Lassa fever virus

How virus-host interactions affect Lassa and Influenza virus replication, virulence and

pathogenesis?

Research Projects in Ly’s & Liang’s Labs

Influenza virus

Lassa Virus Causes Lethal Hemorrhagic Fever

Arenaviridae Bunyaviridae Filoviridae Flaviridae Rhabdoviridae?

2 ambisensessRNAs

3 (-)/ambisense ssRNAs

(-) ssRNA (+) ssRNA (-) ssRNA

Lassa (BSL4)Junin (BSL4)Machupo (BSL4)Guanarito (BSL4)Sabia (BSL4)Chapare (BSL4)Lujo (BSL4)

Crimean-Congo H.F. (BSL4)Hantavirus (BSL3)Rift Valley Fever (BSL3)

Ebola (BSL4)Marburg (BSL4)

Kyasanur Forest Disease (BSL4)Omsk H.F. (BSL4)Yellow Fever (BSL3)Dengue (BSL2)

Bas-Congo (BSL4)

• Severe multisystem syndrome  • Damage to overall vascular system• Severe symptoms often accompanied by

hemorrhages

• Virulence factors & mechanisms• Virus-induced immune suppression• Pathogenesis

Overall Research Objectives & Approaches

proteomics

1. Animal model 2. reverse genetics 3. cell cultures 4. X-ray crystallography 5. Y2H & Pichinde virus infected guinea pigsMass Spect

liver stomach intestine skin

Hemorrhagic fever-like syndromes in virulent Pichinde virus-infected guinea

pigs

Avirulentinfection

Virulent infectio

n

Pichinde virus: BSL2 arenavirus that is non-pathogenic in humans

Multi-functional roles of Lassa nucleoprotein in viral RNA synthesis

and host immune suppression

Qi et al., December 2010, Nature

Cap binding pocket

C domain

N domain

side view

Cap binding pocket

Changjiang Dong, Ph.D., Univ of St Andrews, Scotland

Qi et al., December 2010, Nature

N terminus of Lassa nucleoprotein has a cap binding pocket required for viral transcription

Qi et al., December 2010, Nature

Lassa viral minigenome transcription assay

293T transfection+

CMVp CMVp

5'Luc Luc

LASV mini-genome with luciferase reporter gene

LUC assay

L NP

C terminus of Lassa nucleoprotein has 3’-5’ exoribonuclease activity required for innate immune

suppression

Qi et al., 2010, NatureHastie et al., 2011, PNASBrunotte et al., 2011, JBCHastie et al., 2012, PloS One Jiang and Huang et al., 2013, JBC, in press

3’-5’ exoribonuclease domain

C domain

N domain

Top view

3’-5’ exoribonuclease cavity

L

RIG-1 MDA5

Long dsRNA, higher-order structure

P P PShort dsRNA 5’pppRNA

NP

NP NP NP

Virus replication

Viral RNA synthesis

Roles of NP in mediating innate immune suppression and in enhancing viral replication

IFN-

Host’s pathogen-recognition receptors

Pathogen-associated molecular patterns

Virus-receptor interaction

Qi et al., December 2010, Nature

Summary #1• Pichinde virus-infected guinea pig as a good surrogate animal model for Lassa and other hemorrhagic fever diseases.

• The reverse genetics systems of Pichinde virus (BSL2 pathogen) to identify the virulence determinants and to characterize the virulence mechanisms of Lassa virus infection.

• Structure-function characterization of Lassa nucleoprotein has revealed unexpected roles for NP in mediating viral transcription and immune evasion.

1. Exploit known 3D structure of Lassa nucleoprotein for rational design of novel antiviral compounds

2. Use Lassa minigenome assay to screen for compounds against viral transcription

3. Use guinea pig animal model to test compounds against viral hemorrhagic fevers (coagulopathy, hearing loss, liver hepatitis, congenital viral infection)

4. Use modified PICV reverse genetics system to deliver transgene into cells

5. Use modified PICV reverse genetics system as novel vaccine vector platform

6. Develop antiviral therapeutics targeting virus-host interactions

Possible collaborative projects

FDA-approved drugs for influenza

Problem: Rapid emergence of drug-resistant strains. - Develop novel anti-flu drugs

- Use combination of diverse classes of anti-flu drugs - Targeting host components as an alternative antiviral approach

Crm1

Virus entry

Viral RNA synthesis

fusion

vRNP nuclear import

Proteintranslation

ProteinMaturation

GenomePackaging

Virus budding

Virus release

vRNP nuclear export

Virus and host factors involve in influenza replication

uncoating

vRNAcRNA

mRNA

vRNA

Liang, et al., 2005, J VirolRegan, et al., 2006, J VirolLiang et al.,, 2008, J Virol Kumar et al. 2008, J VirolKumar et al. 2011, J VirolKumar et al., 2011, AACLiang, et al., 2012, PLoS One

NF-κB signalin

g

TrKAReceptor Tyrosine Kinases

TrkA inhibitors strongly inhibit flu virus replication in vitro

Kumar et al. JVI 2011Kumar et al., AAC 2011

TrkA inhibitor inhibited flu virus replication in vivo and protected mice from lethal flu infection

Possible Collaborations • Various convenient cell-culture based assays to screen for possible new drug candidates against different steps of influenza virus replication

• Host signaling pathways (TrkA) play important roles in influenza virus replication and can serve as alternative anti-flu targets for drug screening.

Other viruses of human and veterinary importanceAvian influenza surveillance and host-pathogen interaction (Drs. Carol Cardona

and Zheng Xing), swine influenza virus surveillance, virus-host interaction, and viral evolution in response to vaccination (Drs. Sri Sreevatsan, Montse Torremorell, Maxim Cheeran, Tom Molitor)

Porcine respiratory and reproductive syndrome virus (PPRSV) and circovirus (PCV2): molecular virology & anti-viral immunity (Dr. Michael Murtaugh)

Avian reoviruses (arthritis, runting, stunting syndrome and malabosorption in chickens and arthritis and enteritis in turkeys) (Dr. Sagar Goyal)

Emerging phleboviruses (severe fever with thrombocytopenia – SFTS virus) and Heartland virus (HLV) (Dr. Zheng Xing): host innate immune suppression

Infectious disease ecology: disease transmission and modeling of canine distemper, rabies, and parvovirus infections of African carnivores (lions) in the Serengeti ecosystem (Dr. Megan Craft)

Cardona lab major projects

• Phenotyping of influenza A viruses recovered during surveillance activities in relevant animal hosts. – Determining host susceptibility, infectious doses– Measuring host responses to infection– Goal: which virus or gene segment could contribute to human or

animal disease?

• The host and virus interactions of mixed infections with orthomyxo- and paramyxo–viruses.– Interactions in vivo and in vitro

• The development of the chicken gut virome. Following the flow of the virome from breeders to their progeny through maturity in a farm environment.

Swine influenza researchDrs. Montserrat Torremorell and Srinand Sreevatsan

• Swine influenza transmission and surveillance:– Models for influenza transmission using the pig as a model:

• Evaluate the effect of immunity (i.e vaccination and passive immunity) on influenza transmission and dissemination

• Evaluate the effect of immunity on influenza diversity and evolution• Determine virus genome diversity within and between animals throughout the course

of infection using de novo sequencing techniques• Evaluate the role of fomites on transmission in experimental settings• Development of mathematical models for influenza spread in populations

– Assessing subpopulations, routes and methods of influenza transmission under high risk interspecies transmission settings:

• Assessment of animal subpopulations responsible for maintaining and spreading influenza

• Determining the role of indirect routes of transmission (aerosols and fomites) to influenza exposure in animals and people

– Methods to decrease influenza transmission and assessment of influenza dissemination in populations

Xing, Zheng PhDXing, Zheng PhD Associate ProfessorDepartment of VeterinaryBiomedical Sciences, CVM

Research Interests: Pathogenesis and host-pathogen interaction of 1) avian influenza virus (AIV) & 2) bunyaviruses in human and animals

AIV is a zoonotic pathogen that infects birds and humans. We study host responses and their regulations in infected birds and humans comparatively. Using genomic and other high throughput approaches, we study host-virus infections and identified cellular proteins that are required for viral replication. We expect to develop antiviral strategies based on the identified cellular components essential to viral replication.

The bunyavirus we are studying is the one that causes severe fever with thrombocytopenia syndrome (SFTS) with a high fatality rate up to 15% in China. A similar virus, Heartland virus (HLV) has been identified in the Midwestern region of the US. We are studying the mechanisms how the viral infection suppresseshost innate immunity. We have discovered a unique mechanism that HLV uses its nonstructural protein (NSs) to modulate host antiviral responses.

Murtaugh Laboratorywww.murtaughlab.com

Lab CommitmentTo discover and disseminate knowledge that

enhances animal health; to prepare young scientists for careers in agriculture, biology, and medicine; to

provide training in genomics and biotechnology.

Collaborations: Promote mutual interest and facilitate problem solving.Intellectual Property: Translate fundamental knowledge into useful products and tools.Advanced Training: Share expertise with sponsors and end users.

Molecular Mechanisms of Disease Resistance

Core Technologies

Laboratory Outreach

Porcine ImmunologyB-cell function

Immunoglobulin diversityTh1-Th2 paradigm

Early Warning Biomarkers

PRRSV disease signaturesMetabolic disorder markers

XenotransplantationMolecular mechanisms of engraftment

Pathway identification for stress reductionXenopathogen molecular diagnostics

Mucosal ImmunobiologyFunctional genomics of mucosal immunity

Differential gene expression in the gutOpioids in intestinal innate immunity

Infectious Disease Immunity

Anti-viral immunityPRRSV, PCV2

PRRSV BiologyVirion structural characterizationGlycan structural modifications

Viral evolution and diversity

Graduate Education in Molecular Biosciences

Agricultural BiotechnologySabbatical

Opportunities

Advanced Technical Training

Biotechnology Workshops for End

Users

Biomedical Collaborations

Mass SpectrometryProtein discovery

Glycan characterization

Protein EngineeringGene cloning and expression

Protein modificationBacterial and yeast expression Immuno-cellular

MethodsVirus culture

ELISA and ELISPOT

Massively Parallel Sequencing

Population genomicsExon profiling

Evolutionary mechanisms

ProteomicsBiomarker discoveryFunctional genomics

Antigen discovery

Quantitative Gene Expression

Single gene RT-PCRTranscriptome profiling

Michael Murtaugh

Kyra Martins, Scientist

Diem Ngo, Sr. Lab Tech.

Suzanne Stone, Sr. Lab Tech.

Jenny Zhang, Jr. Scientist

John Schwartz, Graduate StudentSally Robinson,

Graduate Student

Marina Figueiredo, Graduate Student

Xiong Wang, Graduate Student

Jie Zhang, Visiting Scientist

Cheryl Dvorak, Research Associate

Avian reovirusesDr. Sagar Goyal

Chicken reovirus is known to cause: arthritis runting and stunting syndromemalabsorption

Turkey enteric reovirus (TERV) is widespread and is involved in turkey enteritis

Studies on turkey arthritis reovirus (TARV) are at least 20 years old.

We have isolated TARV from recent outbreaks of lameness in turkeys and are studying their pathogenesis in addition to characterizing the viruses