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.