orthomyxovirusorthomyxovirus and and...
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OrthomyxovirusOrthomyxovirus and and ParamyxovirusParamyxovirusI fl C SARS i fl d hI fl C SARS i fl d hInfluenza, Corona SARS, avian flu and other Influenza, Corona SARS, avian flu and other
respiratory virusesrespiratory virusesyyChao ZHAO(赵 超)
MOH&MOE Key Lab of Medical Molecular VirologyShanghai Medical College Fudan UniversityShanghai Medical College, Fudan University复旦大学上海医学院分子病毒学教育部/卫生部重点实验室
Nov 15, 2013,
TOPIC: what is the biggest challenge
• War?• Food safety• Finance crisisFinance crisis• Natural disasters, eg earthquake…
t i f ti di i t• acute infectious diseases, esp respiratory viruses infection (RVI)
• Chronic disease & aging• Biosafety & Bioterror(ism)Biosafety & Bioterror(ism)
Why is RVI
• Easy transmit• large population• Worldwide (Wide geographic spread)• Worldwide (Wide geographic spread)• Emergency• Variant/mutant
EXAMPLE:1918 Influenza Pandemic
1918 Influenza Pandemic1918 Influenza Pandemic
C d 40 illi d th ld id• Caused 40 million deaths worldwide– Mainly the young!
• 80% of US Army deaths in World War I lt d f i f ti ith i flresulted from infection with influenza
virus
• We still do not understand fully why this t i f I fl A istrain of Influenza A virus was so
devastating to the younger population
High Mortality of 1918 Influenza Pandemic
Spanish flu
Infectious Disease Mortality, United States--20th Century
Armstrong, et al. JAMA 1999;281:61-66.
Recent Outbreaks of
NewNew Influenza
2013 H7N92008 H5N1
H5N1 (Avian) InfluenzaAttempt to control: complete or partial slaughter of poultry populationcomplete or partial slaughter of poultry populationin infected areas
† ASM News 70:154 (2004)
h ( i h fl ) ( i fl )1918 2008
What happened what could
Then (Spanish flu) Now (Avian flu)
What happened what could1.8 billion World population 6 billionTroop ships Primary transportation Jets
il d drailroad mode4 months Time for virus to 4 days
circle globeGauze masks Preventative measures Vaccines
disinfectantsBed rest Treatments Some
aspirin antivirals20+ million Estimated dead 60 million?
Learning ObjectivesAt the end of this lecture, the students should be able to:
1 O tli th di ti i hi f t f i fl i1. Outline the distinguishing features of influenza viruses.
2. Outline the basic steps in the pathogenesis of influenza.
3. Discuss the potential complications of influenza infection.
4 Understand why a “flu” shot is needed every year4. Understand why a flu shot is needed every year.
5. Understand how genome structure and viral polymerase impact on appearance of new virus strainsappearance of new virus strains.
6. Outline the impact of animal reservoirs of viruses in the recurrence of influenza.influenza.
Reference:Jawetz, Melnick, & Adelberg's Medical Microbiology, 25tg gyInfluenza virus-an example of virus mutations. key notes on medical molecular virology.
Editors: Yu-mei Wen, et al. Fudan University Press, p64-75, 2005
Respiratory viruses
1 Orthomyxovirus (~idae) :y ( )influenza V
2 Paramyxoviruses (~idae) :influ B
vaccinemeaslesmumpsRSV (respiratory syncytial V)
3 others i id b ll
MMR, measles-mumps-Togaviridae: Rubella V
Coronaviridae: SARS-CoVOth d i hi i (Pi i id )
mumpsrubella vaccine
Others: adenovirus, rhinovirus (Piconaviridae)….
Part I: Orthomyxovirusfl-- Influ V
• orthomyxoviridae #• orthomyxoviridae #Ortho, “standard, correct;” myxo, “mucus”
• Pathogen causing viral influenza• Pathogen causing viral influenza• 3 subtypes(ABC)
I fl A l d d i / id i• Influenza A leads pandemic/epidemic• pandemic/epidemic #
t• 1st finding: in 1933, by a British Doctor Wilson Smith,named H1N1
Influenza Types
• Type A– Epidemics and pandemics– Animals and humans
All– All ages
T B• Type B– Milder epidemics
H l– Humans only– Primarily affects children
1. Properties of influ v
• -ss RNA, envelopecore:RNA(7~8, segmented)、nucleoprotein(NP, A,B,C, category)、RNA polymerase
Matix proteinMatix protein
Hemagglutinin, HA #,15envelope
Neuraminidase, NA #, 9
General Properties of Influenza A Virusp
– Enveloped virus with helical symmetryEnveloped virus with helical symmetry
– ss-RNA (segmented) genome with negative– ss-RNA (segmented) genome with negative polarity
– 8 unique segments, 10 protein products
– All proteins but one (NS1) are structural
– Host nucleus required for replication• Unusual for an RNA virus
2. Morphology & structure
• 80—120nm,envelope• Spikes: glycoproteins--Spikes: glycoproteins
Virion Structure - Diagram
Envelope glycoproteins (spikes):Hemagglutinin (HA)Neuraminidase (NA)
M1: matrix
M2: ion channel
NP: nucleoproteinhelical nucleocapsid
Polymerase complexPA, PB1, PB2
Genome:8 segments, SS, (-) sense
NS1, NS2: non-structural proteins (some NS2 also in virions)
3. Influenza Virus CompositionNomenclature for Designation of Subtypes of Influenzag yp
Type of nuclearmaterial
HemagglutininNeuraminidase
A/Beijing/32/92 (H3N2)
Hemagglutinin
Virus type
Geographic origin
Strain number
Year of Isolation
Virus subtype
j g ( )
type origin number Isolation
3.1 Hemagglutinin (HA)
1 Integral membrane protein1. Integral membrane protein
2. Responsible for binding of virions to host cell via sialic acid receptorsp
3. Responsible for fusion of virus envelope with endosomal membrane
4. Major antigen to which neutralizing (protective) antibodies develop within the infected host
5. HA0 cleaved into HA1 + HA2 by cellular protease, required for fusion function
6 HA bi d t i li id th f f RBC (R d bl d ll)6. HA binds to sialic acid on the surface of RBC (Red blood cell) to cause the “hemagglutination” of RBC’s
Hemagglutinin (HA)
HA1
HA2
3.2 Neuraminidase, NA
• for subtype; catalytic site for neuraminic acid
• function– Release of virion
P h i d– Promote the virus spread
Structure of Neuraminidase
Sialic acid
Specific NA inhibitors as new anti-flu drugs (Tamiflu)
Site of cleavage by Neuraminidase (NA)( )
NA cleavage is required to release influenzavirions bound to SA on cells or other virionsvirions bound to SA on cells or other virions(aggregation) in order to spread to other cells
N-linked OligosaccharideN-linked Oligosaccharide
Chain of GlycoproteinsChain of Glycoproteins
3.3 M1 & M2
M1: matrix, the structure
HA and M2 Collaborate in Releasing RNP from Endosome
M2: Ion Channel
The channel is specifically blocked by the antiviral drug Amantadine
3.4 Other proteins
• Non-structural proteins:NS1: interferon (innate immunity) resistance
• H5N1 virusH5N1 virus• 1918 virus
• PA, PB1,PB2:polymerase
• NP: nucleoproteinNP: nucleoprotein
4. General Scheme of Replication
mRNA proteinmRNA
( RNA)
p
(cRNA) (vRNA)
= NP coating RNA
5 Antigenic drift & Antigenic shift5. Antigenic drift & Antigenic shift
Antigenic variant, occur in HA and NAg ,
• Antigenic drift (minor change)g ( g )
mutation
• Antigenic shift (major change)
reassortment
Antigenic ShiftAntigenic Shift
Appearance of Influenza pp
P d iPandemics
Influenza A Pandemics of the Past 100 Years
Year of Origin Subtype in Circulation1890 -1890 1900 -1918 H1N1 (Spanish flu)1957 H2N2 (A i fl )1957 H2N2 (Asian flu)1968 H3N2 (Hong Kong flu)(1977) H3N2 and H1N1( )
(reintroduction of H1N1 into the population, but did not cause a pandemic)
2009 New H1N1 (Swine flu)
New H1N1
Development of a Pandemic-1
• Every new pandemic is accompanied by a dramatic change in the HA proteindramatic change in the HA protein
• NA may or may not change
• With a new pandemic, the HA changes and th th t li i tib di lthus the neutralizing antibodies no longer react with the new HA molecule
Development of a Pandemic-2
How does the complete change of HA molecule occur?HA molecule occur?
Multiple independent mutations do not occursimultaneously
Rather, the reassortment of the segmented genome of the virus
Reassortment of Influenza Virus Gene Segments
EMERGENCE OF NEW PANDEMIC STRAINS
Human virusAvian virusH1 N1
N2H2
1918 (H1N1), 1997 (H5N1)
rareH2
Peacefulcoexistence
e
Co - in f e c t io nPi i i i Co - in f e c t io nRe assor t m ent
Pig is permissivefor both avianand human strains
N2H2Genetic reassortment
(mixing)
Reassorted virus new HA and/ or
of genome segments
Reassorted virus new HA and/ or NA; other segments (green) enhancereplication/transmission in humans
Antigenic DriftAntigenic Drift
Appearance of InfluenzaAppearance of Influenza
Epidemics
Antigenic Driftg• Explains the “local” epidemics that occur more frequently (every 1-2
yrs)
• Occurs by point mutations in the HA gene that lead to altered antigenicOccurs by point mutations in the HA gene that lead to altered antigenic sites that are poorly recognized by the existing immune response
• Viruses within a single subtype (H1N1 or H3N2) change with time• Viruses within a single subtype (H1N1 or H3N2) change with time
• Can be measured by hemagglutination-inhibition; the number of fantibody units that block HA measures the degree of relatedness
between the two HA proteins. This correlates with neutralizing antibody.
Summary of Antigenic Drift vs. Shift
Antigenic Drift
Antigenic Shift
GENETIC VARIATION IN INFLUENZA VIRUSES
ANTIGENIC DRIFT ANTIGENIC SHIFT
Influenza A and B Influenza A only
Slow accumulation of mutationsin HA and/or NA genes
Complete replacement ofHA or NA genes
Escape fromantibody mediated
viral transcriptase/ li i
HA and NAencoded in
Broad host range
antibody-mediatedneutralization
replicase iserror-prone
HA NA
encoded indifferentgenomesegments
Mixed co-infectionin pigs
HA NA segments
6. Influenza pathogenesis6. Influenza pathogenesis6. Influenza pathogenesis6. Influenza pathogenesis• Transmission• Aerosoles• Aerosoles
• Infection• Epithelial cells
• Replication cycle• 4 –6 h
• Cell death• Cell death• Necrosis,apoptosis
• Incubation• 18 – 72 h18 72 h
• PBMC• Non-productive
7. Clinic: Influenza (learn by yourself)
• Respiratory infection• Transmission: contact with respiratory secretions
from an infected person who is coughing and sneezing
• Incubation period: 1 to 5 days from exposure to onset of symptoms
• Communicability: Maximum 1-2 days before to 4-5 days after onset of symptoms
• Timing: Peak usually occurs December through March in North America
DISEASE CAUSED BY INFLUENZA VIRUSES
INFLUENZA
CLINICAL PRESENTATION EPIDEMIOLOGICAL FORMS
Epidemic PandemicAbrupt onsetHeadacheChillSymptoms
every1 2
every10 20
ChillsDry cough-rapidly followed
by high fever (38-41oC) declines 2nd or 3rd day 1-2 years 10-20 yearsdeclines 2nd or 3rd day
MalaiseSignificant myalgias
symptoms increaseas fever decreases
Respiratorysymptomsdue to
Systemic symptomsas fever decreases necrosis of
respiratoryepithelium
due to releaseof interferon
Uncomplicated Influenza is Limited to Upper Respiratory Tract
HA precursor ( HA0)No fusion Mature HA
fusion
S-S
S S
fusion
HA1TryptaseClara
S
Cleavagesite
S-S
HA2
HA0
Airway lumen
Nonciliated Clara cells in bronchial epithelia secrete the organ specific protease
COMPLICATIONS ASSOCIATED WITH INFLUENZA
PNEUMONIA
Primary Viral Combined BacterialPrimary Viral
Influenza A
CombinedViral and Bacterial
Bacterial
Invade afterInvade afterloss of
Mucocilliarybl k
Persons >65 years (30% fatalities)Pregnant women in 2nd and 3rd trimesterP ith di l di
Influenza A
+ blanketPersons with cardiovascular diseaseImmunodeficient individualsInfants and very young children
Streptococcus pneumoniaeHemophilis influenzaeStaphlococcus aureus
+
Staphlococcus aureus
Excreted bacterial proteases cleave HA0pHost inflammatory proteases cleave HA0
Influenza is a serious illness
• Annual deaths: 36,000*• Hospitalizations: >200 000*• Hospitalizations: >200,000** Average annual estimates during the 1990’s
• Who is at greatest risk for serious complications?persons 65 and older– persons 65 and older
– persons with chronic diseases– infants– pregnant women– nursing home residents
8. Diagnosis, Treatment and Immunization
Clinical Grounds Killed Vaccine
Symptoms Epidemicperiods Reformulated yearly Vaccinateperiods
virus isolation Three antigenic types
Sept. tomid-Nov.
and typing;antibody testing
Three antigenic typescirculating the previous
year 70-90%effective
for epidemiology 2 type A 1 type Bagainstepidemicstrains containedin vaccine
Also, live flu vaccine (nasal spray)
Influenza Influenza -- diagnosisdiagnosis
•Specimens: throat swabs, nasal aspirates, sputump p p
•Culture: conventional, rapid
•Direct detection (EIA, IF, PCR)
•Serologic response: 4 fold or greater•Serologic response: 4-fold or greater rise in IgG titers
Antivirals Against InfluenzaAntivirals Against Influenza
Stocking
Resistance
De elopingDeveloping
“Universal” Flu Vaccine in Development
Elicit antibodies targeting the highlyElicit antibodies targeting the highly conserved, “fusion peptide” of HA, instead of its variable, receptor-binding regionregion.
Potentially active against multiple subtypes.
Vaccine Development (current)
surveillance WHO/CDC)
Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec
select strains
prepare reassortants
standardize antigen
WHO/CDC/FDA
CDC/FDA
FDAstandardize antigen
assign potency
review/license
FDA
FDA
formulate/test/package
vaccinate
manufacturers
clinic
Pandemic Flu TodayDespite . . .
yp
– Expanded global and national surveillance – Better healthcare, medicines, diagnostics– Greater vaccine manufacturing capacity
N i kNew risks:– Increased global travel and commerce
G t l ti d it– Greater population density– More elderly and immunosuppressed
More daycare and nursing homes– More daycare and nursing homes– Bioterrorism
PART II.h i i
P i d R b ll V
other respiratory viruses• Paramyxoviruses and Rubella V
measlesmeasles• Paramyxovirusesy• -ssRNA• One serotype (antigen stable)• Inclusion body in cellInclusion body in cell • Human being is the only host• Cellular immune
l imeasles patient
K likKoplik
Mumps V• paramyxovirueses
+ RNA• +ssRNA• One serotypeyp• vaccine
Corona virus
• SARS-corona virusSARS corona virus• +ssRNA
SARS host
SARS-CoV
Rubella V• Togaviridae, cause rubella, Germen g , ,
Measles+ RNA• +ssRNA
• One serotypeyp• Congenital rubella syndrome (CRS)
Questions
• Why influenza virus is so easy to spread over the world? (Why is the influenza virus easy toworld? (Why is the influenza virus easy to produce variant?) (1.everyone is easy to be infected 2 vaccine failure due to antigen changeinfected, 2. vaccine failure due to antigen change, 3. RNA, mutation; segmented, reassortment)
• What is antigen reassortment? Antigen shift?What is antigen reassortment? Antigen shift? Antigen drift?
• Plz describe the functions of hemagglutinin andPlz describe the functions of hemagglutinin and neuraminidase?
• What should you do when you face to a suspicious outbreak of respire infectious diseases? Supposed you as a virologist, a pp y g ,general physician, a infectionalist (physician in dept of infection) an(physician in dept of infection), an epidemiologist, or the editor in chief of a f i l j l ti lfamous virology journal respectively.