Spring 2002290

Influenza - History

a. Oldest record of epidem

ic probably caused by influenza was recorded by H

ippocrates in 412 BC.

b. Looking back at recorded epidemics prior to 1800s:

1. Epidemics occurred relatively frequently but at irregular intervals

2. Sometim

es the disease appeared as if had disappeared for significant periods of time

3. Epidemics varied in severity but usually caused death in the very young and elderly.

4. Epidemics appeared to radiate from

specific locationsa. Ex: 1781 epidem

ic that spread across Russia from

Asia.

5. Influenza has killed untold millions throughout the centuries

c. Influenza in the 20th century1. 1918-1919 epidem

ic was particularly severe

2. 40 million people died: Significantly m

ore than died in World W

ar I.3. 80%

of US W

.W.I deaths w

ere due to influenza4. A

significant factor in the Germ

an loss was influenza

5. First human influenza virus w

as isolated in 1933.6. R

ecent outbreak in Hong K

ong (1997) H5N

1

d. Different strains cause different epidem

ics, but human strains can recirculate

Spring 2002291

U.S. Life Expectancy

Spring 2002292

1918 Influenza Deaths

Spring 2002293

Influenza N

omenclature

a. Influenza divided into three types: A,B, C

1. Type A: a. Found in m

any animal species, in addition to hum

ansb. C

losely related to Type B but not Type Cc. M

ain Type found in human epidem

icsd. D

emonstrates the greatest antigenic variability (“antigenic drift”)

2. Types B and C:

a. Found almost exclusively in hum

ansb. Type C

can also infect swine

c. Clear m

orphological and antigenic differences between Types B

and Cb. D

esignation of Type A strains: type A

subdivided by:a. H

ost of originb. G

eographic locationc. Strain num

berd. Y

ear of isolatione. D

escription of hemagglutinin (H

A) and neuram

inidase (NA

) virion sufaceglycoproteins. Ex: A

/swine/Iow

a/15/30 (H1N

1)

Spring 2002294

Pathogenesisa. Transm

ission is by person-to-person spread via aerosol route1. H

ighly infectious & contagious

b. Virus replicates throughout the respiratory track

c. Disease tim

e course1. V

irus peak 24-48 Hr. post infection

2. Virus triggers both a host interferon and im

mune response

Antibodies m

ade HA

, NA

, NP, and M

proteins

d. Influenza A virus induces pathological changes throughout the entire respiratory

tract.1. M

ost significant pathology is in the lower respiratory tract.

2. Clinical sym

ptoms can range from

asymptom

atic infection to a primary viral

pneumonia (fatal outcom

e)3. Serious low

er respiratory tract complications include:

Primary viral pneum

onia which can lead to death in 1-4 days.

Com

bined viral-bacterial pneumonia: 3x m

ore comm

on than primary

pneumonia: Influenza infection follow

ed by bacterial pneumonia

Spring 2002295

Pathogenesis (cont’d)4. Extra pulm

onary manifestation of influenza:

a. Infection in the blood (rare)b. M

uscle and cardiac involvement

c. Reye’s Syndrom

e: neurological and metabolic disease of children and adolescents

d. Central nervous system

: encephalitise. Pregnant w

omen have an increased risk of developing fatal influenza disease

5. Influenza B and C virus infections

a. Influenza B: Less comm

on than influenza A (4x less serious)

Effects predominately adolescents and school-age children

b. Influenza C:

Seldom causes sever disease.

By early adulthood, most individuals (96%

) have antibodies to influenza C

Spring 2002296

Epidemiology

The virus undergoes significant antigenic variation which results in epidem

icsa. Tw

o types of antigenic variation:1. A

ntigenic drift: Involves minor changes in H

A and N

A polypetides

2. Antigenic shift: Involves m

ajor antigenic changes in HA

and NA

polypeptides.

b. Nature of A

ntigenic drift1. H

A is the m

ajor surface antigen of influenza2. M

Abs to H

A are used to determ

ine antigenic drift3. Type A

virus undergoes pronounce antigenic drift4. A

ntigenic drift in HA

occurs by point mutations

5. Antigenic drift can also occur in N

A at approxim

ately the same rate as H

A6. M

echanism of antigenic drift:

i. Clearly results from

the accumulation of point m

utations, but multiple point m

utations m

ust occur before the virus is able to escape neutralization by antibodies ii. M

utations are thought to occur during the spread of virus from person-to-person

c. Nature of antigenic shift1. Thus far, all m

ajor antigenic shifts occurred in China

i. Ex: 1957: H2N

2 subtype replaced the H1N

1 1968 H

3N2 appeared to be replaced by H

1N1

ii. Ex: Since the 1890s: three different HA

subtypes have been introduced

There has been a cyclical appearance of three human subtypes:

H2: 1890, 1957

H3: 1900, 1968

H1: 1918, 1977

Spring 2002297

Transmission M

odels2. First M

odel: Antigenic shift m

ay result from genetic reassortm

ent between hum

anand anim

al influenza A viruses in vivo (see diagram

). i. Ex: 1968 a shift occurred from

H2N

2 to H3N

2 H

uman H

3 was 98%

identical on the nucleic acid level to avian influenza A.

ii. Little antigenic drift observed in influenza B and C because they have

principally human hosts

3. Second model: The ‘new

” form of virus m

ay have been hidden.Ex: R

ussian influenza H1N

1 appeared 1977 in northern China that w

as identical tosam

e virus that caused epidemics in 1950.

4. Third model: N

ew virus could appear in the hum

an population because an animal or

bird form of the virus becam

e infectious for humans.

Ex: Virus from

pigs to man on the sam

e farm in W

isconsin

d. The virus is maintained in the hum

an population by person-to-person spread duringacute infection1. School-age children are the m

ajor vectors in the transmission of the virus in

comm

unities2. Increase in m

ortality seen with introduction of new

strain

Spring 2002298

Pathogenesis (cont’d)

Spring 2002299

Virus Structure

a. Influenza types A,B, and C

have similar structures

1. Irregularly shaped spherical particles approximately 120 um

in diameter

2. Enveloped virus with layers of spikes projecting radially outw

ard over the surface.

Spring 2002300

Viral Proteins

b. Surface spikes are of two distinct types:

1. Hem

aggultinin (HA

) 2. N

euraminidase (N

A)

Spring 2002301

Viral Proteins

•c. W

ithin the lipid envelope lies the mem

braneprotein M

1 (see diagram)

•d. Spanning the lipid envelope is the sm

all mem

braneprotein, M

2.

•e. W

ithin the matrix shell are eight single stranded

RN

A m

olecules of the negative sense each associatedw

ith the nucleocapsid protein (NP).

•f. Three large proteins (PB1, PB2, and PA

)responsible for R

NA

replication and transcriptionare clustered at the 3' end of each R

NA

as if poisedto initiate R

NA

synthesis.

•g. O

ne additional virus coded nonstructual proteins(N

S2) isfound in the virion. It is responsible fortransport of nucleocapsids out of the nucleus.

Spring 2002302

Genom

e Organization

a. Eight ss (-) RN

A segm

ents, separately encapsidated by NP, w

hich encode for 10 proteinproducts (except Type C

which only encodes for 8 or 9)

b. Nucleocapsid proteins:1. 3 polym

erase proteins (PB1, PB2, PA)

2. Nucleoprotein (N

P)3. A

ll nucleocapsid proteins function in the cell nucleus and contain specific nucleartransport sequences for transport from

their cytoplasmic site of synthesis.

c. Envelope proteins1. H

emm

agglutinin (HA

):i. M

ediates the initial attachment of virus particle its cellular receptor (sialic acid)

ii. HA

is cleaved post-translationally which enables the virus envelope to fuse

with an intracellular m

embrane so that the virion nucleocapsid can be

delivered into the cell cytoplasm.

2. Neuram

inidase (NA

):i. A

ppears to have an exclusively enzymatic function: rem

oval of sialic acidresidues from

any gylcoconjugate.ii. A

ppears to play a role in virion assembly and release

Spring 2002303

Genom

e Organization (cont’d)

3. Mem

brane protein 2 (M2):

i: Forms an ion channel in the envelope

ii. It is postulated to play a critical role in virus disassembly upon infecting a

new cell.iii. Expressed by R

NA

splicing of RN

A segm

ent 7 of types A, and B

d. Other structural proteins1. M

embrane protein 1 (M

1)i. Links H

A and N

A w

ith NP

e. Nonstructural proteins (N

S1 and NS2):expressed via splicing of R

NA

segment 8

1. NS1: Suppresses cellular m

RN

A splicing &

polyadenylation2. N

S2: Responsible for transport of nucleocapsids out of the nucleus

Spring 2002304

Attachm

ent & Entry

a. The HA

spike protein is a homotrim

er with a m

olecular weight of 110 kD

a.b. Each subunit is synthesized as a 549 am

ino acid precursor glycopolypeptide, termed H

A0,

which is anchored to the envelope m

embrane near the C-term

inus (see diagram).

c. HA

0 is post-translationally cleaved, a process required for infection, into two polypeptides:

1. HA

1 comprised of 328 am

ino acids2. H

A2 com

prised of 221 amino acids

d. From the prim

ary sequence we can deduce several im

portant features of HA

1. At the N

-terminus there is a 16 aa hydrophobic signal peptide for insertion into the ER.

2. The next 550 aa encode, in order, the HA

1 and HA

2 polypeptides3. A

single arg aa separates HA

1 from H

A2 w

hich is lost during proteolytic cleavage (cleavage probably by a cellular trypsin-like proteinase)

4. HA

1 and HA

2 remain tightly associated to each other after cleavage by a disulfide

bridge (position 14 of HA

1 with position 137 in H

A2 )

5. The C terminus of H

A2 contains a 26 aa uncharged m

embrane dom

ain followed by a

10 aa hydrophilic cytoplasmic dom

ain6. The H

A polypeptide is glycosylated at specific asparagine residues

Spring 2002305

e. High resolution structure of the external portion of H

A has been accom

plished(1-175 of H

A2)

1. Each HA

monom

er contains a 80 Å long stalk capped by a large globular

region about 40 Å in diam

eter. At the interface w

ith the mem

brane, asm

aller 10Å globular dom

ain is formed.

2. The amino term

inus of HA

1 begins near the mem

brane, extends throughthe stalk region, form

s the large globular head (aa 63-304), and finallydescends into the stalk and term

inates 20Å from

the mem

brane surface.3. The H

A2 protein is confined entirely to the stalk and approaches the

mem

brane surface.

Spring 2002306

4. The HA

trimer is stabilized by the form

ation of a hydrophobic core formed betw

een the three stalkregions.

5. Attachm

ent sites for the cellular receptors are located near the top of each large globular region6. The large globule also contains all the antigenic sites required for virus neutralization.

a. Sites occur in surface loops which do not contribute significantly to the H

A quater-

nary structureb. These sites are free to m

utate extensively which contributes greatly to the observed

antigenic variability

f. Receptor binding

1. The exact glycoprotein(s) that serve as host cell surface receptors has not been identified, but it is know

n to contain sialic acid2. A

shallow binding pocket has been defined

Attachm

ent & Entry

Spring 2002307

HA

-mediated m

embrane fusion

a.After binding H

A to the cell surface receptor(s) the virus is internalized by

endocytosis.

b. The low pH

of endosomes ( pH

5.0-6.0) activates the mem

brane fusion potential ofH

A.

c. The subsequent fusion of viral and endosomal m

embranes allow

s the release of theviral genom

e into the cellular cytoplasm

d. How

is mem

brane fusion accomplished?

The low pH

results in a irreversible conformational change in H

A w

hich is required form

embrane fusion activity

1. At low

pH there is an extrusion of the highly conserved hydrophobic am

inoterm

inus of HA

2 from its position in the native protein

2. This region can promote m

embrane fusion (term

ed the ‘’fusion peptide’).3. The m

echanism of how

the ‘fusion peptide’ promotes m

embrane fusion is

unknown

Spring 2002308

Structural Changes in HA

Spring 2002309

Fusion

Spring 2002310

M2 Protein

III. Function of the M2 protein

a. M2 is a 97 residue, non-glycosylated transm

embrane protein

b. A tetramer of M

2 appears to functions as a transmem

brane ion channelc. The ion channel appears to play tw

o important roles:

1. During virus entry it may facilitate release of free nucleocapsid into

the cella. How

?:1. It is known that the drug am

antadine acts early ininfection and effects M

2 function.2. The drug does not block virus binding, internalizationby endocytosis, or m

embrane fusion-nucleocapsid

release into the cytoplasm3. Apparently the drug inhibits nucleocapsid uncoatingand/or nuclear transport4. H

ow is a m

embrane protein (M

2) important in N

Pdisassem

bly?a. There is a 25 m

in. lag between virus

appearance in endosomes and m

embrane fusion.

b. During this lag, the M2 protein m

ay allow an

influx of H+ w

hich may induce conform

ationalchanges in the nucleocapsid w

hich allow release

of NP, and M

12. Late in infection it m

ay regulate the pH of G

olgi vesicles involved intransport of viral glycoproteins to the cell surface.

a. How

?: Clue came after analyzing the effect of the drug

amantadine on influenza.

1. All drug resistant mutants m

apped to the M2

polypeptide2. In the presence of the drug, W

T influenza displayedH

A on the surface of the cell in its low-pH

conformation

3. This suggests that in the absence of the drug the M2

polypeptide may alter the pH

of the acidic Golgi vesicles

(to a more basic pH

)4. In the presence of the drug, M

2 function is blocked,and the G

olgi vesicles remain acidic.

IV. Transcription to produce viral mRN

Asa. Influenza requires both viral and cellular activities to m

ake its mRN

As fromits (-) strand RN

As.b. Influenza RN

A transcription occurs in the nucleus, thus:1. RN

A Pol II required for viral transcriptionVirus RN

A transcription can be inhibited by the drugactinom

ycin DViral m

RNA requires a capped/m

ethylated primer synthesized

RNA Pol II

These products are provided by scavenging cellularm

RNAs

2. Cell also provides RNA m

odification machinery (i.e. splicing )

3. The virus provides replicase proteins (PB1, PB2, PA and NP)

These polypeptides must provide signal sequences for nuclear

localization4. M

odel for initiation of viral mRN

A synthesis (see diagram)

Spring 2002311

M2 (cont’d)

Spring 2002312

III. Function of the M2 protein

a. M2 is a 97 residue, non-glycosylated transm

embrane protein

b. A tetramer of M

2 appears to functions as a transmem

brane ion channelc. The ion channel appears to play tw

o important roles:

1. During virus entry it may facilitate release of free nucleocapsid into

the cella. How

?:1. It is known that the drug am

antadine acts early ininfection and effects M

2 function.2. The drug does not block virus binding, internalizationby endocytosis, or m

embrane fusion-nucleocapsid

release into the cytoplasm3. Apparently the drug inhibits nucleocapsid uncoatingand/or nuclear transport4. H

ow is a m

embrane protein (M

2) important in N

Pdisassem

bly?a. There is a 25 m

in. lag between virus

appearance in endosomes and m

embrane fusion.

b. During this lag, the M2 protein m

ay allow an

influx of H+ w

hich may induce conform

ationalchanges in the nucleocapsid w

hich allow release

of NP, and M

12. Late in infection it m

ay regulate the pH of G

olgi vesicles involved intransport of viral glycoproteins to the cell surface.

a. How

?: Clue came after analyzing the effect of the drug

amantadine on influenza.

1. All drug resistant mutants m

apped to the M2

polypeptide2. In the presence of the drug, W

T influenza displayedH

A on the surface of the cell in its low-pH

conformation

3. This suggests that in the absence of the drug the M2

polypeptide may alter the pH

of the acidic Golgi vesicles

(to a more basic pH

)4. In the presence of the drug, M

2 function is blocked,and the G

olgi vesicles remain acidic.

IV. Transcription to produce viral mRN

Asa. Influenza requires both viral and cellular activities to m

ake its mRN

As fromits (-) strand RN

As.b. Influenza RN

A transcription occurs in the nucleus, thus:1. RN

A Pol II required for viral transcriptionVirus RN

A transcription can be inhibited by the drugactinom

ycin DViral m

RNA requires a capped/m

ethylated primer synthesized

RNA Pol II

These products are provided by scavenging cellularm

RNAs

2. Cell also provides RNA m

odification machinery (i.e. splicing )

3. The virus provides replicase proteins (PB1, PB2, PA and NP)

These polypeptides must provide signal sequences for nuclear

localization4. M

odel for initiation of viral mRN

A synthesis (see diagram)

Transcription & Replication

Spring 2002313

a. A viral encoded endonuclease cleaves the donor mRN

A 10-13nucleotides from

its cap, leaving a 3'OH1. The endonuclease requires a cap and usually cleaves3' to a A w

hich can base pair with the 3' U

found at theend of each viral RN

Ab. The resulting oligonucleotide acts directly as a prim

er, thefirst base added is a G

(penultimate in each viral RN

A is a C)c. Elongation of the transcript is then m

ediated by the viraltranscriptase (PB1, PB2, PA)d Transcription term

inates 15-22 nt from the ends of their

template w

here oligo U"s are used as a tem

plate for synthesis ofa 3' term

inal poly A tail

V. V

iral RN

A genom

e replicationa. N

uclear extracts from infected cells can synthesize full-length (+)

viral RNA

in the absence of capped primers

i. Indicates that 5' cap structure regulates termination in viral m

RNA

s ii. N

P is required along with PB1, PB2, and PA

b. The full-length (+) serve as a template for production of full-length

progeny (-) RNA

c. All viral RN

As have identical 3' and 5' term

ini, so each RNA

segment is capable of being efficiently replicated.

d. Both viral mRN

As and (-) full-length progeny are quickly exported

to the cytoplasm, w

hereas (+) full-length progeny remain localized to

the nucleus.

Spring 2002314

Transcription &Replication

(cont’d)

Spring 2002315

Assem

bly

VI. Virion assembly and release

a. Complete virion assem

bly requires two assem

bly modules assem

bled indifferent subcellular locations:

1. The nucleocapsids are assembled in the nucleus

a. The RNA assem

bly sequences for initiating formation of

nucelocapsids are located in the termini of each full-length viral

RNA (but not in viral m

RNA w

hich lack the 5'-termini)

2. The envelope is assembled at the cell-surface m

embrane

b. The M1 protein m

ay serve to bind the nucleocapsid to the C-terminus

cytoplasmic dom

ains of HA and N

Ac. Rem

ember: M

2 may play a critical role in regulating G

olgi vesicle pH.

d. There must be som

e mechanism

(yet to be elucidated) to assure that eachvirion receives a full com

plement of nucleocapsids

e. The role of NA

1. Structure (see diagram):

a. 469 aa glycoprotein which form

s a tetrameric spike on the

virus envelope surfaceb. The catalytic site for neuram

inic acid esterase activity islocated on top of each head ( 4 sites per spike)

Spring 2002316

NA

2. Biological role of NA: To remove term

inal sialic acid from oligosaccharides on cell-surface proteins

and glycolipids.a.

Acts on the neighboring HA such that progeny influenza virus particles cannot aggregate, thuseach particle can at as a separate infectious unit

3. Other possible biological roles for NA include:

a. Separating virus particles from inhibitory m

ucopolysaccharides in the respiratory tractb. Facilitating m

ovement of attached virus to the cell surface by releasing the HA from

its receptor

Spring 2002317

Spring 2002318

Prevention & Control

a. Antivirals:1. D

rugs (amatadine, ribavirin);

new recent round of drugs target N

A (see box 19.6 p 700)

2. Interferon

b. Inactivated vaccines (inactivated with form

alin)1. 60-80 %

effective in most individuals

2. Swine Flu V

accine (1976)