InfluenzaThere are three types of influenza in humans: A,B, C

1. Flu A: Found in many animal species, in addition to humans

Closely related to Type B but not Type C

Main type responsible for human epidemics

Demonstrates the greatest antigenic variability (“antigenic drift”)

Reservoir in nature is waterfowl

2. Flus B and C: Found almost exclusively in humans

Flu C can also infect swine

Flu C is morphologically and antigenically distinct from A, B

3. Flu A strains designated by host from which isolated, where isolated, year of isolation, and type of HA and NA. An isolate (strain) number may also be included if there are multiple isolates.

Example: A/goose/Leipzig/137/79 (H7N2)

ORTHOMYXOVIRIDAE

INFLUENZAVIRUS A

INFLUENZAVIRUS C

“THOGOTO-LIKE VIRUSES”

INFLUENZAVIRUS B

InfluenzaA Humans, birds, swine

Airborne Respiratory disease

WorldwideFLUAV

Tick-borneThogoto virus MammalsTHOV

GENUS/ MEMBERS

USUAL HOST(S)

TRANSMISSION DISEASE WORLD DISTRIBUTION

VIRUS NAME ABBREV.

Influenza B Humans Airborne WorldwideFLUBV Respiratory disease

Influenza C Humans WorldwideAirborneFLUCV Respiratory disease

Influenza

Virus

Structure of Influenza Virus

Influenza A virus is an enveloped particle that when spherical is about 120 nm in diameter

Many particles are not spherical but filamentous in shape

There are two glycoproteins at the surface in surface “spikes”

HA (hemagglutinin) is present as homotrimersNA (neuraminidase) is present as homotetramers

The genome consists of 8 RNA segments present in helical nucleocapsids

Protein M2 forms ion channels in the lipid bilayer

The matrix protein M1 lines the inner side of the lipid bilayer

Influenza NucleocapsidsNP is the major nucleocapsid protein.

PA, PB1, and PB2 are minor components of the nucleocapsid and form the RNA synthesis machinery.

The function of PA is unknown but may be involved in the switch from mRNA synthesis to genome replication

PB1 is an endonuclease that process the mRNA primer; it also is a polymerase that catalyzes nucleotide addition

PB2 recognizes the cap of host cell mRNA required for priming mRNA synthesis

It has a major structural role

It is also required for the switch from mRNA synthesis to genome replication.

M is the matrix protein.

It is a peripheral membrane protein that underlies the viral membrane.

It interacts with the nucleocapsid and with the tails of HA, NA, and M2

Attachment & EntryThe HA spike is a homotrimer with a molecular weight of 110 kDa.

HA is synthesized as a 549 aa precursor called HA0 which is anchored in the membrane near the C-terminus.

HA0 is cleaved into HA1 (328 aa) and HA2 (221 aa)

At the N-terminus of HA is a 16 aa hydrophobic signal peptide for insertion into the ER.

A single Arg separates HA1 from HA2 and cleavage is by a cellular trypsin-like proteinase

HA1 and HA2 remain covalently associated after cleavage by a disulfide bridge

The C terminus of HA2 contains a 26 aa uncharged membrane-spanning domain followed by a 10 aa hydrophilic cytoplasmic domain

The HA polypeptide is glycosylated at specific asparagine residues

HA-mediated membrane fusionThe HA trimer is stabilized by a hydrophobic core formed between the three stalk regions.

Attachment sites for the cellular receptors are located near the top of each large globular region, which also contains neutralization epitopes.

The exact glycoprotein(s) that serve as host cell surface receptors has not been identified, but it is known to contain sialic acid.

After binding of HA to the cell surface receptor(s) the virus is internalized by endocytosis.

The low pH of endosomes ( pH 5.0-6.0) results in an irreversible conformational change in HA which results in the extrusion of the highly conserved hydrophobic amino terminus of HA2

from its position in the native protein.

This region, termed the ‘fusion peptide’, promotes membrane fusion.

The mechanism by which the ‘fusion peptide’ promotes membrane fusion is not completely understood.

The subsequent fusion of viral and endosomal membranes allows the release of the viral genome into the cellular cytoplasm

Activation of the HA Spike

HA0 precursor Cleaved spikeAfter acid treatment

and proteolysis

Activation of Fusion Activity of Flu HA0 by Cleavage

View of One Monomeric Unit in the Spike

S

HA1N

(328aa)16aaC

HA2 (221aa)

TMA.

S - S

B. C.

1(N)

40

175 1(N)

105

153

129

153

40

129

105

B’. C’.

C

A

B

GGG

DF1 S

S

E

153

40

105

76

E

1

H

F SS

B

C

A

C

G

D38

153

105

7676

Structures of the Native and Fusion Active Conformations of the Influenza Hemagglutinin

Change illustrated for one monomeric unit of the trimeric spike

Model for Fusion

NeuraminidaseNA spike consists of a tetramer. NA is a type 2

glycoprotein with the N terminus inside and the C terminus outside.

NA removes sialic acid from oligosaccharides on cell-surface proteins and glycolipids, thus destroying receptors for the virus.

Also removes sialic acid from HA so that progeny influenza virions cannot aggregate.

Separates virus particles from inhibitory mucopolysaccharides in the respiratory tract allowing efficient infection.

Genome Segments of Influenza viruses

Influenza A Influenza C

RNA Segment

Length (nt)

Encoded Protein Name (aa)

Function RNA Segment

Length (nt)

1 2341 PB2 Cap recognition, RNA synthesis

1 2365 PB2

3 2233 PA 3 2183 PA

4 42073 HA Hemagglutinin, fusion, major surface antigen, sialic acid binding. HEF of FLUCV also has esterase activity

2073 HEF

5 1565 NP 5 1809 NP 565

6 1413 NA Neuraminidase

7 1027 M1

M2 Ion channel

6 1180

spliced

spliced M 242

internal initiation

CM2 (139)

139

8 934 NS1

Nucleocapsid protein

??

spliced NS2

Nonstructural protein

Nuclear export protein ??

7 934 NS1 286

spliced NS2 122

Matrix protein

759

716

Encoded Protein Name (aa)

774

2 2341 PB1 RNA synthesis 2 2363 PB1757 754

709

566

498

454

252

97

230

121

655

RNA synthesis

vcRNA

mRNA

15-22ntReplication

mRNA synthesis

ppp-AGC AAAGCAGGA

G

HO-UCG UUUCGUCCCU

CCUUGUUUCUACU

GGAACAAAGAUGA

5'

3'

3'

5'

3'

AAAAAAAAAAAAA(PolyA)

10-13 nt

"Cap-snatching"

GC AAAGCAGGA GG A

m GpppX Y

7 m

5'

UUU UUU Genome RNA

Synthesis of mRNAs and RNA Replication

M1 mRNA

M2 mRNA

Genome Segment 7

Cap-snatching, mRNA synthesis, splicing

Translation

Translation

M2 protein

M1 protein

(97aa)

(252aa)

CAP

CAP

Cap-snatching, mRNA synthesis

5'

3'

3'

5'

3'5'

Poly(A)

Poly(A)

Splicing to Produce Influenza A mRNAs

Since influenza RNA synthesis occurs in the nucleus, the cellular splicing machinery can be used

In Flu A two mRNAs are produced from both segments 7 and 8

One mRNA is unspliced, the second is spliced

Flu C lacks NA and has only 7 segments

It has HEF that performs the functions of HA and NA in Flu AB

The receptor for Flu C is 9-O-acetyl-N-acetyl neuraminic acid

The Flu C esterase removes the 9-O-acetyl group to destroy the receptor

The HEF gene is also present in some coronaviruses, which must have obtained it by recombination with Flu C at some time in the past

Influenza C Has an Esterase

M2M2 tetramers form ion channels in viral and cellular membranes

Exposure to low pH is required to dissociate the nucleocapsid from the matrix protein, allowing the nucleocapsid to be transported to the nucleus

M2 also prevents premature activation of the fusion activity of HA

Amantadine interferes with the function of M2 and is an effective flu antiviral

Virus Assembly

Nucleocapsids assemble in the nucleus during genomic RNA synthesis

The encapsidation signal is at the end of the RNA and not present in mRNAs

Glycoproteins are synthesized on the ER and transported to the plasma membrane

Nucleocapsids bud through the plasma membrane to form virions

More than 8 segments may be packaged: Ten segments randomly selected would result in ~3% of progeny virions having at least one each of the 8 segments

Random selection of segments would mean efficient reassortment during mixed infection, which is known to occur

Nucleocapsids are exported to the cytoplasm in a process that requires NS2 and M1

Influenza - Some HistoryOldest record of an epidemic probably caused by flu: Hippocrates, 412 BC.

Epidemics have occurred relatively frequently but at irregular intervals

Epidemics vary in severity but the very young and elderly are most at risk.

Epidemics appear to radiate from specific locations

Example: 1781 epidemic that spread across Russia from Asia.

Influenza has killed untold millions throughout the centuries

1. 1918-1919 epidemic was particularly severe

2. 20-1000 million people died, more than died in World War I.

3. 80% of US WWI deaths were due to influenza

4. A significant factor in the German loss was influenza

First human influenza virus was isolated in 1933.

Different strains cause different epidemics, but human strains can recirculate

Antigenic Shift and Drift in Flu A

HA and NA are the major surface antigens of the virus

Antigenic drift describes the selection of variants by the immune system

Relatively slow

Resistance is only partial

Antigenic shift describes the results of recombination (reassortment)

There are 15 different subtypes of HA

There are 9 different subtypes of HN

A reassortant with a different HA and/or HN may cause a pandemic

Only a few of the subtypes have been isolated from humans

Subtypes differ by 30% or more in amino acid sequence

The reservoir of influenza A in nature is birds

In particular, migratory ducks are important in the maintenance and spread of influenza

All 15 HA and 9 NA have been found in aquatic birds

Influenza infection of birds is usually asymtomatic

Influenza replicates in the respiratory tract and the intestinal tract of birds

It is excreted in the feces and high concentrations have been found in waters in which migratory ducks congregate

The virus appears to be in equilibrium in birds--little or no sequence drift has been found in bird viruses and disease seldom results from infection

In contrast, the virus drifts rapidly in humans and vaccines must be reformulated yearly, and serious illness is produced

Influenza A in Birds

Year Virus

1889 H2N2

1900 H3N8

1918 H1N1

1957 H2N2

1968 H3N2

1977 H1N1

Common Name

Spanish

Asian

Hong Kong

Russian

Epidemic Influenza Strains

At present, H3N2 and H1N1 continue to cocirculate in humans

When a new strain appears the previous strain usually dies out

An H1N1 strains has circulated continuously in pigs in the U.S. since 1918

Sialic Acid (N-Acetyl Neuraminic Acid)

Different HAs prefer one or the other linkage

Avian intestine contains predominantly 2,3 linkages

Terminal NANA is attached to galactose by 2,3 or 2,6 linkages

Human trachea contains predominately 2,6 linkages

Pig trachea contains both linkages and serves as an efficient intermediate host in which reassortment can take place--pigs are often referred to as mixing chambers

Other components also contribute to host specificity, best studied for NP

U.S. Life Expectancy

1918

1900 1910 1920 1930 1940 1950 1960

Year

Lif

e E

xp

ecta

ncy

(years

) 70

62

54

46

38

U. S. Life Expectancy

1918 Influenza Deaths

Age Brackets (years)

0-9 10-19 20-29 30-39 40-49 50-59 >600

10

20

30

40

50

60

Influenza and Pneumonia 1918

Pneumonia 1917

Influenza 1917P

erc

en

t of

Death

s b

y A

ge B

rack

et

Deaths due to:

The 1918 Flu in America

0

2

4

6

8

Year

Exc

ess

Mort

ali

ty (

Death

s X

10

-4)

Cocirculating B and A

Influenza A type H1N1

Influenza A type H2N2

Influenza A type H3N2

Influenza B

1935 1940 1945 1950 1955 1960 1965 1970 1975 1980 1985 1990

Type H2N2 appears

Type H3N2 appears

Excess Mortality Caused by Influenza A and B Virus in the United States Between 1934 and 1990.

Influenza affects 10-20% of U.S. population each year, causing up to 70,000 deaths. Average death rate in people over 65 is 1/2200 but in 1957-8 it was 1/300.

Influenza virus infects superficial cells throughout the respiratory tract.

There is little or no spread to other organs.

Extensive destruction of epithelial cells of the LRT can result in primary viral pneumonia.

Influenza infection can result in secondary bacterial infection of the LRT resulting in bacterial pneumonia.

Immunity following influenza infection is incomplete and appears to fade in time.

It has been suggested that the high death in young adults in the 1918 pandemic could have resulted from a more active immune response to the virus.

Death following influenza infection is usually due to pneumonia, whether viral or bacterial or combined.

High temperatures often accompany the infection, 38-41 C, that last 3-6 days.

Cough and weakness can last 1-2 weeks longer.

Illness Induced by Influenza Virus

Bird Influenza

An epidemic of influenza in chickens occurred in Hong Kong in 1997

The virus was highly virulent, killing 70-100% of infected chickens

Bird viruses are not normally transmitted to humans but the 1997 Hong Kong virus resulted in 18 humans becoming infected

This virus was highly virulent in humans--6 of 18 infected people died

The virus was H5N1 and did not spread in humans--no person to person transmission occurred

To eradicate the virus and to prevent new reassortants from arising that might give rise to epidemic virus by direct person to person transmission, 1.6 million chickens were slaughtered

Defenses against Influenza

Inactivated vaccines are in widespread use

These vaccines must be reformulated every year because of shift and drift

They are 60-80% effective

An emergency response to swine flu in 1976 demonstrates the difficulties in preparedness decisions

Attempts being made to develop attenuated virus vaccines that could be reformulated yearly by reassortment

Vaccines

Antivirals

Amantadine and Rimantadine licensed for use and ameliorate symptoms

Inhibitors of NA being developed

Bunyaviridae

Sin Nombre Virus La Crosse Virus

BUNYAVIRIDAE

BUNYAVIRUS ( ~150 types)

HANTAVIRUS

NAIROVIRUS

PHLEBOVIRUS (~50 types)

TOSPOVIRUS

Rattus speciesSeoul Eastern Asia, Eastern Europe

Hemorrhagic fever

Sin Nombre Pulmonary syndrome

Western US and Canada

Peromyscus maniculatus

GENUS/ MEMBERS

USUAL HOST(S)

TRANSMISSION/ VECTOR

HUMAN DISEASE

WORLD DISTRIBUTION

Snowshoe hare Mosquitoes (Culiseta and Aedes)

Lagomorphs Northern USRarely infects humans

California encephalitis

Western US, Canada

Aedes melanimon A. dorsalis

Rodents, rabbits Encephalitis (rare)

WorldwideHantaan Feces,urine, saliva

Apodemus agrarius

Hemorrhagic fever

Prospect Hill Microtus pennsylvanicus

None? United States

Rift Valley fever Mosquitoes, also contact, aerosols

Hemorrhagic fever

Sheep,humans, cattle, goats

Africa

Aedes triseriatis Midwest USLa Crosse EncephalitisHumans,rodents

Feces,urine, saliva

Bunyamwera Rodents, rabbitsAedes mosquitoes WorldwideFebrile illness

Feces,urine, saliva

Jamestown Canyon North AmericaAedes species, C. inornata

white-tailed deer Increasing

Crimean-Congo hemorrhagic fever

Hemorrhagic fever

Tick-borne Africa, EurasiaHumans, cattle, sheep, goats

Dugbe AfricaTick-borneSheep, goats

Sandfly fever Sicilian Phlebotomous flies

MediterraneanNonfatal febrile illness

Humans

Tomato spotted wilt Plants Australia, Northern hemisphere

Thrips None

Uukuniemi Tick-borne FinlandBirds ??

Nairobi sheep disease.Sheep, goats Tick-borne Africa

G1 G2

G2 G1NSm

BUNYAVIRUSN

NSsNested reading frames

L(BUNV)Post-translational cleavage

G1/G2 G1/G2

PHLEBOVIRUS

NSm

LN

Ambisense transcription and translation

C NSs N

Post-translational cleavage(RVFV)

G1G2

NAIROVIRUSLN Post-translational cleavage(C-CHFV)

HANTAVIRUSLN(HTNV) Post-translational cleavage

S RNA M RNA L RNA

3’ 5’ 3’ 5’ 3’ 5’

Minus strand genome segments

( range of sizes in kb)(0.94 - 2.9 kb) (3.6-4.8 kb) (6.4-8.9kb)

TOSPOVIRUSC N

G1G2Ambisense transcription and translation

LN

C NSs N

(TSWV)

NSm

Genome Organization of the Bunyaviridae

5'

3'

5'

3' 5'

3'

3'

5'

CAP

CAP

ReplicationGenome RNA

NmRNA

vcRNA

NS mRNAs

NS protein s

Translation

mRNA synthesis

Translation

N protein

mRNA synthesis

Ambisense Coding Strategy of Bunyavirus S RNA

Rodent-borne Hantaviruses Rodent hosts

Murinae(Old world rats and mice,found in Europe and Asia)

Arvicolinae(Voles; found in Europe, Asia, and the Americas)

Sigmodontinae(New World rats and mice, found only in the Americas)

76-118cumc-b11

hojoleehv114

Hantaan isolates

b1

sr-1180-39

Seoul isolates

*Thailand

Dobrava

Puumala isolates

TulaProspect HillBayouBlack Creek Canal

New YorkEl Moro Canyon

vindelnvranica

cg1820

sotkamo90-13

Laguna Negra

Sin Nombre

Phylogenetic Tree of Rodent-borne Hantaviruses

Latvia

Serbia

Ukraine

Romania

Distribution of Various Hantaviruses in Eurasia

Hantaan virus

Variant Hantaan

Puumala virus

Variant Hantaan and Puumala

Bayou

Monongahela

New York

Black Creek Canal

Sin Nombre

Juquitiba

Laguna Negra

Rio Mamore

Oran

AndesLechiguanas

United States

Canada

Bolivia

Argentina

Paraguay

Brazil

Chile

Uruguay

1-10

11-50

51-150

>150

Number of HPS cases

Hantavirus Pulmonary Syndrome in the Americas

Arenaviridae

Lassa Virions

Budding Machupo Virion

Tacaribe Virion

5'

3'

5'

3' 5'

3'

3'

5' Genome RNA

NmRNA

vcRNA

GPC mRNA

Translation

Translation

N protein (570 aa)

Replication

Cleavage

G2 (234aa)G1 (256aa)

Lassa fever virus S RNA (3417nt)

Lassa fever virus L RNA (7279 nt) 5'3'5'

Genome RNA

LmRNA

vcRNA

Z mRNA

5'

3'

5'

Translation

Replication

L protein (2218 aa)

Translation

Z (99aa)

mRNA synthesis

mRNA synthesis

mRNA synthesis

mRNA synthesis

Genome Organization and Replication Strategy of an Arenavirus

Representative Arenaviruses

Old World

Lymphocyticchoriomeningitis

Lassa

Mobala

New World

Tamiami

Whitewater Arroyo

Pichinde

Guanarito

Junin

Machupo

Sabia

Tacaribe

Rodent Host Disease Where Found

Mus musculus

Mastomys sp.

Praomys sp.

Sigmodon hispidus

Neotoma albigula

Oryzomys albigularis

Zygodontomys brevicauda

Calomys callosus

?

?

Calomys musculinus

Meningitis

HF

?

None?

3 fatal ARDS

None?

Venezuelan HF

Argentine HF

Bolivian HF

3 severe cases

?

Worldwide

West Africa

CAR

Florida

Western U.S.

Colombia

Venezuela

Argentina

Bolivia

Brazil

Trinidad

Arenaviruses in the New World

Virus IsolatesBefore 19601960 to 196919751990 on

Tamiami (1964)

Guanarito (1990)

Tacaribe (1956)

Pirital (1995)

Amaparí (1964)

Sabiá (1990)

Oliveros (1990)

Whitewater Arroyo (1995)

Pichindé (1965)

Flexal (1975)

Machupo (1963)

Latino (1965)

Paraná (1965)

Junín (1958)

(Sigmodon hispidus)

(Zygodontomys brevicauda)

(Artibeus Bats)

(Sigmodon alstoni)

(Oryzomys capito)

host unknown

(Bolomys obscurus)

(Neotoma albigula)

(Oryzomys albigularis)

(Oryzomys spp.)

(Calomys callosus)

(Oryzomys buccinatus)

(Calomys musculinus)

(Calomys callosus)

Virus Disease Geographic Range

Vector transmission Treatment (Prevention)

ARENAVIRIDAEJunin Argentine HF Argentine pampasInfected field rodents,

Calomys musculinusAntibody effective, ribavirin probably effective; preventive vaccine exists

Machupo Bolivian HF Beni province, Bolivia

Infected field rodents, Calomys callosus

Ribavirin probably effective

Guanarito Venezuelan HF Venezuela Infected field rodents, Zygodontomys brevicauda

No data for humans, ribavirin probably effective

Sabiá HF Rural areas near Salo, Brazil

Unidentified infected rodents

Intravenous ribavirin effective in one case

Lassa Lassa fever West Africa Infected Mastomys rodents

Ribavirin effective

BUNYAVIRIDAERift Valley fever

Sin Nombre and others

Americas (See Fig 4.25)

HPS, also rare HF

As for viruses causing HFRS

Rapid course makes specific therapy difficult

FILOVIRIDAE

FLAVIVIRIDAEYellow fever

Yellow fever Africa, South

AmericaAedes mosquitos Very effective

vaccine

Omsk hemorrhagic fever

OHF Western Siberia Poorly understood cycle involves ticks, voles, muskrats??

Needs further study

a

aAbbreviations used: HF - hemorrhagic fever; HFRS - hemorrhagic fever with renal syndrome; HPS - hantavirus pulmonary syndrome; DHF - dengue hemorrhagic fever; DSS - dengue shock syndrome; KFD - Kyasanur Forest disease; OHF - Omsk hemorrhagic fever

Case Mortalityb %

15-30

15

Rift Valley feverSub-saharan Africa

Aedes mosquitos Rapid course; ribavirin or antibody might be effective

50

Crimean-Congo HF

Crimean-Congo HF

Africa, Middle East, Balkans, Russia, W. China

Tick-borne Ribavirin used and probably effective

15-30

bIn humans.

Hantaan, Seoul, Puumala, and others

HFRS Worldwide (See Fig 4.24)

Each virus maintained in a single species of infected rodents

Ribavirin useful; supportive therapy is mainstay

Variablec

cHantaan is 5-15% fatal, while Puumala is <1% fatal.

40-50

Marburg, Ebola

Filovirus HF Africa Unknown No effective therapy, barrier nursing prevents spread of epidemics

Marburg -25EbolaZ 30-90

20

Dengue DHF,DSS Tropics and subtropics worldwide

Supportive therapy useful; vector control

Aedes mosquitos <1

Kyasanur forest disease

KFD Mysore State, India

Tick-borne ????0.5 - 9

?

This table includes data from Nathanson et al. (1996) Table 32.1 on p. 780.

Some Viruses

Hemorrhagic fever

Causing

Representative Viruses Causing Encephalitis

Flaviviridae

St. Louis encephalitis

Japanese encephalitis

West Nile

Murray Valley enceph.

Tick-borne enceph.

Bunyaviridae

La Crosse

California enceph.

Alphaviruses

Eastern equine enceph.

Western equine enceph.

Venezuelan equine enceph.

Herpesviridae

Herpes simplex

Paramyxoviridae

Mumps

Measles