequences and could be considered novel viruses. The presence of viruses from5families(familiesCaliciviridae,Coronaviridae,Papillomaviridae,Parvoviridae,Pneumoviridae)wereconfirmedbyPCRin6samples and complete genome sequencing and epidemiological investigations are ongoing. The successfulcharacterizationof3AIVnear-completegenomes(99.0%,90.4%and99.6%coverage)showedourapproachcanbealsouseful as an amplicon-based complete genome sequencing system in a streamlined and multiplex-capable setting.Furthermore,CACTUSassembliesconsistentlyproduced longcontigs spanning theentire lengthofeachviral segment,demonstrating its strong performance in complete genome assembly. Lastly, ViDiT-based shotgun libraries weresuccessfully used to profile microbial diversity in a deep-sea sponge. Phylum-level composition showed a diverseassemblageofbacteria,includingProteobacteria,Bacteroidetes,Cyanobacteria,ActinobacteriaandFirmicutes,whilethedetected Archaea phyla represented the Euryarchaeota and Thaumarchaeota andwe detected both double-stranded(Caudovirales,MimiviridaeandPhycodnaviridae)andsinglestranded(Microviridae)DNAviruses.Comparingourdatatopreviously published 16S rDNA-based data of the same sponge showed ViDiT-CACTUS can capture all but the lowestabundancebacterialtaxa.Furthermore,ViDiTbasedtaxonomicprofilesrevealedadditionaltaxaandlongercontigsthatcould be used to assemble a preliminary functional metagenome of the sponge. In conclusion, ViDiT-CACTUSdemonstrated its validity in a wide range of microbiology applications. Valuable information was produced at anextremelylowcost(<$5USDperlibrarypreparation)andthehigh-sensitivityofourmethodallowedforinvestigationofsamples with low to unmeasurable NA concentrations. The simplicity andmodularity of ViDIT-CACTUSmake it easilyimplementableinanymolecularbiologylaboratory,forvariousresearchgoalsandwithdifferentsequencingplatforms.


88-Molecularcharacterizationandpathogenicityanalysisofafowladenovirus4isolateYanlongPei1,PeterKrell1,ÉvaNagy11UniversityofGuelph

Somefowladenovirus4(FAdV-4)isolatescauseseverehepatitis-hydropericardiumsyndrome(HHS)withmortalityupto80%, especially in 3-5week-old broiler chickensworldwide. However little is known about virulence determinants ofFAdV-4.Preliminaryinovoexperimentsshowedthat10-day-oldspecificpathogenfree(SPF)chickenembryosinoculatedwith 1x105 of an Ontario FAdV-4 isolate, FAdV-4ON2, had 33%mortality (2 out of 6) post-inoculation on day 5 andshowedtypicalHHSlesions,suchasnecrotichepatitis.IncontrasttotheON2group,noclearlesionsandmortalitywereobservedatthesamedoseofinoculationwiththenonpathogenicFAdv-4ON1andthePBSmockgroups.Inthisstudy,wesequencedtheentiregenomeof theputativevirulentstrain,FAdV-4ON2.Compared toFAdV-4ON1,FAdV-4ON2hasagenomesizeof45,577bpwhichis57bpshorterthanthatoftheON1isolate.Boththeleftend(7,088bp)andcenterpart(25,717bp)oftheON2genomeare99%identicaltoON1.However,therightendsequence(12,772bp)isonly96%identicaltothatofON1.ThesignificantdifferencesareinTR-EandORF19.TheON2TR-Esequenceis144bpshorterthantheON1TR-EandthereisastopcodonintheON2ORF19thatwouldmaketheON2lipase209aminoacidshorterthantheON1 lipase.Ourdata indicatethat thedeletionofTR-EandthemutationofORF19arepotentiallyassociatedwithFAdV-4CAVpathogenicity.90-BlockingtheRNAinterferencepathwayimprovesoncolyticvirotherapyMathieu J.F. Crupi1,2, Donald Bastin1,2, Amelia S. Aitken1,2, Adrian Pelin1,2, Larissa A. Pikor1,2,Marie-Claude Bourgeois-Daigneault1,2,MichaelS.Huh1,2,JohnC.Bell1,2,CarolinaS.Ilkow1,21CentreforInnovativeCancerResearch,OttawaHospitalResearchInstitute,Ottawa,K1H8L6,Canada,2DepartmentofBiochemistry,MicrobiologyandImmunology,UniversityofOttawa,Ottawa,K1H8M5,Canada

Antiviralresponsesarebarriersthatmustbeovercomeforefficacyofoncolyticvirotherapy.Inmammaliancells,antiviralresponses involve the interferon pathway, a protein-signaling cascade that alerts the immune system and limits viruspropagation. Tumour-specific defects in interferon signaling enhance viral infection and responses to oncolyticvirotherapy,butmanyhumancancersarestillrefractorytooncolyticviruses.Giventhatinvertebrates,fungiandplantsrelyonRNAinterferenceprocessesforantiviralprotection,weinvestigatedthepotentialinvolvementofthisalternativeantiviral mechanism in cancer cells. In our studies, we detected viral genome-derived small RNAs, indicative of RNAinterference-mediated antiviral responses in human cancer cells. To combat RNA interference-mediated antiviralresponses, only a fewmammalian viruses encode viral suppressors of RNA silencing (VSR) that interact with cellularsilencingmachinery.Assuch,weengineeredanoncolyticvesicularstomatitisvirusvarianttoencodeanectopicVSRthatis known to bind double-stranded RNA and inhibit processing by Dicer to prevent the production of anti-viral smallinterfering RNAs. Our virus showed enhanced viral replication and cytotoxicity, impaired viral genome cleavage andalteredmicroRNAprocessing in cancer cells.Our data establish the improved therapeutic potential of our novel viruswhichtargetstheRNAi-mediatedantiviraldefenseofcancercells.


94-TheviralproteinKaposinBelicitsprocessingbodydispersalbyactivatingthecytoskeletalkinaseROCKElizabethLCastle1,CarolynRobinson1,JenniferACorcoran1,2,31DepartmentofMicrobiologyandImmunology,DalhousieUniversity,Halifax,NS,Canada,2DepartmentofSurgery,DalhousieUniversity,Halifax,NS,Canada,3BeatriceHunterCancerResearchInstitute,Halifax,NS,Canada.

Kaposi’s sarcomaherpesvirus (KSHV) is theetiologic causeofKaposi’s sarcoma, ahighly inflammatoryendothelial cellcancer that is most severe in immunosuppressed individuals. During latent KSHV infection, the virus promotes twonotable phenotypes: cell elongation (spindling) and increased expression of inflammatory and angiogenic molecules.EctopicexpressionofthelatentviralproteinKaposinB(KapB)recapitulatesthesefeaturesbypromotingtheformationofactinstress fibers (SF)andthedispersalofprocessingbodies (PBs).PBsareribonucleoprotein-containinggranulesthatmediate degradation or translational suppression of cellularmRNA. Because PBs are amajor site for the constitutivedecayof a class ofmRNA that encode cytokines and angiogenic factors, their size andnumber controls inflammatorygeneexpression.KapB inducesbothSFandPBdispersionbyactivatinga stress-responsivesignalingaxis that links thekinaseMK2 to the cytoskeletal regulator,RhoA.Previouswork inour labusedknockdowns todemonstrate thateachcomponent of this signaling axis is required for both these phenotypes. Therefore, we have not yet been able touncoupleKapB-mediatedeffectsontheactincytoskeletonfromKapB-mediatedPBdispersal.Thishasledustoposethefollowing research question: Is actin SF formation required for the dispersal of PBs during KapB expression? Toinvestigate this question, we investigated the effects of shRNA knockdown of downstream RhoA effectors known tocoordinateSFformation(ROCK1,ROCK2,mDia1,mDia3)onKapB-inducedactinSFformationandPBdispersal.IfactinSFformationwaseliminatedusingtheseknockdownsbutPBdispersionwasnotaffected,wewouldknowthatactinSFwerenotrequiredforPBloss.KnockdownofROCK1andROCK2eliminatedactinSFformationandpreventedPBdispersal incellsexpressingKapB.Notably,thoughknockdownofbothisoformsincreasedthesizeandnumberofPBs,knockdownofROCK1restoredPBsonly inKapB-expressingcellswhileknockdownofROCK2 increasedPBnumbers inbothKapBandcontrolcells.KnockdownofmDiaproteinsalsoeliminatedSFformationbutdidnotaffectPBdispersalinKapB-expressingcells.Thesedatasuggest thatKapB-mediatedactivationof thekinasesROCK1/2 is required forPBdispersionand thatactinSF formation isnot required forKapB-mediatedPBdispersal. These resultsprovidemechanistic insight intohowKapBmanipulatescellularstressresponsesinordertocreateanimmunologicalnichethatfacilitatesviralsurvival.


95-EnhancingoncolyticadenovirusefficacybyimprovingviralspreadRyanClarkin1,2,3,JoshDelPapa1,2,3,RobinJ.Parks1,2,3,41RegenerativeMedicineProgram,OttawaHospitalResearchInstitute,2DepartmentofBiochemistry,Microbiology,andImmunology,FacultyofMedicine,UniversityofOttawa,3DepartmentofMedicine,TheOttawaHospital,4CentreforNeuromuscularDisease,UniversityofOttawa

Conditionally-replicating adenoviruses (CRAds) have shown promise in preclinical studies, based on their ability toselectively replicate in and kill cancer cells, but leave healthy cells unharmed. However, CRAds have demonstratedlimitedtherapeuticefficacyinhumanclinicaltrials,inpartduetotheirpoorabilitytospreadthroughoutatumormass.Typically, CRAds only diffuse a few millimeters from the site of injection, leaving much of the tumor unaffected.Overexpressing theAddeathprotein (ADP),which improvesvirus-mediatedcell lysisandreleaseofviralprogeny,andincorporating the p14 FAST protein,which causes host cell-cellmembrane fusion,may help overcome these physicalbarriers.WepreviouslydevelopedCRAdFAST,a fusogenic vector inwhich theearly region3 (E3)of theviral genome,whichencodesADP,was removedand replacedbyap14FASTexpressioncassette.CRAdFASTefficiently fusedcancercellsandimprovedoncolyticactivitycomparedtoanon-fusogenicCRAdintissuecultureandmousemodelsofcancer,buthadonlyamodesteffectonoverall tumorgrowthandmouse survival.Wehypothesize thatexpressionof theAddeathproteinandp14FASTprotein fromaCRAdvectorwill furtherenhanceviral replication,oncolysis,andspread inpreclinicalmodels of cancer. Toevaluate theeffect of includingADP in a fusogenicCRAd,wedeveloped two vectors,CRAdRC109(E3+)andCRAdRC111(E3-).Intheseviruses,thep14FASTexpressioncassettewasrelocateddownstreamofthe viral late 5 (L5) region, allowing reinsertion of the entire E3 region including ADP. CRAdRC109 and CRAdRC111replicateefficientlyinA549cancercellsyethaveadrasticreductioninp14FASTproteinexpressionrelativetoCRAdFAST.Consequently, CRAdRC109 and CRAdRC111 have reduced fusogenic and oncolytic properties compared to CRAdFAST.However, inclusion of the E3 region in CRAdRC109 increased viral plaque size, suggesting ADP did improve viralreplication and spread. We subsequently developed CRAdRC116, in which the E3 region of the viral genome wasremovedandreplacedwithabicistronicexpressioncassettecontainingp14FASTandADPseparatedbyaself-cleavingP2A peptide. Preliminary results suggest CRAdRC116 efficiently replicates in and fuses A549 cancer cells. We arecurrentlyevaluating thisnewvector in tissuecultureandmousemodelsof cancer. In summary,wehavedevelopedaCRAd vector with improved viral replication and spread, which may improve therapeutic efficacy of oncolyticadenovirotherapyincancerpatients.97-AnalysisandmappingofsubgenomicRNAsofGrapevinerupestrisstempitting-associatedvirusHuogenXiao1,BaozhongMeng11UniversityofGuelph

Grapevinerupestrisstempitting-associatedvirus(GRSPaV)isamemberofthegenusFoveavirusinthenewlyestablishedfamilyBetaflexiviridae,whichcontainspathogenicvirusesofimportantwoodyfruitcrops.Duetodifficultiesworkingwithwoodyplantsandunavailabilityofeffectiveexperimentalsystems,themolecularbiologyofthegenusFoveavirusandthefamily Betaflexiviridae is scarce. GRSPaV is prevalent among commercial grapevine cultivars with a worldwidedistribution. GRSPaV comprises a family of sequence variants and has been associated with three distinct diseases:Rupestris Stem Pitting, Vein Necrosis and Syrah Decline, although its etiological role in these diseases remains to beestablished. TheGRSPaVgenome is apositive sense, ssRNA,8,725nucleotides in lengthandencodes5-6ORFs.ORF1encodes the replicase polyprotein;ORFs2-4 constitute the triple gene block (TGB), encoding TGBproteins 1, 2 and 3;ORF5 encodes the capsid protein. However, how these proteins are expressed from the GRSPaV genome remainsunknown.Bydefault,ORF1wouldbetranslateddirectlyontheviralgenomicRNAtoproducethereplicasepolyprotein.ORFs2-5arelikelyexpressedfromsub-genomic(sg)RNAsthatsharethe3’-terminalsequencewiththegenomicRNA.Anadditional ORF, ORF6,was identified via in silico analysis at the 3’-terminal end of the viral genome,with a putativetranslationproductof11kDa.ThepresentstudyaimstodeterminetheexpressionprofileofgenomicandsubgenomicRNAsofGRSPaV throughnorthernblotting analysis, and tomap the transcription start sitesof these sgRNAs throughRLM-RACE.WehaveconfirmedthepresenceofthreesgRNAs,correspondingtoORF2,ORF3plusORF4,andORF5.WealsohavemappedthetranscriptionstartsiteofthesethreesgRNAs.WearefurtherexaminingthepresenceofthefourthsgRNA for ORF6. Information generated from GRSPaV would help advance the study of other members of theFoveavirus.ACKNOWLEDGEMENTS:ThisresearchwasfundedbytheNaturalScienceandEngineeringResearchCouncilofCanada,DiscoveryGrantprogram(RGPIN-2014-05306).


98-StatisticalandFunctionalAnalysesRevealSubtype-specificConstraintsonHIV-1EscapefromHostCellularImmunityNatalieN.Kinloch1,GuinevereQ.Lee2,3,StevenW.Jin1,JonathanM.Carlson4,ChansonJ.Brumme2,HelenByakwaga5,6,Conrad Muzoora5, Bosco Bwana5, Kyle D. Cobarrubias1, Mark A. Brockman1,2, Peter W. Hunt6, Jeff N. Martin6, MaryCarrington7,DavidR.Bangsberg8,P.RichardHarrigan2,9,ZabrinaL.Brumme1,21FacultyofHealthSciences,SimonFraserUniversity,2BritishColumbiaCentreforExcellenceinHIV/AIDS,3RagonInstituteofMGH,MITandHarvard,4MicrosoftResearch,5MbararauniversityofScienceandTechnology,6UniversityofCalifornia,SanFrancisco,7CancerandInflammationProgram,LaboratoryofExperimentalImmunology,LeidosBiomedicalResearchInc.,FrederickNationalLaboratoryforCancerResearch,8OregonHealthSciencesUniversity,9DepartmentofMedicine,UniversityofBritishColumbia

Background: Pandemic HIV-1 group M strains, which comprise nine subtypes and dozens of circulating recombinantforms, are extraordinarily diverse both at the individual and population scales. Selection of viral variants harbouringmutations enabling the virus to evade an individual host’s Human Leukocyte Antigen (HLA) class I-restricted CD8+cytotoxic T-cell (CTL) response is reproducible based on their HLA profile. Termed HLA-associated polymorphisms or‘escapemutations’,patternsofadaptationhavebeencomprehensivelymappedindifferentglobalpopulations,howeverthe extent to which viral genetic context constrains HLA-mediated escape remains incompletely understood. Wecombine statistical analyseswith in vitro functional assessments to investigateHLA-driven adaptation in a populationwheremultipleHIV-1subtypesco-circulate(Uganda).Methods:HLA-associatedpolymorphismsinHIV-1Gag,Polymerase(Pol), and Nef were identified in 200 subtype A1- and 135 subtype D-infected individuals using published statisticalassociation approaches that take into consideration the phylogenetic relationships between HIV-1 sequences. Thestrengthofselectionofeachpolymorphismwascomparedbetweensubtypesusingaphylogenetically-informedlogisticregressionapproach.ThefunctionalimpactofselectHLA-associatedpolymorphismsineachsubtypewasassessed,intheGag protein, by generating VsVg-pseudotyped virus stocks expressing parental/mutant subtype A1 or D gag-proteasesequencesinanNL4.3referencestrainbackboneusingGibsonenzymaticassembly,followingwhichinvitroreplicationcapacitywasdeterminedusingamulti-cycleGFP-reporterassay. IntheNefprotein, theabilitiesofmutantsubtypeA1and D Nef sequences to downregulate host cell surface proteins CD4, HLA and SERINC5 were evaluated by flowcytometry.Results:Atotalof83Gag,198Poland105NefHLA-associatedpolymorphismswereidentifiedinsubtypeA1and/orDatq<0.2.Ofthese,34%exhibitedsignificantdifferentialselectionbetweensubtypes(q<0.1;p<0.05).Moreover,experimentalconfirmationrevealedthatsomeinstancesofdifferentialselectionresultfromsubtype-specificmutationalconstraints. For example, in the HIV-1 subtype D p24 capsid protein, HLA-B*57:03 strongly selected the Gag-T242Nmutation(OddsRatio[OR]=250).Incontrast,HLA-B*57:03didnotselectGag-T242NinsubtypeA1(OR=1.8,p=0.8).ThisintersubtypedifferenceinthestrengthofselectionofGag-T242NbyB*57:03ishighlystatisticallysignificant(p=8x10-6).The lack of selection of B*57:03-T242N in subtype A1 is likely because in this subtype the consensus proline at theadjacentcodon243 is incompatiblewithT242N.Molecularmodelingpredictedmarkedconformationalchanges inp24structurewhen the 242N escape variantwas adjacent to proline-243, but not leucine-243, the subtype D consensus.Indeed, in vitro replication was abolished when these residues were engineered together (p=0.0009), while T242Nminimally impacted replication when leucine was at the adjacent codon. Other viral polymorphisms that weredifferentiallyselectedacrossHIV-1subtypesonlyminimally impactedprotein function.Forexample, theHLA-A*74:01-selected Nef 196Kmutation, which represents one of the strongest examples of subtype-specific selection observed(subtype D OR=2.08, p=3.8x10-6; subtype A1 OR=-0.4, p=0.7; inter-subtype comparison p=3.6x10-5) had a negligibleimpact on Nef-mediated downregulation of CD4, HLA and SERINC5 in both subtypes A1 and D, suggesting othermechanisms besides mutational constraints contribute to subtype-specific HLA-mediated adaptation. Conclusions:Results highlight the influence of viral subtype on HLA-mediated selection of immune escape mutations in HIV-1.Mechanisticelucidationofdifferentialimmuneescapepathwaysmayhelpidentifymutationally-constrainedviralregionsforthedesignofsubtype-specificand/oruniversalHIV-1vaccines.


99-Identificationofpharmacologicaltargetsandsmallmoleculesinhibitingadenovirusreplication.BritiSaha1,2,OliverVarette2,3,Jean-SimonDiallo2,3,RobinJ.Parks1,2,41RegenerativeMedicineProgram,OttawaHospitalResearchInstitute,2DepartmentofBiochemistry,MicrobiologyandImmunology,UniversityofOttawa,3CancerTherapeuticsProgram,OttawaHospitalResearchInstitute,4CenterforNeuromuscularDiseases,UniversityofOttawa

Thehumanadenovirus(Ad)causesminorrespiratoryillnessesinmostpatients,butcanleadtoseverediseaseanddeathinpediatric,geriatricandimmunocompromisedpatients.Noapprovedantiviraltherapycurrentlyexistsforthetreatmentof severe Ad-induced diseases.Within the first few hours of infection, the Ad DNA enters the host cell nuclei andassociates with cellular proteins including histones, adopting a nucleoprotein structure similar to the cellular DNA.Assemblyoftheviralgenomeintothisrepeatingnucleosome-likestructureisrequiredforefficientexpressionofvirus-encoded genes. Consequently, one approach to treating Ad-induced disease may be to prevent the viral DNA fromtransitioningtothistranscriptionallyactivestate.Thus,ourobjectiveistoidentifynovelsmall-moleculeinhibitorsofAdreplication and to investigate the molecular mechanism underlying the inhibition, including effects on epigeneticregulationof the viral genome.Wehavegeneratedawild-type-likeAd construct encoding the red fluorescentprotein(RFP)withintheviralgenome.RFPfromthisconstructisonlyexpressedfollowingAdDNAreplication,whichallowsustoeffectivelymonitorvirusreplicationbyfluorescencemicroscopy.Usingthisconstruct,wedesignedanefficientmethodfor screening small-molecule libraries. We have screened two small molecule libraries consisting of over 1300compounds to identifydrugs thatexhibitanti-Adactivity. Severalpositivehitshavebeenvalidated toeither inhibitorconsiderablydelayAdgeneexpression,andreducevirusyield.Inparticular,thepan-histonedeacetylase(HDAC)inhibitorvorinostatwasfoundtosignificantlyreduceRFPexpression.Follow-upstudiesrevealedthatvorinostatdelaystheonsetof viral gene expression and replication, and decreases virus yield from infected cells. Vorinostat was also effectiveagainst more virulent and clinically relevant Ad serotypes. The drug’s inhibitory effects were found to be mediatedthrough the inhibition of HDAC2 activity. Further elucidation of the underlying mechanism and in vivo studies areunderway.The costs associatedwithAd-induceddiseaseare significant in termsofmedical expenses, lostworkhoursandlossoflifeinsomepopulations.IdentificationofnovelAdinhibitorswillallowthedesignanddevelopmentofmoreeffectiveantivirals,ultimately leadingtodecreaseddiseasepathogenesisandhighersurvival rates insevere infections.InvestigationofthemechanismbywhichthesecompoundsimpactAdreplicationwillprovideusefulinsightsonvirus-cellinteractions,andallowustoidentifynewpharmacologicaltargetsfortherapeuticintervention.100-CardiotonicsteroidssuppressadenovirusreplicationbyaffectingearlygeneexpressionFilomenaGrosso1,3,MarthaBrown1,2,3,AlanCochrane1,21UniversityofToronto,2DeptofMolecularGenetics,3DeptofLaboratoryMedicineandPathobiology

Humanadenovirusescancausesevere,evenlife-threatening,infectionsyettherearenoapprovedagentsfortreatment.Wehave shown that cardiotonic steroids, in particular, digoxin and digitoxin, reduce the yield ofmultiple adenovirusspeciesby2-4logsinhumanlungepithelialcells(A549)bysuppressingviralDNAreplicationatoneormorestepsbeyondimmediateearlyE1Aexpression.ThecurrentstudyaimedtocharacterizethenatureoftheantiviralblockbyanalyzingtheeffectofdigoxinanddigitoxinonspecificviralproteinsnormallyexpressedpriortoDNAreplication.A549cellswereinfectedwithhumanadenovirusC5under conditions sufficient to infectmostof the cells.Afteronehour adsorption,virusinoculumwasremovedandreplacedwithfreshmediumwithandwithout100nMdigoxinordigitoxin.Cellswereharvested at different times post infection for analysis of specific viral proteins by immunoblot and by fluorescencemicroscopy.Immunoblotanalysisat14hourspostinfectionshowed~60%reductioninexpressionofE272KDNAbindingprotein(DBP),incontrasttoE1B55Kproteinwhichwasbarelydetectableintreatedcells.DespiteappreciablelevelsofE272KDBP, itsstainingpatternwasaltered.Thoughstillexclusivelynuclear,thesignal inmostcellswasdiffuseratherthanclusteredatviralreplicationcentres,consistentwithablockinviralDNAreplication.AnalysisoftranscriptsbyRT-qPCRshowedareductioninlevelsofE1BandE4expressionto~20%oflevelsinuntreatedcells.E2Btranscriptsencodingthepre-terminalprotein,pTp,however,werebarelydetectable.Overall,digoxinanddigitoxincompromisetranscriptionofearlyregionsE1B,E2andE4,afterE1Aexpression.TheblockingenomereplicationlikelyreflectsinsufficientlevelsoftheessentialpTPandviralDNApolymerase.Theantiviraleffectofdigoxin,butnotdigitoxin,wasovercomebyincreasingthe extracellular concentration of potassium. Overall, this work supports the idea that cardiotonic steroids could bedevelopedfortreatmentofsevereadenovirusinfections.


101-SUMOWrestlingwithKSHV:CouldDisruptionofaPost-TranslationalModificationContributetoKS?SarahVeinot1,JenniferA.Corcoran1,2,31DepartmentofMicrobiologyandImmunology,DalhousieUniversity,Halifax,NS,Canada,2DepartmentofSurgery,DalhousieUniversity,Halifax,NS,Canada,3BeatriceHunterCancerResearchInstitute,Halifax,NS,Canada

Background:Tomaintainbalance,cellsneedwaystorespondtostress,andusesignalingpathwaystocopewithstress.Onestressresponsivepathwayisthep38mitogen-activatedproteinkinase(MAPK)cascade;inthiscascade,p38MAPKphosphorylatesMAPK-activatedproteinkinase2(MK2).MK2playsaroleinactindynamicsandcytokineproduction,andis of interest to our lab because it can promote inflammation and tumorigenesis. Our lab studies Kaposi’s sarcoma-associatedherpesvirus(KSHV),whichcausestheendothelialcell(EC)cancerknownasKaposi’ssarcoma(KS).KSlesionsarecharacterizedby inflammation,proliferation,angiogenesisandcytoskeletaldysfunctionssuchascell spindling.TheviralgeneproductKaposinB(KapB)canrecapitulatetwoofthesephenotypesinvitro(cellspindlingandstabilizationofinflammatory mRNAs) by directly binding to and activating MK2. However, KapB does not act as a kinase, so themechanism of MK2 activation remains unclear. Recently, it was discovered that the post-translational modificationknownasSUMOylationregulatesMK2activity.WhenoneofthekeySUMO-associatedlysinesinMK2wasmutatedtoanarginine,kinaseactivityandstressfibreinductionincreased,suggestingthatSUMOylationdampensMK2kinaseactivity.ThesephenotypesaresimilartothoseobservedduringinvitroKapBexpression;therefore,IhypothesizethatKapBbindsto MK2 and reduces its SUMOylation. Methodology: I will address this hypothesis using two approaches: 1) MK2SUMOylationwillfirstbereaffirmedinvitro.HEK293Tcellswillbeco-transfectedwithmyc-MK2,Ubc9-HAandHA-taggedSUMOproteins.Standardco-immunoprecipitationapproacheswillbeusedtopull-downmyc-taggedproteincomplexes,and this precipitantwill be immunoblotted for HA-tagged protein complexes. After successful reaffirmation, cellswillthenbeco-transfectedwithandwithoutKapB.2)MK2constructswillbecreatedthat lackpredictedSUMOmotifs,asdeterminedusingtheonlinealgorithmJASSA(JoinedAdvancedSUMOylationSiteandSimAnalyser).PrimershavebeendesignedtomutatethreepredictedSUMO-associatedlysines(K64,K188,andK353)toarginine.NewEnglandBiolab’sQ5site-directedmutagenesis protocol will be used to create theMK2mutant constructs, whichwill be verified throughsequencing.ExpressionandSUMOylationstatusesof theseconstructswillbeverified inHEK293Tcells.Thesemutantswill then be subcloned into lentivirus expression vectors to generate recombinant lentiviruses, whichwill be used totransduceECs.Results:ExpressionoftheKapB,myc-MK2,HA-SUMO(1and3)andUbc9-HAconstructshasbeenverifiedinHEK293TcellsusingWesternblot.CreationoftheK353RmutantMK2constructhasbeeninitiated,withcoloniesbeingactively screened for themutationby sequencing.Co-immunoprecipitationof controlshasbeenperformed.However,lowexpressionoftheHA-taggedSUMO3constructhaslimitedsuccess.IpredictthatthepresenceofKapBwillreduceMK2 SUMOylation. I also predict that expression of themutantMK2 constructswill recapitulate the KapB associatedphenotypesof stress fibre inductionand inflammatorycytokinestabilization. Impact:This researchwillprovide insightinto how KapB contributes to viral pathogenesis during KSHV infection by elucidating the precise details of themechanismofMK2activationbyKapB.Thisresearchwillalsorevealthecentral importanceofSUMOylationtonormalcellular function, and how viruses can disrupt this post-translational modification to cause cellular dysfunction anddisease.


102-ViralProteinEngagementofSelectiveAutophagyPathwaysControlsInflammatoryGeneExpressionGillianKSingh1,CarolynRobinson1,JenniferACorcoran1,2,31DepartmentofMicrobiologyandImmunology,DalhousieUniversity,Halifax,NS,Canada,2DepartmentofSurgery,DalhousieUniversity,Halifax,NS,Canada.,3BeatriceHunterCancerResearchInstitute,Halifax,NS,Canada

Kaposi’s Sarcoma-associated herpesvirus (KSHV) is the etiological agent of the angiogenic and highly inflammatoryendothelialcell(EC)cancer,Kaposi’sSarcoma(KS).KaposinB(KapB)isaviralproteinexpressedduringthelatentlifecycleofKSHVthatcontributestotheestablishmentoftheinflammatorycharacteroftheKSlesion.WhenexpressedinprimaryECs,KapBpromotesthedispersalofcytoplasmicribonucleoprotein(RNP)granulescalledprocessingbodies(PBs).PBsarethemajorsitefortheconstitutivedegradationofAU-richelementmRNAs(ARE-mRNAs),aclassofcellularmRNAsthatcode for inflammatorymolecules.PBdispersalbyKapBcorrelateswith increases in steady-state levelsofendogenousARE-mRNAs (IL-6, IL-8, COX-2) by droplet digital PCR(ddPCR) and qPCR.More recently, our lab has shown that KapBupregulatesbulkautophagicflux,aprocessbywhichcellsdegradecytoplasmiccomponentssuchasorganellesinvesiclescalled autophagosomes. Inhibiting autophagosome maturation (BafilomycinA) and knocking down the expression ofessentialautophagygenes(Atg5)reversesKapB-mediatedPBdispersal.Furthermore,treatmentofKapB-expressingECswith autophagy-inducing drugs (Torin, Thapsigargin) increases steady-state levels of endogenous ARE-containingtranscripts via ddPCR and qPCR. These findings indicate that KapB-mediated PB dispersal requires upregulatedautophagy;however,wedonotunderstandpreciselyhowautophagicfluxmodulatesARE-mRNAlevelsorhowPBsarerecruitedtonascentautophagosomesfordegradation.Recently,PBsandotherRNPgranuleswereshowntobetargetedspecifically toautophagosomes inaprocess termed selectiveautophagy.During selectiveautophagy,proteins suchasNDP52actasmolecularbridgestobindandtargetcargotogrowingautophagosomes.Givenrecentevidenceimplicatingselectiveautophagy inRNAgranuledegradationandtheroleofPBs inregulatingARE-mRNAexpression, IhypothesizethatKapBupregulatesselectiveautophagytofacilitatePBdegradationandtopromotetheexpressionof inflammatoryARE-mRNAsthataretypicallydegradedinPBs.Mypreliminarydatasuggeststhatknockdownoftheselectiveautophagyreceptor,NDP52reversesbothKapB-mediatedPBdispersal (via immunofluorescence)andKapB-mediated increases inARE-mRNAs(viaqPCR).Collectively,thesedatasuggestKapBisco-optingselectiveautophagymachinerytomodulatePBsand ARE-mRNA expression thus promoting an inflammatory environment beneficial for tumourogenesis and viralinfection.


103-DifferentialRestrictionofParamyxovirusesbytheLongIsoformofZAPPatriciaA.Thibault1,AndrewP.Ryan2,Chuan-tienHung1,MatthewDaugherty2,BenhurLee11DepartmentofMicrobiology,IcahnSchoolofMedicineatMountSinai,NewYorkNY,USA,10029,2SectionofMolecularBiology,DivisionofBiologicalSciences,UniversityofCalifornia,SanDiego,LaJollaCA,92093

Paramyxoviruses (PVs) are negative sense, non-segmented single-strand RNA viruses that infect a wide range ofvertebratehosts,andareresponsibleforendemichumanandanimaldiseaseslikemeaslesandmumps,ordistemperandNewcastle disease. Nipah and Hendra viruses are recently emerged zoonotic viruses, and surveillance has identifiednumerousotherPVsinbatsandrodentswithunknownpotentialforzoonoticspillover.UnderstandinghostfactorsthatpermitorrestrictPVsinhumansanddomesticanimalsisafirststepindeterminingtherisksposedbybothfamiliarandnew PVs. ZAP (zinc-finger antiviral protein) exists as two splice-variant isoforms that have differential expression,localization,andantiviralactivity.BothmediateantiviralactivityprimarilybybindingviralRNAsthroughtheircommonzinc-finger domain and directing the RNA to the exosome complex for degradation. The ZAP-S (short) isoform is aninterferon-stimulatedgene;itwasidentifiedfirst,andsoisknowntorestrictawiderangeofviruses,fromnegative-andpositive-senseRNA viruses to someDNA viruses, retroviruses, and retroelements. ZAP-L (long) ismuchmore recentlydiscovered, but appears to have a more potent antiviral activity when compared to ZAP-S. ZAP-L is constitutivelyexpressed, and bears a prenylation motif not found on ZAP-S; this localizes ZAP-L to endosomal membranes and isthought to enable better interaction with viral RNAs. Activity of either ZAP isoform has not been reported againstParamyxoviruses, sowe generated inducibleHEK-293 cells to overexpress ZAP-S or ZAP-L, aswell asmutants of eachisoformtoadd(ZAP-S+pren)orremove(ZAP-LΔpren)theprenylationmotif.WetheninfectedthesecellswithapanelofPVs: Human parainfluenza virus 3 (HPIV3),Measles (MeV),Mumps (MuV), Sendai (SeV), and Newcastle disease virus(NDV).ReplicationofHPIV3,MeV,andMuVwasreducedover100-foldbyoverexpressionofZAP-L,butthiseffectwasabolishedintheZAP-LΔprencells,indicatingthatprenylationofZAP-Lwaskeytoitseffectontheseviruses.Thiscanbeseen early during infection, in both reporter gene expression and viral RNA levels. ZAP-S did not impact any PV, butaddition of the prenylationmotif in the ZAP-S+pren cells conferred intermediate antiviral activity against these threeviruses.Interestingly,SeVandNDVwerenotstronglyaffectedbyoverexpressionofanyZAPprotein.SeVandHPIV3areclosely-relatedviruses in the samegenus,and so it isnot clearpreciselywhyZAP-L restrictsonlyHPIV3,andnotSeV.Conversely, knockdown of endogenous ZAP-L positively impacts HPIV3 replication, without affecting SeV replication.Thus,thelongisoformofZAPbearsantiviralactivityagainstsome,butnotallparamyxoviruses; itsprenylationmotif isnecessaryforthisactivity,andcanconferintermediateactivitywhenaddedtoZAP-S.Additionally,ofthevirusestested,human ZAP-L appears to only restrict the human pathogens (HPIV3, MeV, MuV), and does not affect the murinepathogen SeV, despite the fact that SeV and HPIV3 are so closely related, nor the avian pathogen NDV.We plan toexpand our panel of PVs to further explore potential implications for the role of ZAP in tropism. Mechanistically,overexpressionof ZAP-L impactsHPIV3RNA levelsearly in infection, suggesting that its restrictionoccursbeforevirusassembly and budding.While previouswork defining the effects of ZAP-S on a wide range of viruses showed that ittargeted specific viralmRNAs for degradation, no commonmotif or structurehas yet beendefined.Weare currentlyexploring whether ZAP-L activity can be similarly linked to degradation of specific HPIV3 mRNAs, and are in parallelpassagingvirusinthepresenceofoverexpressedZAP-LtoidentifypotentialescapemutantsthatmayprovideinsightintotheinteractionsbetweenPVsandZAP.


104-FromtheArctictoAntarctica:ProbingthegeographicdistributionofmarineRNAvirusquasispeciesMarliVlok1,AndrewS.Lang2,CurtisA.Suttle11UniversityofBritishColumbia,2MemorialUniversityofNewfoundland

RNAviruses,particularly thegeneticallydiversemembersof theorderPicornavirales,arewidespreadandabundant inthe ocean. Gene surveys suggest that there are spatial and temporal patterns in the composition of RNA virusassemblages, but there are limited data on their diversity and genetic variability in different oceanographic settings.Here, we show that specific RNA virus genomes have widespread geographic distributions and that the dominantgenotypes areunderpurifying selection. Threepreviouslyundescribedpicorna-like viruses (BC-1, -2 and -3) and threepreviouslydescribedmarineRNAviruses (JP-A, -BandHeterosigmaakashiwoRNAvirus),alloriginally identifiedat thesame location in coastal British Columbia, exhibited different biogeographical patterns, indicating that RNA virusdistributionsareaffectedbybioticfactorssuchashost-specificityandvirallifecycle,andnotjustabioticprocessessuchasdispersal.Sequencedifferences relative to the referencegenomes imply thatvirusquasispeciesareunderpurifyingselection, with geographically distinct genomes dominated by synonymous substitutions and conserved amino acidsequences.ThisisthefirstbiogeographicalanalysisofmarineRNAvirusesacrossoceanographicprovincesandevaluationof the selection pressures acting on them. These data add to the collection of knownmarine RNA viruses, show theimportanceofdispersalandpurifyingselectionformarineRNAviruses,anddemonstratethatcloselyrelatedRNAvirusesarepathogensofeukaryoticmicrobesacrosstheglobalocean.105-Examiningviralpopulations,CRISPR-resolvedvirus-hostinteractions,andCRISPR-CassystemdiversityinamunicipallandfillNikhilA.George1,AngusS.Hilts1,LauraA.Hug11UniversityofWaterloo

Recent culture-independent approaches have unveiled a staggering diversity of viruses from a wide variety ofenvironments. The comparatively poor representation of viruses within reference databases, despite their numericalabundance inecosystems, clearly illustrateshowcharacterizing thevirospherehashistoricallybeenamethods-limitedendeavor.Understudied environments, likemunicipalwaste sites, are likely to house novel, diverse viral populations,whosehostinteractionsmayimpactnutrientcyclingandcontaminantdegradation.Usingmetagenomics,weprobedtheviraldiversitywithinalandfillandadjacentaquiferinSouthernOntario.Metagenomicsequencesweregeneratedfromsamplescollectedfromthree leachatewells,a leachatecollectioncistern,andfromanaquiferadjacenttothe landfill.DNAwasextractedfromfilteredbiomass,andsequencedbytheJointGenomeInstitute(JGI).UsingFastViromeExplorer,avirusandphageidentificationpipeline,alongwithNCBI’sRefSeqdatabaseandtheJGI’sEarthViromeProjectdatabase,weidentifiedtheviralsignaturesinallsixmetagenomes.Thedifferentsitesvariedintheirviralpresence,withthecisternsamplescontainingthehighestviralabundancesanddiversities,andtheaquifersampleshowingthelowest.Toestablishvirus-prokaryotic host interactions within the landfill, we used Crass to first identify CRISPR (Clustered RegularlyInterspacedShortPalindromicRepeats)withinourmetagenomes.ThespacerswithintheCRISPRarrayswereextracted,and searched againstNCBI’s Refseq database and ourmetagenomes using BLASTn, in order identify the viruses fromwhichthespacersoriginated.ThevastmajorityofidentifiedspacershadnohitsinRefseq,suggestingthatmanyoftheecologicallyimportantviruseswithinoursystemarenotpresentwithinthisreferencedatabase.Thefewspacersthatdidhave hits to Refseq had homology predominantly to Pseudomonad-infecting phage genomes. Spacer hits to ourmetagenomes were identified as spacer-viral element matches or spacer-host CRISPR array matches, allowingconnectionsbasedonprior infectionstobedetermined.CRISPR-Cassystemsareuseful inelucidatingvirus-prokaryotichost interactions, and are of huge value in the agricultural and health industries. Current biotechnology applicationsutilize CRISPR-Cas systems adapted from isolated bacteria, while novel Cas proteins may offer improvements oralternativestocurrentgene-editingtools.WesoughttoidentifytheCasproteindiversitywithinourmetagenomes.UsingprofileHiddenMarkovModels(HMMs)generatedfromspecificCasproteinalignments,wescreenedourmetagenomesfor the presence of Cas proteins. Numerous Cas proteins types and subtypes were detected, aligned, and homologymodeled in order to identify novel Cas proteinswithin the landfillmicrobial communitywith potential biotechnologyapplications.


107-PMLIsoformSwitchingDuringKSHVInfectionKatrinaBouzanis1,Carolyn-AnnRobinson1,JaymeSalsman2,EricPringle1,3,GrahamDellaire2,3,CraigMcCormick1,31DepartmentofMicrobiologyandImmunology,DalhousieUniversity,2DepartmentofPathology,DalhousieUniversity.,3BeatriceHunterCancerResearchInstitute.

Promyelocytic leukaemia nuclear bodies (PML-NBs) are dynamic and heterogeneous protein complexes, involved inregulationofdiversecellular functions.Thepromyelocytic leukemia (PML)protein is themaincomponentofPML-NBsand is essential for their formation. PML is alternatively spliced to generate six nuclear localized, functionally distinctprotein isoforms. PML-NBs mediate an intrinsic antiviral defence, following virus entry into the cell, inhibiting viralgenome replication and protein production. Accordingly,many viruses encodemechanisms to disrupt PML-NBs, PMLmRNAandproteinexpression.OnehypothesizedmechanisminvolvesmodulationofPMLpre-mRNAsplicing,thereforeinfluencing the production of PML protein isoforms to disrupt PML-mediated antiviral responses. Kaposi’s sarcoma-associatedherpesvirus (KSHV)hasbeenshowntomodulatehostsplicingandmRNAexportduring infection, thereforewehypothesizedthatKSHVlyticreactivationfromlatencywouldresult inmodulationofPMLsplicing,resultinginPMLisoform switching.Using immunofluorescence andPCRbasedmethods,we characterizedPMLexpression and splicingduring KSHV latency and lytic reactivation. PML-NBs were quantified by immunofluorescence. Following KSHV lyticreactivation,weobservedadisruption inPML-NBs.PML2-to-PML5 isoformswitchingwasdetectedusinga fluorescentreporter construct specific for this isoform-switching event andqRT-PCR.We furtherobservedPML isoform switchingfromtheantiviralPML2proteintoPML5andidentifiedKSHVORF57asamediatorofPMLsplicingandsubsequentPMLisoformswitching.Together,thesefindingsadvanceourunderstandingofPMLisoformfunctionduringviralreplicationandthemechanismsemployedbyvirusestoantagonizehostantiviralresponses.


108-DiscoveryandmolecularcharacterizationofthreenovelavianpapillomavirusesMartaCanuti1,AndrewS.Lang11BiologyDepartment,MemorialUniversityofNewfoundland

Papillomaviruses (PVs)are small,non-envelopedvirusescharacterizedbya circulardsDNAgenomeof~7.5-8Kb.Thesevertebrateviruses infectepithelialcellsandinfectioncanbeeitherasymptomaticorresult inneoplasias,whichvary inseverity frombenign lesions (warts) to invasive tumorsandcancer.PVsbelong to the familyPapillomaviridaeandareclassifiedbasedonnucleotide sequence identitiesof the capsidprotein gene, L1, into genera (>60% identity), species(>70%identity)andtypes(>90%identity).Currently,over200PVtypes(belongingto52genera)havebeenidentifiedandmorethan50%ofthesearehumantypes.Inbirds,avianPVs(APVs)cancausecutaneousormucosalproliferativelesionson the feet, legs and near the beak. Eight APV sequences (1 partial and 7 complete genomes) are present in publicdatabases and have been obtained from various bird species from cutaneous lesions, healthy skin, fecalspecimens/cloacal swabs, mesenchymal tissue and oral swabs. In this study, we applied an in-house virus discoverymethod(ViDiT-CACTUS) insearchforknownandunknownviruses inwildbirds,and identifiedPVgenetic fragments inpairedoropharyngeal/cloacalswabscollectedfromamallard(Anasplatyrhynchos),anAtlanticpuffin(Fraterculaarctica)andanAmericanherringgull(Larussmithsonianus).ToobtainmoregeneticinformationaViDiT-basedgenomewalkingtechnique (ViDiWa) was applied and the viruses were molecularly characterized and phylogenetically analyzed. Thecompletegenome(7699bp)oftheLarussmithsonianusPV(LsPV1)wasobtainedandwecouldlocateallofthetypicalPVopenreadingframes(ORFs)forthecapsid(L1andL2)andregulatory(E1,E2,E6andE7)proteins.Onlypartialgenomicsequenceswereobtainedfortheothertwoviruses:approximately4000bpoftheAnasplatyrhynchosPV(ApPV1)and5000bpof theFraterculaarcticaPV (FaPV1).All threevirusescontainedanE9ORF, foundexclusively inAPVs.TheL1nucleotidesequencesofFaPV1andLsPV1were69%identicaltoeachotherbut<60%identicaltotheirclosestrelativesinGenBank.Phylogeneticanalyses revealed these twoviruseswere includedwithin theAPVclade,where theyclusteredtogetherbutseparatelyfromothergenera.Theseresultsindicatethatthesetwovirusesconstitutetwodifferentnovelspecies within the same genus. The L1 sequence was not available for ApPV1 but preliminary sequence identityinvestigations involving partial E1 sequences indicated that ApPV1 could also represent a novel species. The closestrelativetothisvirus,whichsharedapproximately68%nucleotideidentity,wasapartiallysequencedvirusidentifiedinacloacal swab collected fromducks in India. Phylogenetically, these two viruses formeda distinct clade, separate fromothergenera,withintheAPVclade, indicatingtheycouldbepartofthesamegenus. Inconclusion,wehave identifiedand partially molecularly characterized three novel APV species, belonging to two novel genera within thePapillomaviridae. These viruses were identified in three birds from three different families, indicating that a greatdiversityofPVsispresentamongbirdsandthatmanymorePVgeneraandspeciespotentiallyremaintobediscovered.Itis our priority to complete the genome sequences of FaPV1 and ApPV1 and perform screening to evaluate thedistribution of all three viruses among wild birds. Future investigations will also aim to reveal if these viruses areassociatedwithcutaneouslesionsininfectedbirds.109-AconservedroleforahistidinetriadinoldworldarenavirusglycoproteinduringviralentryGrahamGouldMaule1,JoshuaBrost1,YuxiaBo1,CorinaWarkentin1,MarcelineCôté21UniversityofOttawa,2UniversityofOttawa

Oldworld arenaviruses, suchas lassa fever virus (LFV), and lymphocytic choriomeningitis virus (LCMV), areenvelopedvirusesthatcancauseseverediseasesinhumans.DespitetheextensiveuseofLCMVinstudyingimmuneresponsestoviral infection, very little is known about its entry pathway. A critical step for entry is interaction between the viralglycoprotein(GP)andareceptor.PreviousstudieshaveshownthatLFVGPcontainsatriadofhistidines(His)that,whenprotonated in the acidic environment of the endosomes, allows binding with its intracellular receptor, LAMP1.Interestingly,theHistriadisalsofoundinLCMVGPsuggestingthatprotonationofGPmightberequiredforinteractionwithanunknownintracellularreceptor.Usingsite-directedmutagenesis,wefoundthatwhilemutationoftheHistriadhadnoeffectonGPexpressionandincorporationontoviralparticles,viralentrywasabrogated.TotesttheimportanceoftheHistriadinsurfacebinding,weengineeredsolubleGPsandfoundthatbothproteinswereabletoattachtocells,suggesting that the His triad is critical for a step following cell attachment, presumably binding to an intracellularreceptor.Takentogether,mystudiesrevealaconservedroleforaHistriadinarenavirusentry.


110-HostinfluenzahistorydictatesvaccineresponsesthroughamemoryBcellmechanismAlysonKelvin1,3,4,LuolingXu2,AmberFarooqui2,AdnanKhan21CanadianCentreforVaccinology,2UniversityHealthNetwork,3DalhousieUniversity,4IWKHealthCentre

Influenza viruses areconstantly changing. Antigenicshifted and driftedviruses re-emergeevery yearto infect thenownaïve human population and cause disease. As people are susceptible to multiple infections over a lifetime, eachinfluenzaexposureshapesfutureimmuneresponsestoinfectionsandvaccinations.Unfortunately,vaccinesareneitherdeveloped or testedwith the human influenza history inmind.We have developed a preimmune ferretmodel thatincorporates influenza history as it is seen in humanswith the purpose of understanding themechanisms ofvaccineresponses in the previously infected host. Ferrets are the most accepted animal model for studying influenzapathogenesis and evaluating vaccines. Using ferrets, the immune responses of preimmune ferrets to influenzavaccinationandchallengewereinvestigated.Toestablishpreimmunity,adultferretswereinfectedwithasublethaldoseof thehistorical seasonalH1N1A/USSR/90/1977 (USSR/77).Following recovery (67days), ferretswerevaccinatedandboostedwiththeSanofiQIVsplitvirionvaccine(FluZone®Sanofi-Pasteur,PA,USA).Toevaluateprotection,theferretswere challenge with a currently circulating 2009 H1N1 pandemic virus on D123. This virus, A/California/07/2009(Cal/2009),isoneofthecomponentsoftheQIVvaccine.Followingchallenge,ferretsweremonitoredforsignsofclinicaldisease including weight loss, fever, and lethargy. Preimmune-vaccinated ferrets lost minimal weight and did notexperience a temperature increase. Interestingly, the control group, naïve-vaccinated ferrets that did not have aninfluenza history, developed the most severe disease with significant weight loss and high temperature increases.Hemagglutinationinhibition(HAI)assaysshowedsignificant,sustainedincreasesinantibodytitersinpreimmuneferretspostvaccination forantigens in thevaccinecompared to titers fromnaïve-vaccinated ferrets. Furthermore,early timepointspostvaccinationshowedthatCal/09HAantibodieswerepresentat7dayspostvaccinationinpreimmuneferretsandabsentinthecontrolnaïve-vaccinatedgroup.Thisindicatingthatanimalswithanimmunebackgroundwereabletogeneratevaccinespecificantibodiesmorequickly.InvestigationoftheimmunoglobulinisotypeprofilesshowedahigherlevelofvirusspecificantibodiesoftheIgGisotypeintheserumofthepreimmune-vaccinatedferretssuggestingmaturityin antibody producing B cells. Taken together,my results showed that preimmune animals had greater responses tovaccination and were more protected during challenge. The early generation of antibodies toward an antigenicallydistinctvirusandthepredominantIgGvirusspecificantibodiesincirculationsuggestplasticityinanexistingmemoryBcellclone.Theseresultsareimportantandshouldbeappliedtoinfluenzavaccinedevelopment.Byunderstandingthemechanismsofmoreeffectivevaccinationresponses,wecandesignthenextgenerationofsmartervaccinesthatbothincorporatesinfluenzahistoryandmechanismsofimmunogenicity.


111-Province-widesurveyformajorviralpathogensincommercialvineyardsinOntarioHuogenXiao1,MehdiShabanian1,ClaytonMoore1,CaihongLi1,Wend2,BaozhongMeng11DepartmentofMolecularandCellularBiology,UniversityofGuelph,2OntarioMinistryofAgriculture,FoodandRuralAffairs,Ontario,CanadaGrapesrankasthesecondlargestfruitcropinOntario,withafarmgatevalueof$100millionannually.Ontariowineshavegarneredprestigiousrecognitionintheinternationalmarket,andthegrapeandwineindustrytogetherproducesaneconomic impact estimated at $4.1 billion. Viruses are highly detrimental pathogens of grapes, causing devastatingeconomiclossesduetoreductionsinvigor,yield,quality,andproductivelifespanofvineyards,aswellaspooryieldandqualityofwineproducts. Inrecentyears, the industryhasexperiencedoutbreaksofviraldiseasesacrosstheprovince,resultinginsevereandeventotalcroplosses.Theultimateandmosteffectivewaytofightagainstvirusesisthebroaduseofcleanpropagatingstocks.However, little isknownabouttheprevalenceofvirusesandviraldiseases inOntario.Since2015,wehaveconductedlarge-scalesurveysformajorvirusesincommercialwinegrapesaswellasinothertypesofgrapesinordertoobtainacomprehensiveunderstandingoftheprevalenceandseverityofviraldiseasesinOntario.Over657compositegrapeleafsampleswerecollectedfrom3,285vinesinthe137vineblocksof33vineyardsfromthethreeprimaryappellations:Niagarapeninsula,LakeErieNorthShoreandPrinceEdwardCounty.Thesesamplescovered5major red grape varieties (Cabernet Franc, Cabernet Sauvignon, Pinot noir,Merlot, Syrah) and 5majorwhite grapevarieties(Chardonnay,Riesling,Pinotgris,Sauvignonblanc,Gewürztraminer).WehavedevelopedandusedmultiplexRT-PCRforthesimultaneousdetectionofmultipleviruses inasingleassay.Compositesamplingandtheuseofmultiplexassays result in substantial time and cost-savings. We tested for 17 major viruses including all viruses involved ingrapevineleafroll(GLRaV-1,2,3,4,7),Grapevineredblotchvirus(GRBV),GrapevinePinotgrisvirus(GPGV),Grapevinefleckvirus(GFkV),Grapevinerupestrisstemsitting-associatedvirus(GRSPaV),GrapevinevirusA(GVA),GrapevinevirusB(GVB),Arabismosaicvirus(ArMV),Tomatoringspotvirus(TomRSV),Grapevinefanleafvirus(GFLV),amongothers.14ofthemhavebeendetected,whichincludeGLRaV-3,GLRaV-1,GLRaV-2,GRBaV,GPGV,GVA,GVB,GRSPaVandGFkVandtheir incidencewere 47.9, 2.1, 4.4, 18.3, 21.6, 6.2, 3.0, 84.0 and 21.8% , respectively.Mixed infectionswithmultiplevirusesarecommon.95.6%of thesamples testedwere infectedwithat leastonevirus;67%of thesampleswith2-4virusesand4.7%ofthesampleswith5-6viruses.ThemajorgrapecultivarstestedwereallsusceptibletotheinfectionofGLRaV-3,GRBaV,GPGV,GRSPaVandGFkV.Theresultsalsosuggestedthatplantingof infectedmaterials isoneof themost important factors responsible for the recent outbreaks of viral diseases across the province. Findings from thissurveyprovidesabaselineforthegrapeandwineindustryindevelopingstrategiesformanaginggrapevineviraldiseasesinOntariovineyards.ACKNOWLEDGEMENTS:Thisprojectwas fundedbyOMAFRA-UoGPartnershipsEMprogram.Wethankthewineriesandgrapegrowerswhohaveparticipatedinthisproject.WealsothankR.Monteith,O.Thompson,S.Kim,G.Lees,J.Easlick,J.Goltz,S.Thomas,A.Kaur,V.TuronandL.Ishakyfortechnicalhelp.112-IdentificationandCharacterizationofImmunodominantMHCClassI-restrictedEpitopesofOncolyticReovirusYouraKim1,DerekClements1,ErinHelson1,ShashiGujar1,21DalhousieUniversity,2IWKHealthCentre,Halifax,NS,Canada

Background: Reovirus, a benign human pathogen, selectively targets and kills cancer cells by direct oncolysis andinductionofanti-tumorimmunity.Unfortunately,anti-viralimmuneresponseseliminatereovirusfromthehostbeforeitcan completely fulfill its anti-cancer effects. Thus, measuring the magnitude and specificity of anti-reoviral T cellresponses is critical forunderstanding thegenerationand regulationofadaptive immunityduring reovirus-basedanti-cancertherapies.Methods:Computerizedalgorithms(SYFPEITHI)wereusedtopredictMHCclassI-restricted8-or9-merepitopesequences.AsubsetofepitopeswassynthesizedandtestedfortheinductionofCD8+Tcellinterferon-gammaresponses by intracellular cytokine staining using splenocytes harvested from reovirus-infected C57BL/6 mice andstimulated in vitro with the individual peptides. A panel of MHC class I tetramers loaded with the identifiedimmunodominantpeptidesweregeneratedandusedtostainreovirus-specificCD8+Tcellsbyflowcytometry.Results:Based on the SYFPEITHI-predicted MHC binding affinity, we identified >1,500 MHC class I-restricted (H-2Kb, H-2Db)epitopesfoundwithinthe12-13reoviralproteins.Wesynthesized118peptideswiththehighestMHCbindingaffinity,andobservedthatonly20%ofthepeptideswerecapableofinducingTcellresponses.Interestingly,MHCaffinitydidnotalways correlatewith immunogenicity, andmajority of the repertoire of anti-viral CD8+ T cell responsewas directedagainst8-10peptidesinahierarchicalfashion.Conclusion:Theidentificationoftheimmunodominantepitopeofreovirusprovidesapromisingcandidatethatcanbeusedfortheselectiveinhibitionofanti-viralimmunity,therebyenhancingtheefficacyofreovirus-basedanti-cancertherapy.


113-Influenzaburdenofdiseaseandpreliminary2016/17end-of-seasoninfluenzavaccineeffectivenessestimatesforpreventinginfluenza-associatedhospitalizationamongCanadianadults:ananalysisfromtheCanadianimmunizationresearchnetwork(CIRN)seriousoutcomessurveillance(SOS)networkMichaela Nichols1, Melissa K Andrew1, Todd Hatchette1, Ardith Ambrose1, Guy Boivin2, May ElSherif1, Karen Green3,Jennie Johnstone4, Kevin Katz5, Jason LeBlanc1, Mark Loeb4, Donna MacKinnon-Cameron1, Anne McCarthy6, Janet EMcElhaney7,AllisonMcGeer3,AndrePoirier8, JeffPowis9,DavidRichardson10,MakedaSemret11,Daniel Smyth12, SylvieTrottier2,LouisValiquette13,DuncanWebster14,LingyunYe1,ShellyMcNeil11CanadianCenterforVaccinology,IWKHealthCentreandNovaScotiaHealthAuthority,DalhousieUniversity,Halifax,NovaScotia,CA,2CentreHospitalierUniversitairedeQuébec,Québec,Québec,CA,3MountSinaiHospital,Toronto,Ontario,CA,4McMasterUniversity,Hamilton,Ontario,CA,5NorthYorkGeneralHospital,Toronto,Ontario,CA,6TheOttawaHospital,Ottawa,Ontario,CA,7HealthSciencesNorthResearchInstitute,Sudbury,Ontario,CA,8CentreIntégréUniversitairedesantéetservicessociaux,Quebec,Quebec,CA;,9MichaelGarronHospital,Toronto,Ontario,CA,10WilliamOslerHealthSystem,Brampton,Ontario,CA,11McGillUniversity,Montreal,Québec,CA,12TheMonctonHospital;Moncton,NewBrunswick,CA;,13UniversitédeSherbrooke,Sherbrooke,Québec,CA,14HorizonHealth,SaintJohn,NewBrunswick,CA

Background:The influenzaviruscontributestothousandsofhospitalizationsand influenza-relatedseriousoutcomes inCanada each season. To inform public health decision making around influenza prevention and treatment, ongoingsurveillance of the influenza burden of disease and assessment of influenza vaccine effectiveness (VE) is critical. TheCanadian Immunization Research Network (CIRN) Serious Outcomes Surveillance (SOS) Network conducts activesurveillanceeachinfluenzaseasontocharacterizetheburdenofinfluenzadiseaseinCanadaandtoprovideestimatesofinfluenza VE to prevent influenza-relatedhospitalization in adults.Methods: The CIRN SOSNetwork conducted activesurveillance for influenza infection inadults (≥16yearsofage (y))beginningonNovember15th,2016andendingApril30th,2017at13adultacademicandcommunityhospitalsitesinfourprovinces(Ontario,Quebec,NewBrunswick,NovaScotia). Patients with an admitting diagnosis of community acquired pneumonia (CAP), exacerbation of chronicobstructivepulmonarydisease(COPD)/asthma,unexplainedsepsis,anyrespiratorydiagnosisorsymptomwereeligiblefor enrolment. Nasopharyngeal swabs obtained from eligible patients were tested for influenza by polymerase chainreaction (PCR). Patients whotested positive for influenza were considered cases and enrolled; patients who testednegativeforinfluenzawereeligibletobecomecontrols.Detaileddemographicandmedicalinformation,courseofillnessdetails, and outcomeswere obtained from the patient’smedical record. To calculate influenza VE, influenza positivecaseswerematched to influenza-test negative controls on admission date (within 14 days of date of admission), agestratum(≥65yor<65y)andsiteofenrolment.Usingconditionallogisticregression,overallinfluenzaVEandinfluenzaAVEwasestimatedas1-oddsratio(OR)ofinfluenzainvaccinatedvs.unvaccinatedpatientsx100%,withcorresponding95% confidence intervals (CIs) for all patients, and patients ≥65y and <65y. Results: During the 2016/2017 influenzaseason there were 1431 influenza cases enrolled in the SOS Network. Themajority of these cases were influenza A(n=1299),andamongsubtype-knowninfluenzaAcases,100%(n=546)wereinfluenzaA/H3N2.Amonginfluenzapositivecases,144patientswereadmittedtotheintensivecareunit(ICU)and91patientsdiedwithin30dofdischarge.OverallinfluenzaVEforpreventionof influenza-relatedhospitalizationinallageswas29.8%(95%CI:12.0-43.9%),withslightlylowerVEobservedinpatients≥65y(VE:21.3%;95%CI:-1.1-38.8%)andhigherVEobservedinpatients<65y(VE:55.6%;95% CI: 25.2-73.5). Influenza VE estimates for prevention of influenza A hospitalization were similar to overall VEestimates.Conclusions:The2016/2017influenzaseasonwascharacterizedbydominantcirculationofinfluenzaA/H3N2.Amoderately effective influenza VE of 30%was demonstrated for the prevention of influenza-relatedhospitalization.Given the substantial burden of disease caused by influenza and the modest influenza VE observed, continuedassessmentofinfluenzaVEiscrucialtoinformimmunizationpolicyinCanada,andtoemphasizetheimportanceofthedevelopmentandutilizationofimprovedinfluenzavaccines.


114-AgeneticapproachtoinvestigatetheintrinsicantiviralpropertiesofstressgranulesDanikaKnight1,AndreaMonjo1,MarielKleer1,DenysKhaperskyy1,3,CraigMcCormick1,3,41DalhousieUniversity,2DalhousieUniversity,3CanadianCenterforVaccinology,IWKHealthCentre,4BeatriceHunterCancerResearchInstitute

Protein synthesis is a tightly regulated and energy-intensive process. In response to diverse environmental stresses,including the stress of viral infection, sentinel kinases phosphorylate eukaryotic initiation factor 2-alpha (eIF2a) andprevent the formation of the eukaryotic initiation factor 2 (eIF2)–GTP–Met-tRNAMetiternary complexrequired fortranslation initiation. Stalled messenger ribonucleoprotein (mRNP) complexes are bound by aggregation-prone RNA-bindingproteinsthatdrivetheformationofcytoplasmicstressgranules(SGs).Allvirusesrequirehostproteinsynthesismachinery todecode viralmRNAs, anddiverse viruses havebeen shown to regulate SG formation,with SG inhibitionbeingthepredominantoutcome.Inmanycases,viralsuppressionofSGformationhasbeenshowntoactdownstreamofstress-induced translation arrest, suggesting that SGs may have independent antiviral properties that reinforce theantiviral effectsof protein synthesis arrest.Moreover, theobservationof SG-localizedpathogen recognition receptors(PRRs) has prompted some to suggest that SGs aid antiviral responses by providing a platform for detection of viralpatterns.Thishypothesisremainsunproven.TouncoupletheeffectsofSGformationfromproteinsynthesisarrest,wecreatedlentivirusesbearingdoxycycline(dox)-inducibleredfluorescentprotein(RFP)fusedtoa40amino-acidfragmentoftheubiquitinspecificpeptidase10(USP10)fusionprotein.ThisRFP-USP10fusionconstructhadpreviouslybeenshowntoactasadominantnegativeprotein that inhibitsSGaggregation irrespectiveofproteinsynthesisarrestandwithoutaffecting levelsofendogenousSG-nucleatingproteins.U2OSosteosarcomacellsweretransducedwith lentivirusesandtreatedwithdoxto induceRFP-USP10expression.Treatmentofuninducedcontrolcellswith500mMsodiumarsenitecausedtherapidformationofSGsasexpected,whereastheRFP-positivedox-treatedcellsfailedtoformSGsinresponseto treatment. This inducible cell linewill beused to complementexistingapproaches to investigatepotential antiviralpropertiesofSGsagainstawidevarietyofviruses.115-HIVlatencyiscontrolledbyanovelhistonedeacetylasecomplexEmmanuelleWilhelm1,MikaëlPoirier1,PierreLavigne2,BrendanBell11Départementdemicrobiologieetinfectiologie,UniversitédeSherbrooke,2Départementdebiochimie,UniversitédeSherbrooke

Human immunodeficiency virus (HIV) latency is amajor obstacle for the development of anHIV cure or vaccine. ThetherapeuticcontrolofHIVlatencyisthereforeanimportantbiomedicalgoal.Viraltranscriptionisarate-limitingstepinthecontroloflatencyandoccursviathehijackingofthehostcellRNApolymeraseII(PolII)machinerybythe5’HIVLTR.AttheheartoftheLTRofHIVliesaparticularlycrucialDNAregiontermedthecorepromoterthatisspecificallyrequiredfor the reactivationofHIVby theviral trans-activatingproteinTat. Tat controlsHIV latencyaspartofapositive feed-forward loop that stronglyactivatesHIV transcription.The recognitionof thecorepromoterbyhost cellpre-initiationcomplexesofHIV(PICH)hasbeenshowntobenecessaryforTattrans-activation,yettheproteincompositionofPICHhasremainedobscure.WeemployedDNA-affinitychromatographytoidentifyanewdeacetylasecomplexasacomponentofPICHthatselectivelyrecognizetheHIVcorepromoter.OurdatarevealthreenewPICHproteinsthatactinacomplextoreactivatelatentHIVincludingonethatdirectlyrecognizestheHIVcorepromoter,onethatregulateschromatin,andathird that binds directly to the HIV Tat protein. Our data fill a significant and long-standing mechanistic gap in theunderstandingoflatencyandidentifypotentialnewdrugtargetsforHIVcurestrategies.


116-InvestigationofInfluenzaAVirusNucleoprotein-MediatedStressGranuleInhibitionviaProximity-LabelingProteomicsJacobSicheri1,DenysKhaperskyy1,BrittanyPorter1,CraigMcCormick1,2,31DalhousieUniversity,2BeatriceHunterCancerResearchInstitute,3CanadianCentreforVaccinology

Introduction: Each segment of the influenza A virus (IAV) genome is associated with many copies of the viralnucleoprotein (NP). During replication, NP association is essential for synthesis of new viral genomes. Recently, wediscoveredthatIAVNPalsoparticipatesinsuppressionofhostinnateimmuneresponsesbyinhibitingtheformationofcytoplasmicmRNPaggregatesknownasstressgranules(SGs).SGinhibitionrequiresNPoligomerization,butRNA-bindingandnuclearlocalizationaredispensable.WehypothesizethattheinteractionofoligomericNPwithspecifichostproteinsis required for SG suppression.Methods:Aproximity-labelingproteomicmethodknownasBioIDwasused to identifyhostNP-interactingproteins.TheBirA*biotinligaseenzymewasfusedtoNPproteinspredictedtoinhibitSGs(wildtype(WT),nuclear localizationmutant,RNA-bindingmutant), aswell as anoligomerizationmutantprotein that shouldnotinhibitSGs.EffectsofBirA*tagonsubcellularlocalizationandSGinhibitionbyNPwasverifiedbytransfectionintoeitherHEK293A or U2OS cells and immunofluorescence microscopy. Results: As predicted, the oligomerization mutant NPproteinfusedtoBirA*failedtosuppressSGformation,whereasotherBirA*-NPfusionproteinssuppressedSGformation.Biotinylatedhostproteins largelyaccumulatedinthesamecellularcompartmentsastransfectedBirA*fusionproteins,except that proteins labeled by proximity to the cytoplasmically-restricted oligomerization mutant NP fusion proteinaccumulatedinthenucleus.Distinctbiotinylatedhostproteinswereobservedbyimmunoblotting.Conclusions:BioIDisapowerfulmethodtodiscovertransientandstableprotein-proteininteractions.ThisworksetsthestageforidentificationofhostproteinsthatselectivelyassociatewithNPisoformsthatcansuppressSGformationusingmassspectrometry.118-Establishmentofanovelanimalmodelforahumantumorvirus,Epstein-BarrvirusJanaHundt1,GiaLuu1,JaredRowell1,MarkusCzub11UniversityofCalgary

Morethan90%oftheworld’spopulationisinfectedwithEpstein-BarrVirus(EBV),amemberofthegammaherpesvirussubfamily. EBV is the cause of 2% of all neoplasms worldwide, and responsible for the development of infectiousmononucleosis also known as glandular fever, a disease affectingmainly young adults. Despite 50 years of research,thereisnopreventivemeasureavailableagainstEBVinfectionandEBV-cancerformationinhumans.Thisdeficiencycanbe attributed to the lack of a suitable animal model for studying EBV associated immune responses and diseasemanifestation.We intend to establish a novel pig model, in which transmission, prevention, immune response andtreatmentofaporcinevirus,verysimilartoEBV,willbestudied.ThisapproachopensnewdimensionsfortacklingEBV.Porcine lymphotropic herpesvirus (PLHV) is a virus that is closely related to human EBV. In pigs, PLHV causes aproliferativediseaseoflymphocytesthatresemblesEBV-lymphomainEBV-infectedhumans.Westartedtocharacterizethenovelpigvirus,and identifiedseveralpig tissuesandaporcinetumorcell linetobe infectedwithPLHV.Basedonpreviousdatafromourandotherresearchgroups,wehypothesizethatPLHViscapableofinfectingpigs,establishinganinfection, inducingspecific immunity,andcausecancerofwhitebloodcells, likeEBV.Before testing thishypothesis invivo,webegan tocharacterize theporcine gammaherpesvirus in vitro.Next generation sequencing is currently beingemployedtogather fullsequence informationonPLHV,andweaddednew informationon40000bpofPHLVgenome.We are currently establishing a reverse genetic system for PLHV. A major goal of our proposed research is themanipulationoftheviralgenomebyexchangingporcinegammaherpesvirusgenesagainsthomologousEBVgenes.Thechimericviruseswillbeusedinimmunologicalandpathogenesisstudiesinpigs.Around200.000casesofEBV-associatedmalignanciesarereportedeveryyearworldwide.OurworkutilizesanewstrategyforgatheringvaluableknowledgeonEBVinfectionanddiseasemanifestation.ThiswillaidinthedevelopmentofanEBVvaccineand/orantiviraltherapy,andthus,havingasubstantialpublichealthandeconomicimpact.


119-AVSV-MeaslesChimericVrusCanTargetNectin-4PositiveBreastCancerTumoursInAnImmuneCompetentMouseandIsEnhancedByTransientInhibitionofAnti-ViralInnateImmunityChristopherD.Richardson1,AngelitaAlcos1,NatalieMishreky1,GarySisson11DepartmentsofMicrobiology&Immunology/Pediatrics,DalhousieUniversity

OurlaboratorypreviouslydiscoveredthatthetumormarkerNectin4(PVRL4)istheepithelialcellreceptorformeaslesvirus,andisexpressedonmanyadenocarcinomas.Usingabreastcancercohortandanalysisoftissuefrom176patients,we showed thatNectin 4 is upregulated onmetastatic breast tumors. Breast cancer cells expressingNectin 4 can beinfectedwithmeaslesvirusoravesicularstomatitisvirus(VSV)hybridviruscontainingtheHandFproteinsofmeaslesvirus.TumorscanbereadilyinfectedwithmeaslesandVSV-measlesvirusesthatexpresseithergreenfluorescentprotein(eGFP)orfireflyluciferase(Luc)reportergenes.HistologicalanalysisofMD468orpatientderivedtriplenegativetumorsshowed that they expressed eGFP reporter and viral proteins. Although the viruses replicate in the tumors engraftedonto immunedeficientmice, the infectiousagentswerenotoncolyticandonlyslowedtumorprogression.To test thecontribution of the host immune system,mouse 4T1 and 4T1-Nectin 4 tumorswere produced in immune competentBALB/Cmice.ThehighlyconservedNectin-4protein increasedgrowthandmetastasistoproduceaggressive300cubicmmmouse breast tumors within 6 days. 4T1 and 4T1-Nectin 4 tumors on xenographic (NIH III nude) and syngeneic(BALB/C)micewere injectedwithmeasles and VSV-measles viruses andmonitored over 24 days. The growth of 4T1-Nectin4tumorsintheimmunecompetentBALB/Cmicewasslowedbyintra-tumoralinjectionofmeaslesvirusorVSV-measleschimericvirus.TumorregressionwiththeVSV-measleschimericviruswasfurtherenhancedusingeitherBX795or vanadate chemical inhibitors, which transiently inhibited anti-viral innate immunity. Infection of tumors wasdocumented using confocal immune microscopy. We concluded that the anti-tumor properties of measles and VSV-chimericvirusesaredependentonboththeinnateandadaptivehostimmunesystems.


120-PhylodynamicinsightintoHIVepidemicdynamicswithinCanadaJeffrey Joy1,2, Richard H. Liang1, Brenner Bluma3,4, Lynch Tarah5, John Gill5, Jared Buller6, David Alexander6, ZabrinaBrumme7,AnnBurchell8,SeanRourke9,MonaLoutfy9, JanetRaboud9,CurtisCooper10,DeborahKelly11,ChrisTsoukas4,NimaMachouf12,Marina Klein4, AlexanderWong13, Paul Levett14, SeanHosein15,MarkWainberg3,4, Paul Sandstrom16,JulioS.G.Montaner1,2,RobertS.Hogg1,6,ArtF.Y.Poon17,P.RichardHarrigan1,2,CANOCCollaboration11CenterforexcellenceinHIV/AIDS,2DepartmentofMedicine,UniversityofBritishColumbia,3LadyDavisinstituteforMedicalResearch,4McGillUniversity,5AlbertaHealthServices,6UniversityofManitoba,7SimonFraserUniversity,8St.Michael’sHospital,9UniversityofToronto,10UniversityofOttawa,11MemorialUniversity,12UniversitédeMontreal,13UniversityofSaskatchewan,14UniversityofRegina,15CATIE,16PublicHealthAgencyofCanada,17UniversityofWesternOntario

Background:HIVisoneofthemostdevastatinginfectiousdiseasestoimpactCanadainhumanhistory,withanestimated75,500 people living with HIV in Canada. However, despite the national importance of HIV, the early transmission,geographicdissemination,anddynamicsoftheviruswithinpopulationsandaggregateriskfactorsacrossCanadaremainunclear.EpidemiologicalprocessessuchasgeographicspreadandpopulationgrowthstampameasureablesignatureonHIV-1 genomes sampled from infected individuals at different places and times. Using statistical phylodynamicapproaches applied to HIV-1 sequence data sampled from across Canada we test hypotheses concerning past andpresent HIV epidemic dynamics. Methods: We compiled a dataset of 51,493 doubly-anonymized HIV protease andreversetranscriptasesequencessampledfrommorethan20,000patientsannotatedwithclinicalandsocio-demographicparameters. Datawere available from 6 Canadian provinces: British Columbia, Alberta, Saskatchewan, Ontario, and alimitedamountfromQuebec.Tomitigatetheinfluenceoftherapeuticinterventionsweremovedcodonsassociatedwithknowndrug resistance fromthesequencesprior toanalysis.Weperformedphylogeneticclusteringanddiversificationrateanalysesontheentire“Canada-wide”dataset.Bayesianphylogeographicanalysesutilizedreduceddatasets,downsampled fromthe fulldataset, toavoidpotentialbias inspatial inferenceestimates thatarise fromoversamplingoneparticularlocationoverothers.Alldatasetswereanalyzedusinggeneraltime-reversiblenucleotidesubstitutionmodelsspecifyingagammadistributionasaprioroneachsubstitutionrate.Weemployedanuncorrelated lognormal relaxedmolecularclockinordertoinferthetimescaleofHIVevolutionwhileaccommodatingamonglineageratevariation.Toreconstruct the spatial dynamics of HIV-1 we employed a Bayesian discrete phylogeographic approach usingMarkovchain Monte Carlo sampling. Results: We observed substantial variation among provinces in the proportion of non-subtype B infections (P<0.05) with the Prairie Provinces having relatively greater numbers of non-B infections.Phylogeneticclusteringoccurspredominantlywithinprovincesthroughlocaltransmission.Mostprovincescontainlargeprovincespecificclustersdominatedbytransmissionthroughintravenousdruguse.Somebetweenprovinceclusteringisobserveddominatedbytransmissionamongstmenwhohavesexwithmen.ConsistentwithothersourcesofevidenceSaskatchewanandManitobaaretheprovincesobservedtohavethehighestratesofHIVdiversificationinourdataset.We also observe variation in timing of the geographic spread of HIV within Canada with the Prairie Provincesexperiencinglaterperiodsofepidemicignitionandgrowth.Conclusion:Secondaryanalysisofgenotypicresistancedataprovidesusefulepidemiologicalinferencesonanationalscale.Differentcombinationsofcircumstanceshavepermittedestablishment, spatial dissemination, and epidemic growth of the HIV epidemic in Canada at different times withincomponentsubpopulations.OurresultsemphasizethevariedchallengesfacingdifferentregionsofCanadaincontrollingthe HIV epidemic going forward. Similar patterns may underlie past emergence of other blood-borne pathogens,particularlyHCV.


121-Cell-mediatedimmuneresponsesdirectedagainstZikavirus(ZIKV)envelopepeptidesinacohortofZIKV-exposedwomen.Agnès Gérémy-Depatureaux1,2, Samuel Fortin-Dion1,2, Hinatea Dieumegard1,2, Doris G. Ransy1, Martine Caty1, ValérieLamarre3,4,ChristianRenaud2,5,MarcBoucher6,FatimaKakkar3,IsabelleBoucoiran6,HugoSoudeyns1,2,41Unitéd’immunopathologievirale,CentrederechercheduCHUSainte-Justine,Montreal,QC,Canada,2DepartmentofMicrobiology,Infectiology&Immunology,UniversitédeMontréal,Montreal,QC,Canada,3PediatricInfectiousDiseasesService,CHUSainte-Justine,Montreal,QC,Canada,4DepartmentofPediatrics,UniversitédeMontréal,Montreal,QC,Canada,5DepartmentofMicrobiology&Immunology,CHUSainte-Justine,Montreal,QC,Canada,6DepartmentofObstetrics&Gynecology,UniversitédeMontréal,Montreal,QC,Canada

Background:ZIKVinfectionduringpregnancyhasbeenassociatedwithadramatic increaseintheincidenceofcasesofcongenitalmicrocephalyinBrazilsince2015.Littleisknownaboutcell-mediatedimmuneresponsestoZIKV,particularlyinpregnantwomen.TheaimofthisstudywastoassessZIKV-specificcell-mediatedimmunity inpregnantwomenwhowere exposed to the virus. Methods: Pregnant women suspected of ZIKV infection according to WHO criteria wereenrolledatCHUSainte-Justine(n=12).ZIKV infectionwasconfirmedbyserologyandPCR.Screeningforotherendemicflaviviruses(e.g.Dengue)wasalsoperformed.Peripheralbloodmononuclearcells(PBMC)wereobtainedatthetimeofenrolment.NewbornswerescreenedforZIKVinfectionbyPCRinblood,CSF,urine,andthroat.IFN-gammaELISpotwasperformedonPBMCandcordbloodmononuclearcells(CBMC)usingpoolsofsyntheticpeptidesbasedonthesequenceofZIKVcore(C),matrix(M),envelope(E),andnon-structural-1(NS1)proteins.Resultswereexpressedasspot-formingunits(SFU)per106cells.Results:Threeof12women(25%)werenotinfected.Eightof12(75%)exhibitednosymptoms.ZIKV-specificIFN-gresponseswerehigherinZIKV-infectedwomenascomparedtouninfectedcontrolsubjects(p<0.05).ResponsesdirectedagainstEproteinpeptideswerethelargestandmostfrequentlyobserved.NoreactivitywasseeninCBMC(n=3)despitethepresenceofoneconfirmedcaseofmother-to-childZIKVtransmission(positivePCRinCSF).Noimpairment in growth and neurological development was observed in this child. Conclusion: To our knowledge, thisreport isthefirstdescriptionofcell-mediated immuneresponsesagainstZIKV inpregnantwomen.TheseresultscouldimproveourunderstandingofZIKV-specificantiviralimmunityandinformvaccinedesign.122-QuasispeciesdiversityandneutralizingantibodyresponsesinverticalHCVtransmission.Ariane Larouche1,2, Kmberly-Ann Milton-McSween1,2, Virginie Calderon1,2,3, Sébastien Fauteux-Daniel1,2, JonathanBoulais4, Doris G. Ransy1, Marc Boucher5,6, Valérie Lamarre5,7, Deborah Money8, Mel Krajden8, Normand Lapointe5,7,IsabelleBoucoiran5,6,ArmelleLeCampion1,HugoSoudeyns1,2,71Unitéd’immunopathologievirale,CentrederechercheduCentrehospitalieruniversitaire(CHU)Sainte-Justine,2DepartmentofMicrobiology,Infectiology&Immunology,FacultyofMedicine,UniversitédeMontréal,3DepartmentofInformaticsandOperationsResearch,UniversitédeMontréal,4CentrederechercheduCHUSainte-Justine,5CentrematerneletinfantilesurleSIDA(CMIS),CentrederechercheduCHUSainte-Justine,6DepartementofObstetrics&Gynecology,FacultyofMedicine,UniversitédeMontréal,7DepartmentofPediatrics,FacultyofMedicine,UniversitédeMontréal,Montreal,Quebec,Canada,8Women’sHealthResearchInstitute,9BCCenterforDiseaseControl,Vancouver,BritishColumbia,Canada

Background: Vertical transmission is themajor cause of pediatric HCV infectionworldwide. To better understand thepathogenesis of hepatitis C during and after pregnancy and to provide insights into the risk factors andmechanismsinvolved in vertical HCV transmission, the evolutionary dynamics ofHCV variant spectra andHCV-specific neutralizingantibody responses were examined using high throughput sequencing and pseudoparticle-based assays in pregnantwomen monoinfected with HCV (n = 17) or coinfected with HCV and HIV-1 (n = 15). Results: Overall, statisticallysignificant associations were found between HCV quasispecies diversity, selective pressure exerted on the HCV E2envelope protein, and neutralizing activity of maternal immunoglobulins. Women with low quasispecies diversitydisplayed significantly higher mean AST and ALT levels throughout pregnancy, but this difference was restricted tomonoinfected participants. Low quasispecies diversity and inefficient neutralizing activity were also significantlyassociated with vertical transmission, but only in the monoinfected group. Discussion: These results indicate thatmaternalneutralizingantibodyresponsesplayanimportantroleinthepreventionofverticalHCVtransmission,butnotin presence of coinfection with HIV-1, and suggest that themode/route of vertical HCV transmission differ betweenmonoinfected and coinfected women. These findings could inform clinical management strategies for prevention ofverticalHCVtransmission.


125-HighContentScreeningImplicatesthePi3kisoformp110αinFASTProtein-MediatedCell-to-CellFusion.DuncanMacKenzie1,RoyDuncan11DalhousieUniversity

The reovirus fusion-associated small transmembrane (FAST) proteins are the smallest known viral membrane fusionproteins.Unlikeenvelopedvirusfusionproteins,FASTproteinsevolvedspecificallytomediatecell-cellfusion,notvirus-cellfusion.Assuch,theyrepresentasimplemodelsystemtodissecthostcellmachinerynecessaryforcellfusion.High-content siRNA and small molecule screens of FAST protein-mediated cellular fusion, quantified using an automatedfluorescencemicroscopy assay, were employed to identify endogenous proteins and pathways involved in syncytiumformation. In this fashion,phosphoinositide3-kinase (PI3K)p110α subunit inhibitorPik-75andAKTkinase inhibitorA-443654wereidentifiedasagentsthatdramaticallyinhibitedsyncytiogenesis.Thenecessityandisoformspecificityofthep110apha/AKT signaling pathway to FAST protein-mediated cell fusionwas validated using isoform-specific shRNAs, atransgenic AKT1-/-/AKT2-/- knockout cell line, and pharmacologic inhibition using PI3K isoform-specific inhibitors.Interestingly, Pik-75 and A-443654 also inhibited syncytiogenesis mediated by the measles virus F+H fusion proteincomplex, implying p110α/AKT involvement in a common cell-cell fusion mechanism. Fluorescence videomicroscopyrevealedcolocalizationofAKT1-PH-GFP(abiosensorofphosphatidylinositol3,4,5-phosphate)andthereptilianreovirusp14FASTproteinatareasofcell-to-cellcontact,andthiscolocalizationincreasedimmediatelypriortotheformationofacell-cell fusionsynapse.TreatmentwithPik-75disruptedp14/AKT-PH-GFPcolocalizationandalteredactindynamicsatareas of cell-to-cell contact resulting in a failure to form a cell fusion synapse. These studies identify the PI3K-AKTpathwayascriticalplayerinvolvedinvirus-mediatedcell-cellfusion.126-TH2CytokinesModulateHumanMastCellResponsestoVirusesLilianaPortales-Cervantes1,OwenCrump1,CraigMcCormick1,JeanS.Marshall11DalhousieUniversity

Introduction:Mastcellsareresidentinmucosalandepithelialtissues,wheretheyserveassentinelcellsagainstparasiticandbacterialinfections.Mastcellscanalsorespondtomultipleviruses,includingrespiratoryvirusessuchasinfluenzaAvirus (IAV), reovirus, and respiratory syncytial virus through the selective production of pro-inflammatory cytokines.AllergicasthmaisassociatedwithaTh2driveninflammationandincreasedsusceptibilitytoviralinfectionswhicharethemain triggers of asthma exacerbations. Interferons (IFNs) have a key role in controlling virus replication and theirproductionhasbeenreportedtobelowerinasthmaticcomparedtohealthysubjects.Th2cytokinesmaybeinvolvedinthisdifferenceinresponse.MastcellshavetheabilitytorespondtoTh2cytokinesandareanimportantsourceofIFNs.TheobjectiveofthisworkistoanalyzewhethermastcellIFNresponsestoreovirusandIAVareimpairedinthepresenceofTh2cytokines.Methods:Cordblood-derivedhumanmastcells(CBMC)wereculturedinmediumaloneorstimulatedwithIL-4(10ng/ml),IL-5(10ng/ml),IL-9(10ng/ml),IL-13(10ng/ml),andIL-33(1ng/ml)for48hfollowedbyinfectionwith reovirus type3Dearing (5MOI) or IAV (H1N1A/CA/07/2009, 5MOI). Supernatantswereharvested24hp.i. forELISAanalysis.mRNAgeneexpressionwasanalyzedbyqPCR.Results: IFNproductionbyreovirus infectedCBMC(Reo-CBMC)wasnotimpairedbypretreatmentwithTh2cytokines.Strikingly,IL-4selectivelyenhancedtheproductionoftypeIandtypeIIIIFNs.AlthoughIL-4andIL-13shareacommonreceptor,theseresponseswerespecificforIL-4.Reo-CBMCstimulatedwith IL-4onlyconcurrentlywithviral infectiondidnotenhance IFNproduction. Inaddition, IL-4pretreatedCBMC showed an increased production of IL-6, CCL-3, and CXCL10 in response to reovirus. Similar to Reo-CBMC, IL-4pretreated CBMC showed an enhanced IFN production in response to IAV. Conclusions: Our data suggest that IL-4modifiesmastcellIFNresponseswhenadministeredpriortoreovirusorIAVinfection.IL-4actionsonmastcellsarelikelytobe induced through type I IL-4 receptors.Wesuggest thatmastcellsmightbeof importance in thecontrolofviralinfectioninasthmaticsbyIL-4-enhancedproductionofIFNs,whicharecriticalforviralclearance.


127-EnteroviralInfectionInhibitsAutophagicFluxbyDisruptingSNAREComplexFormationandthroughTargetingAutophagyReceptorsYasirMohamud1,3,YuanChaoXue1,3,JunyanQu3,HuitaoLiu2,3,HaoyuDeng1,3,HonglinLuo1,2,31DepartmentofPathologyandLaboratoryMedicine,UniversityofBritishColumbia,2DepartmentofExperimentalMedicine,UniversityofBritishColumbia,3CentreforHeartLungInnovation,St.Paul'sHospital

Background:Ourrecentevidencesuggeststhatenteroviralinfectionisaputativecausal/riskfactorinthedevelopmentofsporadicALS(>90%ofallcases).Enteroviruses,includingpoliovirus,coxsackievirus,EV-A71andEV-D68,cantargetmotorneurons and cause polio or polio-like flaccid paralysis. Analogous to other neurodegenerative diseases, ALS ischaracterizedbytheaccumulationofmisfoldedproteins.WerecentlyshowedthatEV-inducedpathologyresemblesthatof ALS, including TDP-43proteinopathy and aberrant accumulation ofmisfolded/ubiquitinatedproteins.However, themolecular mechanisms involved are unclear. Autophagy is a fundamental cellular process by which misfoldedproteins/damagedorganellesandinvadingmicrobesaresequesteredwithinanautophagosomeanddegradedfollowingfusionwithlysosomes.Disruptionoftheautophagicpathwayhasbeensuggestedtoplaykeyrolesinneurodegeneration.WehypothesizethatenterovirusinfectioncontributestoALS-likepathologybytargetingautophagyreceptorsandfusionadaptorproteins, leadingtoenhancedviralgrowthand impairedclearanceofproteinaggregates/damagedorganelles.Methods/Results: To delineate the possible mechanism involved, we focused on proteins previously reported to beinvolved in autophagosome fusion. Notably, we found that the autophagosomal SNARE protein SNAP29 and thetetheringproteinPLEKHM1,twocriticalproteinsknowntoregulateautophagosome-lysosomefusion,werecleaveduponcoxsackievirus B3 (CVB3) infection. Further in vivo (in cells transfected with protease constructs) and in vitro (usingrecombinant proteases) cleavage assays demonstrated that CVB3-encoded proteinase 3Cpro, not 2Apro or caspases, isresponsibleforthesecleavages.WeidentifiedthecleavagesitesonSNAP29(Q161)andPLEKHM1(Q668),respectively,leadingtoimpairedSNAREcomplexformation.Moreover,weshowedthatgene-silencingofSNAP29/PLEKHM1inhibitedautophagic flux, resulting in a significant increase in viral growth, likely due to enhanced accumulation ofautophagosomes that provide sites for viral RNA replication and assembly. Finally, we identified the xenophagicreceptorsNDP52andTAX1BP1asbonafidesubstratesofviralproteinases,complementingourpreviousfindingthatp62andNBR1arealsotargeted.Conclusion:EVinfectioninhibitscargorecruitmentandautophagicfluxbyviralproteinase-mediated cleavage of autophagy receptors (p62, NBR1, NDP52, TAX1BP1) and fusion adaptor proteins (SNAP29 andPLEKHM1). Blockage of autophagic flux results in accumulation of misfolded protein and enhanced viral growth topromoteALSpathogenesis.Futurestudieswillexaminethesignificanceofdisruptingthevirus–autophagyinterfaceasapotentialtherapeuticstrategy.


LISTOFPARTICIPANTSMohamedAbdel-HakeemUniversityofPennsylvania(UPenn)[email protected]édeMontréallevon.abrahamyan@umontreal.caNadineAhmedUniversityofOttawanahme036@uottawa.caTommyAlainChildren'sHospitalofEasternOntarioResearchInstitute,UniversityofOttawatommy@arc.cheo.caCarolinaAlfieriResearchCentreoftheCHUSainte-Justine,andDeptofMicrobiology,Infectiology,Immunology,UniversityofMontrealcarolina.alfieri@umontreal.caEricArtsUniversityofWesternOntarioearts@uwo.caAndraBaneteQueen'[email protected]'[email protected]@mcmaster.caStephenBarrWesternUniversitystephen.barr@uwo.caLisaBarrettABBVIE,GILLND,ViiV,BMS,[email protected]

BrendanBellUniversité[email protected]@[email protected]éanneBlondeauCHUdeQuébecResearchCenter-UniversitéLaval,CancerResearchCenterandreanne.blondeau@crchudequebec.ulaval.caJeanetteBoudreauDalhousieUniversityjeanette.boudreau@dal.caKatrinaBouzaniskatrina.bouzanis@live.caJulieBrassardAgricultureetAgroalimentaireCanadajulie.brassard@agr.gc.caWilliamBremnerUniversityofCalgary,FacultyofVeterinaryMedicinewilliam.bremner1@ucalgary.caMarthaBrownUniversityofTorontomartha.brown@utoronto.caZabrinaBrummeSimonFraserUniversity;BCCentreforExcellenceinHIV/[email protected]&BiomedicalSciencesandtheMichaelG.DeGrooteInstituteforInfectiousDiseasesResearch,McMasterUniversityburrowl@mcmaster.caMartaCanutiMemorialUniversityofNewfoundlandmarta.canuti@gmail.com


ElizabethCastleDalhousieUniversity,Halifax,NS,[email protected]@plos.orgRyanClarkinUniversityofOttawarclar063@uottawa.caJenniferCorcoranDalhousieUniversityjcorcora@dal.caMarcelineCôtéUniversityofOttawamarceline.cote@uottawa.caMathieuCrupiOttawaHealthResearchInstitutemcrupi@ohri.caAlexanderCulleyUniversitéLavala13xcu113y@gmail.comJoshDelPapaOttawaHospitalResearchInstitutejdelp034@uottawa.caStephanieDeWitte-OrrWilfridLaurierUniversitysdewitteorr@wlu.caJean-SimonDialloOttawaHospitalResearchInstitutejsdiallo@gmail.comJimmyDikeakosUniversityofWesternOntariojimmy.dikeakos@[email protected]

AlanDoveScienceJournalistalan.dove@gmail.comBrettDuguayDalhousieUniversitybduguay@dal.caRoyDuncanDalhousieUniversityRoy.duncan@dal.caMayElSherifNovaScotiaHealthAuthority&DalhousieUniversitymay.elsherif@iwk.nshealth.caDarrylFalzaranoVIDO-InterVacdarryl.falzarano@[email protected]élieFradet-TurcotteCHUdeQuébecResearchCenter-UniversitéLaval,CancerResearchCenteramelie.fradet-turcotte@crchudequebec.ulaval.caLoriFrappierUniversityofTorontolori.frappier@utoronto.caCarlA.GagnonUniversitédeMontréalcarl.a.gagnon@umontreal.caPamGallantDalhousieUniversitypamela.gallant@dal.caLaurenGarnettUniversityofManitobagarnettl@myumanitoba.caGerardGaspardDalhousieUniversitygerard.gaspard@dal.ca


KatrinaGeeQueen'sUniversitykgee@queensu.caNikhilGeorgeUniversityofWaterloonageorge@uwaterloo.caMatthiasGotteUniversityofAlbertagotte@ualberta.caGrahamGouldMauleUniversityofOttawaggoul096@uottawa.caTysonGraberChildren'sHospitalofEasternOntarioResearchInstitutetyson@arc.cheo.caNathalieGrandvauxUniversitédeMontréal/CRCHUMnathalie.grandvaux@umontreal.caArlenGuanDalhousieUniversityguanjian0208@126.comShashiGujarshashi.gujar@dal.caChristinaGuzzoUniversityofTorontoScarboroughchristina.guzzo@utoronto.caToddHatchetteNovaScotiaHealthAuthority&DalhousieUniversitytodd.hatchette@nshealth.caJiangyiHeMemorialUniversityofNewfoundlandjiangyih@[email protected]

AlexanderHynesMcMasterUniversityhynes@mcmaster.caZafrinIslamDepartmentofBiochemistry,UniversityofTorontozafrin.islam@mail.utoronto.caEricJanUniversityofBritishColumbiaej@mail.ubc.caBenJohnstonDepartmentofMicrobiologyandImmunology,DalhousieUniversityben.johnston@dal.caBradleyJonesBCCentreforExcellenceinHIV/AIDS;UniversityofBritishColumbiabjones@cfenet.ubc.caCaseyJonesDepartmentofPharmacology,DalhousieUniversitycasey.jones@dal.caJeffreyJoyUniversityofBritishColumbia,BCCentreforExcellenceinHIV/AIDSjjoy@cfenet.ubc.caRachelKampenDalhousieUniversityRachel.Kampen@iwk.nshealth.caDacquinKasumbaCRCHUMdacquinkasumba@gmail.comDavidKelvinDalhousieUniversity;CanadianCentreforVaccinology;ShantouUniversityMedicalCollegedkelvin@[email protected]


DenysKhaperskyyDalhousieUniversityd.khaperskyy@dal.caYouraKimDepartmentofPathology,DalhousieUniversity,Halifax,NS,[email protected],SimonFraserUniversitynkinloch@sfu.caMarielKleerDepartmentofMicrobiologyandImmunology,DalhousieUniversitymariel.kleer@dal.caDanikaKnightDalhousieUniversitydanika.knight@dal.caYumikoKomatsuKyotoUniversityyumikokomatsu56@gmail.comMarikaKöszegiUniversitédeMontré[email protected]@uoguelph.caChristianLalondeFacultédemédecinevétérinairedel'UdeMchristian.lalonde@umontreal.caAlainLamarreINRS-InstitutArmand-Frappieralain.lamarre@iaf.inrs.caAmandaLangSaskatchewanDiseaseControlLab&NovaScotiaHealthamandalslang@[email protected]

DanielleLanoueImmunovaccine,[email protected]@ualberta.caJasonLeBlancNovaScotiaHealthAuthority&[email protected]édeMontré[email protected]@[email protected]éUniversitédeMontréalroger.lippe@umontreal.caPierre-YvesLozachUniversityofHeidelbergpierre-yves.lozach@med.uni-heidelberg.deYaoLuUniversityofSaskatchewanyao.lu@usask.caDuncanMacKenzieDalhousieUniversityduncan.mackenzie@dal.caGrantMacNeilDalhousieUniversitygrant.macneil@[email protected]


AndrewMakrigiannisDalhousieUniversityAmakrigiannis@dal.caJonathanMapletoftMichaelG.DeGrooteInstituteforInfectiousDiseaseResearch,DepartmentofBiochemistryandBiomedicalSciences,McMasterImmunologyResearchCentre,McMasterUniversity,Hamilton,ON,Canadamapletoj@mcmaster.caKarenMaxwellUniversityofTorontokaren.maxwell@utoronto.caCraigMcCormickDalhousieUniversitycraig.mccormick@dal.caJaclynMcCutcheonUniversityofAlbertajgmccutc@ualberta.caAshleyMckibbonAtlanticVeterinaryCollege,UniversityofPrinceEdwardIslandamckibbon@upei.caAnneMcMillanDalhousieUniversityAnn.MacMillan@iwk.nshealth.caNicholeMcMullenDalhousieUniversitync843105@dal.caVanessaMeier-StephensonUniversityofCalgaryandUniversityofLethbridgevmeierstephenson@gmail.comYsabelMenesesMemorialUniversityofNewfoundlandylm017@[email protected]

NatachaMerindolUniversitéduQuébecàTrois-Rivièresnatacha.merindol@uqtr.caMatthewMillerMcMasterUniversitymmiller@mcmaster.caVikramMisraUniversityofSaskatchewanvikram.misra@usask.caYasirMohamudUBCYasir.Mohamud@hli.ubc.caSylvainMoineauUniversitéLavalSylvain.Moineau@bcm.ulaval.caAndreaMonjoDalhousieUniversityAndrea.Monjo@Dal.caClaytonMooreUniversityofGuelphcmoore11@uoguelph.caKarenMossmanMcMasterUniversitymossk@mcmaster.caTylerMrozowichUniversityofLethbridgetyler.mrozowich@uleth.caCaitlinMullarkeyMcMasterUniversitymullarkc@mcmaster.caRoryMulloyDalhousieUniversityrr561522@[email protected]


NicolasNantel-FortierUniversityofMontrealnicolas.nantel-fortier@umontreal.caMichaelaNicholsDalhousieUniversityMichaela.Nichols@Dal.CaRyanNoyceUniversityofAlbertanoyce@ualberta.caQuentinOssemanCRCHUM,universitédeMontré[email protected]@zoology.ubc.caEdwardPattersonUniversityofTexasMedicalBrancheipatter@utmb.eduMartinPelchatFacultyofMedicine,UniversityofOttawampelchat@uottawa.caAdrianPelinOttawaHospitalResearchInstituteapelin20@gmail.comDaniellePetersUniversityofAlbertadlpeters@ualberta.caLilianaPortales-CervantesDalhousieUniversitylportales@dal.caSarahPoynterUniversityofWaterloopoyn7090@mylaurier.caEricPringleDepartmentofMicrobiologyandImmunology,[email protected]

ShirleyQiuUniversityofOttawa,OttawaInstituteofSystemsBiologysqiu096@uottawa.caVincentRacanielloColumbiaUniversity,NewYork,USAvrr1@cumc.columbia.eduMdNiazRahimNationalMicrobiologyLaboratory,PublicHealthAgencyofCanada.MedicalMicrobiology,UniversityofManitoba.mdniaz.rahim@canada.caChristopherRichardsonDalhousieUniversitychris.richardson@dal.caCarolyn-AnnRobinsonDalhousieUniversitycrobinson@[email protected]ÉlisabethRousselUniversitédeMontréalelisabeth.roussel@umontreal.caJaredRowellUniversityofCalgaryrowellj@ucalgary.caAnnetteRushtonDalhousieUniversityhenneberry5@hotmail.comRodRussellMemorialUniversityrodney.russell@med.mun.caDaveSafronetzPublicHealthAgencyofCanadadavid.safronetz@[email protected]


BritiSahaOttawaHospitalResearchInstituteandUniversityofOttawabsaha021@uottawa.caJaymeSalsmanDalhousieUniversityjsalsman@dal.caHeleneSanfaconSummerlandResearchandDevelopmentCentre,AgricultureandAgri-FoodCanadaHelene.Sanfacon@agr.gc.caMayaShmulevitzUniversityofAlbertashmulevi@ualberta.caJacobSicheriDalhousieUniversitysickairy@gmail.comGillianSinghDepartmentofMicrobiologyandImmunology,DalhousieUniversity,Halifax,NS,[email protected]ébecResearchCenter-UniversitéLaval,CancerResearchCenterjustine.sitz0@gmail.comPatrickSlaineDalhousieuniversityPatrick.Slaine@Dal.CaHugoSoudeynsCHUSainte-Justine;UniversitédeMontréal.hugo.soudeyns@recherche-ste-justine.qc.caHannahStaceyDepartmentofBiochemistryandBiomedicalSciences,MichaelG.DeGrooteInstituteforInfectiousDiseasesResearch,McMasterImmunologyResearchCentre,McMasterUniversity,ON,[email protected]@imvaccine.com

JeromeTannerResearchassociate,ResearchCentre,CHUSainte-Justine,Montrealjetanner@videotron.caPatriciaThibaultDepartmentofMicrobiology,IcahnSchoolofMedicineatMountSinai,NewYorkNY,USA,10029patricia.thibault@mssm.eduMackenzieThornburyDalhousieUniversitymackenzie.thornbury@dal.caFrankvanderMeerUniversityofCalgary,FacultyofVeterinaryMedicinefrank.vandermeer@ucalgary.caSarahVeinotDalhousieUniversitysr618131@dal.caJoostVerhoevenMemorialUniversityofNewfoundlandjverhoeven@mun.caHannahL.WallaceMemorialUniversityhlwallace@mun.caLingyanWangMemorialUniversityofNewfoundlandlingyanw@mun.caMichelleWarhuusNovaScotiaHealthAuthority&DalhousieUniversitymichelle.warhuus@iwk.nshealth.caCorinaWarkentinDepartmentofBiochemistry,Microbiology,andImmunology,UniversityofOttawacwark095@[email protected]


DavidWillerGlaxoSmithKline(GSK)[email protected]@uoguelph.caDanyangXuMemorialUniversityofNewfoundlanddanyangx@mun.caShuangYangDepartmentofZoology,UniversityofBritishColumbia,BritishColumbia,[email protected]

YimingYangDalhousieUniversityym275344@dal.caAliZhangMichaelG.DeGrooteInstituteforInfectiousDiseasesResearch,McMasterImmunologyResearchCentre,DepartmentofBiochemistryandBiomedicalSciences,McMasterUniversity,Hamilton,ON,[email protected],Pathologymzhang@[email protected]

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