Virology: Influenza Virus

Front 1

How is influenza transmitted?

Back 1

Aerosol droplets .: influenza is a Respiratory virus

Front 2

Why can we keep getting colds throughout our lives?

Back 2

Many different sources for infectious cold syndrome.
Rhinovirus, coronavirus, Parainfluenza virus, respiratory syncytial virus etc
Over 300 viruses cause upper respiratory syndrome (cold)

Front 3

Why is there no vaccin for a cold?

Back 3

Once we get infected by one virus, develop immunity vs that serotype but there are many, many others (the more antigens you put in a vaccine, the less antibodies are produced by your body)

Front 4

When can you get a cold?

Back 4

Anytime during the year

Front 5

Why are colds more frequently associated with winter?

Back 5

All these viruses transmitted by phlegm. In fall/winter, children at school and they act as vectors for the viruses

Front 6

How is Influenza virus different then the common cold viruses?

Back 6

-More serious
-36-48 hr period when you can't get out of bed
-High fever (101), headache, fatigue etc
-Only get better after day 7 when you have more antibodies
-Influenza is not an endemic disease, all the others are (endemic= around all the time in the population at relatively low virulence)

Front 7

What are the 3 major differences between Influenza and the common cold?

Back 7

1) Influenza is an epidemic disease (not around all the time). The only virus that still has a steady mortality rate
2) It can kill ppl
3) It is also a pandemic disease (world wide disease): this however does not say anything about the severity of the virus
=> Haven't had a pandemic since 1967 (Hong Kong flu). Had a case fatality rate (CFR)of 0.5%

Front 8

What does the flu do to your body?

Back 8

-Infects and kills cells of the upper respiratory tract
-Wipes out mucociliary defenses
-After 1 day: most mucous secreting cells are destroyed and you see flat (basal) cells
-1.5 days: disorganization of cilia cells
-3 days: most ciliated cells are dead .: no local defense mech that can trap and kill inhaled particles, a lot of non-specific defenses are breached, can get bacterial pneumonia
-5 days: buds of new cilia emerging from diff. basal cells
-10 days: get more globular cells and cilia is more pronounced
-14 days: everything goes back to normal

Front 9

What are the 2 surface glycoproteins?

Back 9

Hemagglutinin: H
Neuraminidase: N
-these 2 ptns are immunogens (what antibodies target)

Front 10

What are the hosts of Influenza A virus? How many of each family (H, N) does this virus have?

Back 10

All mammals and avian species
-16 H families
-11 N families
All are antigenically different (no relationship)

Front 11

What was the first influenza virus isolated? What was the efficacity of its vaccine?

Back 11

-1933 H1N1
- 1st year,vaccine 80% effective
-2nd year, 60% effetive
-3rd year, 40 % effective
=> Effectiveness of the vaccine decreases with time

Front 12

Why does the effectiveness of a vaccine decrease with time?

Back 12

-Drifting
-Slow change in the antigenic properties of the viruses cuz of pt mutations in the glycoptn
-.: new vaccines must be made every year

Front 13

What was the problem with the 1957 Asian Flu?

Back 13

-H2N2 strain
-Used a vaccine from the virus of the previous year, which had been an H1N1, .: 0% efficacy => lead to pandemic
-Antigenic shift

Front 14

What is antigenic shift?

Back 14

Genetic recombination event btw human and othe animal strains which have different H and N families

Front 15

What happened with the 1967 Hong Kong flu?

Back 15

-new strain: H3N2
-again led to a pandemic (like in 1957)

Front 16

When did H1N1 first make an appearance?

Back 16

-1918
-circulated until the 1933 strain was first isolated
-H1 from 1918 strain similar to H1 in pigs (swine flu)

Front 17

Which influenza virus caused a pandemic before the 1918 flu?

Back 17

1880: H3N2 virus was circulating
-disappeared in 1918 and then came back in 1967 (Hong Kong H3N2)

Front 18

What are 4 pandemics cuased by influenza?

Back 18

-1880 (H3N2)
-1918 (H1N1)
-1957 (H2N2)
-1967 (H3N2)

Front 19

What happened in 1976 that still occurs at present?

Back 19

-Two different influenza strains circulating at the same time (H3N2 and H1N1)
-There were 2 dif strains of the H1N1 going around at the same time also. This other H1N1 had an H1 only seen in swine, couldn't really dev'p a vaccine vs it caused neurological problems)

Front 20

How susceptible are H and N to antigenic shift by point mutation?

Back 20

~1% aa change/year
-The H1N1 from 1956 disappeared in 1957
-The H1N1 that hit in 1976 only had 1-2% aa dif from he H1N1 from 1956 (.: probly came from someone's lab)

Front 21

Why don't influenza viruses that infect a human from an animal spread to other humans?

Back 21

Inoculate effect: viruses are host adapted to replicate in its own species (unless you get a high enough inoculum in your respiratory tract)

Front 22

Which previous H1N1 is more related to the current H1N1?

Back 22

The H1N1 from the early 1950's (NOT the one from 1976)

Front 23

Structure of the infuenza virus.

Back 23

-ssRNA virus
-segmented (8 pieces)
=> segments act as mini CHR

Front 24

What do the 8 strands of RNA code for?

Back 24

-1 encodes HA
-1 encodes NA
-1 encodes NP (nucleocapsid)
-3 encode 1 of 3 dif Pol
-1 encodes M1 (major structural ptn 1) that underlies the plasma mb
-last segment encodes non-structural ptn that's expressed in infected cells, but not the viral particle

Front 25

Why is it hard for ssRNA viruses to undergo genetic recombination when it co-infects a cell?

Back 25

-If you nick a ssRNA genome, it will fall apart
-Influenza, however, readily undergoes genetic recombination cuz it has 8 dif segments.: easy for these segments to be sorted and get encapsulated into new particles (50% recombination)

Front 26

What does Influenza A Virus infect?

Back 26

-All known mammals
-All known avian species

Front 27

What are the true reservoir host of Influenza A?

Back 27

Waterfowl (aquatic birds)
-99.99% of influenza viruses infect water fowl

Front 28

What does the avian flu do in waterfowl?

Back 28

-Multiplies in CLOACA channel without causing disease
-causes birds to excrete large amounts of the virus into the water
-multiple strains coexist in individual birds .: high level of genetic recom happens
-there is little/no selective P vs the virus since the birds don't get any disease
-New H and N's emerge from this reservoir and infect humans

Front 29

What happened with the 1918 strain of influenza?

Back 29

-Worst outbreak of influenza in history
-caused lotof disease in young, healthy victims (mostly age 20-45)
-Many of the ppl who contracted the flu would die within 36 hours

Front 30

Symptoms of the 1918 flu?

Back 30

-Cyanosis: Blueness cuz can't get O2 into blood
-Hemoptysis:spitting up blood
-ARDS (adult respiratory distress syndrome): uncontrolled hemorrhage and edema

Front 31

What was the cause of mortality in fast progressing cases?

Back 31

ARDS-like disease, got a cytokine storm when immune cells secreted every cytokine possible

Front 32

What was the cause of mortality in slower progressing cases?

Back 32

-Mostly from secondary bacterial pneumonia (cilia is destroyed and bacteria can get further into the lungs)
-could have neural involvement, since there was a frequency of psychiatric disorders
=>Encephalitis letharica: brain gets infected, can't move, also causes Parkinson-like tremors
=> treated with L-dopa, worked for a bit, infected could move, but only temporarily, before effect of drug wore off

Front 33

How widespread was the 1918 flu?

Back 33

-Infected ` 60% of the world's population
-Killed 25 million in 6 months
-global mortality= 2.5-6 %

Front 34

How did the 2009 H1N1 originate?

Back 34

Three strains:
-Classic Swine H1N1
-Human H3N2
-Avian strain

Front 35

What progeny developed from the 3 original strains involved in the 2009 H1N1?

Back 35

2 Progeny:
-North American H3N2
-Swine H1N2 which combined with classic H1N1 to give Eurasian Swine Virus

Front 36

Which strains does the 2009 flu virus now have? (4 segments)

Back 36

-HA gene from classic H1N1
-NA from Eurasian Swine Virus
-Pol from Avian and Human viruses

Front 37

Why do most influenza pandemics start in China?

Back 37

-They eat a lot of pork and poultry
-live in close proximity to their livestock, so virus could easily have passed between human-avian-swine

Front 38

How did the 1956 (H2N2) and 1967 (H3N2) strains arise?

Back 38

-Recombination between human and avian strains
-> possible pig intermediate host

Front 39

How was the 1918 strain sequenced?

Back 39

-Taubenberger found RNA fragment in a soldier's lung and used PCR to amplify it
-Hultin went to Alaska and found a body preserved by permafrost. Got a lung tissu sample
-Whole sequence was obtained

Front 40

What did sequencing the 1918 strain show?

Back 40

-the 1918 strain was a pure AVIAN strain (no similar genetic human material)
-23 aa changes out of 5,672
-The sequence did NOT explain the virulenceof the strain
-decided to use reverse genetics on the seq to get a live flu virus

Front 41

How did the resurrected 1918 strain compare with recent human strains?

Back 41

-50X as many viral particles as released from human lung cells
-32% more body weight is lost by mice after 2 days of infection
-39,000X more viral particles inmouse lungs 4 days after infection
-all mice infected with the 1918 strain died within 6 days, as compared to none that were killed by recent human strains
-coordinate expression of the 1918 virus genes confers the high virulence pandemmic phenotype

Front 42

What 1996 influenza virus strain first detected in China?

Back 42

H5N1
-caused moderate # of deaths

Front 43

What happened in 1997 with a goose strain in China?

Back 43

-Goose virus got internal gene segment from a quail virus
A/Quail/Hk/G1/[H9N2] and NA from duck virus [H6N1]
-This virus became widespread and killed 1 in 3 infected ppl (but only severely
ill ppl went to get medical attention, so stats aren't perfect)
-In 1997, the H5N1 was eradicated by killing all the domestic poultry in Hong Kong (haven't seen this H5N1 again)
-btw 1997-2002: H5N1 reassortment continued to come from duck and goose reservoirs (all had same H5N1, but dif internal genes) .: avian H5N1 continued to evolve
2002: a single H5N1 killed most of the water fowl in Hong Kong (odd cuz waterfowl aren't usually killed by avian virus)
-2003: this H5N1 transferred to humans and killed 1 in 2
-> precursor of Z genotype virus that is still in the populaton
-to date, many birds have died

Front 44

What is different about the recent H5N1 virus strains?

Back 44

-Unprecedented ability to infect humans
-Ability to cause neurotopic disease and high mortality in waterfowl
-ability to cause death and be transmitted among felid (cat) species
-ability to cause neurotropic disease/death in rodents

Front 45

What is required to develop an influenza virus that is as pathogenic as the 1918 strain?

Back 45

-HA ptn that humans have little/no immunity against
-HA mutation 1:easy infection of non-respiratory tissues so virus can quickly spread outside the lung
-HA mutation 2: Highr affinity of oligosaccharide binding (to the human sialyl-6-lactose receptor then to the avian sialyl-3-lactose receptor)
-Pol ptns direct facile RNA synthesis in human cells
-Viral NS ptn has mutations that direct: high level induction of apoptosis, high evel inhibition of IFN production
-Induction of severe pro-inflammatory effects (cytokine storm)
-ability to infect humans easily
-ability to easily spread human-human

Front 46

How is the current H1N1 different from the current H3N2?

Back 46

Same, except current H1N1 has HA with absent human immunity
(But its not a very good pandemic, since it can't infect outside the lungs, NA doesn't induce high lvl of apoptosis, doesn't induce severe pro-inflammatory effects (cytokine storm) and doesn't cause severe disease in humans)

Front 47

What are some inhibitors of virus uncoating?

Back 47

Amantadine
Rimantidine
-good for the currentH3N2 and H1N1
-H5N1 strain can be resistant

Front 48

What are some neuraminidase inhibitors?

Back 48

Tamiflu: effective for prophylaxis, but emerging resistance in H5N1 and H1N1
Zanamivir: Mainly used for prophylaxis

Front 49

What economic benefits can be gained from a treatment only

Back 49

-Stockpiles of antiviral agents for 40% of the population
-Save 100s of lives and 100s of millions of $
-get a lot of stockpilling of the vaccine

Front 50

What kind of viruses is influenza?

Back 50

(-) sense RNA viruses
-this is opposite sense of mRNA
-x: Orthomyxoviruses (flu), Bunyaviruses
-only (-) strand ssRNA viruses can cause disease in humans (+ ssRNA can't)

Front 51

What are examples of non-segmented RNA genomes?

Back 51

(+) sense RNA viruses: Coronaviruses, Picornaviruses
(-) sense RNA viruses:
Paramyxovirus and Rhabdovirus

Front 52

What is the only family of dsRNA?

Back 52

Reovirus

Front 53

What is (+) ssRNA?

Back 53

-Genome can act as mRNA, which can be directly translated to make RNA-dep-RNA-Pol
-RNA viruses must encode RNA-dep-RNA-pol cuz mammalian cells don't have the enz for this
-these pol can transcribe the genome RNA into (-) strand RNA (replicative intermediate), which can be used as a templae to make new (+) strand genome

Front 54

What is (-) ssRNA?

Back 54

-Genome cannot act as mRNA
-must encapsidate an RNA-dep-RNA-pol into the virus particle itself, since it can't be immediately translated in the cell
-after infection, the 1st event that takes place is RNA TRANSCRIPTION (not translation, like with + ssRNA)
-1st step of (-) sense RNA virus replication isn't inhibited by inhib of ptn translation (but the 1st step of (+) sense rep is inhibited by this)

Front 55

What are the 2 phases of RNA transcription?

Back 55

-Early phase: leads to mRNAs
-Late phase: transcripton of (+) sense replicative intermediates that will ass't with the nucleocapsid ptns
-The RNAs as well as the 3 Pol ptns will be encapsidate

Front 56

What do the 3 largest segments on the viral RNA code for?

Back 56

The 3 polymerases
-P1 or PA (pol acidic ptn)
-P2 or PB1 (pol basic ptn 1)
-P3 or PB2 (pol basic ptn 2)

Front 57

What are RNA segments 4-8?

Back 57

4: Encodes HA
5: Encodes NP
6: Encodes NA
7: Encodes M1
8: Encodes NS1
The first 6 RNAs are monocistronic, while segments 7 and 8 are bi-cistronic (both encode 2 mRNAs that can be expressed through mRNA splicing in the nucleus)
-Splice seg 7: MS1 and MS2
-splice seg 8: NS1 and NS2

Front 58

What ptns do RNA viruses that don't go through an RNA intermediate encode?

Back 58

All the ptns req'd for ptn translation, RNA replication and production of new virus particles (don't care if the cell has no nucleus because they have everything they need)

Front 59

Where does RNA replication occur for influenza?

Back 59

In the NUCLEUS

Front 60

What is NS1? Where is it in the cell?

Back 60

-An immunomodulatory ptn, since it has the effect to inc apoptosis and dec the induction and synthesis of IFN
-Goes to the nucleus, since it has a nuclear localization signal

Front 61

What is MS2?
Where is MS1?

Back 61

MS2
Transmb ptn
Functions as an ion channel protein
MS1: present in the cell mb

Front 62

What is HA?

Back 62

Receptor binding ptn that causes RBC to clump when it binds multiple RBC (since virus particle has multiple HA molec on its surface)
-Has mb fusion activity that allows uncoating of the virus

Front 63

What is NA?

Back 63

-Enz that digest neuraminic acid type molec
-receptors for the virus are neuraminic acid-type residues on glycoptns
-viruses budding from infected cells almost immediately bind and clump tog cuz of strong HA activity
-.: it's NA's job to release and disperse newly budding viruses by cleaving receptors (keeps virus infective)

Front 64

How does influenza enter the cell?

Back 64

Endocytosis
-HA binds (at pH=7.4, virus is just stuck to the mb)
-virus is endocytosed into a vesicle (HA still attached to the mb)
-Endosome fuses with lysosome
-pH drops to 4, get conform change of HA so that it can start its mb fusion activity
-virus particle fuses w/ mb of endosomal vesicle and the RNA segments are released into the cell's cytoplasm

Front 65

What is the budding process?

Back 65

-HA and NA are inserted into the mb of the cell (they have cytoplasmic tail that attracts viral M1 ptn)
-M1 attracts encapsidated RNA NP segments to the fusion area (uses ionic attraction)
*prob: ionic interactions btw M1 and NP so strong, NP can't diss't from M1, .: need M2
-M2 ion channel pumps H+ into the virus particle when its in the endocytotic vesicle, acidifying it, which diss't NP and uncoats virus particle

Front 66

What do neuraminidase blockers do?

Back 66

Block virus release from infected cells (ex: Tamiflu, Zanamivir)

Front 67

Where do immunoglobulins bind to the hemagglutinin?

Back 67

Bind globular domains that are close to the receptor binding pocket, but these adjacent domains to the receptor-binding pocket are the most antigenically variant portions of the molec (go through antigenic drift rapidly)
-Receptor binding domain has structural fct, is a conserved region that is immunologically protected

Front 68

What does Tamiflu (Oceltamivir) do?

Back 68

-Analog of neuraminic acid
-binds neuramindase with high affinity and is not degraded
-when it binds to the active site, poisons neuraminidase, which can no longer fct

Front 69

What does the endosomal H+ pump do?

Back 69

-drops the pH of the endosome by an influx of H+ ions
-M2 H+ pump than pumps these H+ into the interior of the virus particles
-this acidification diss't the nucleocapsids from the M1 ptns. allowing for the infectivity of the virus by the entry into the cytosol

Front 70

How were different genera of influenza differentiated?

Back 70

By the varying antigenicity of their nucleocapsid (NP) and matrix (M1) proteins

Front 71

How are subtypes of each influenza virus distinguished?

Back 71

Variation of the structure of their 2 surface glycoproteins

Front 72

What do the Thogotovirus (4th genus of Orthomyxoviridae) infect/

Back 72

Ticks (tick-borne virus)

Front 73

Describe the virion of influenza.

Back 73

-Enveloped particles
-Diameter: 80-120 nm
-13.5 kb (13,500 nucleotides)
-Sperical/ovoid shape

Front 74

How is the genome segmented in:
1) Influenza A
2) Influenza B
3) Influenza C
4) Thogotovirus

Back 74

1) A: 8 segments
2) B: 8 segments
3) C: 6 segments
4) Thogot: 7 segments

Front 75

How does the genome RNA segment exisr when its in a virion?

Back 75

Exists as part of an RNP complex (Also contains, NA, PA, PB1, PB2)

Front 76

Why is influenza constantly able to cause death, despite the availability of vaccines?

Back 76

Its ability to vary its antigenicity slowly and continuosly, also dramatically and suddenly

Front 77

When do epidemics of influenza occur?

Back 77

Winter, usually btw December and March for us

Front 78

When were the 3 worst epidemics?

Back 78

Spanish flu: 1918-1920 (H1N1)
Asian Flu: 1956 (H2N2)
Hong Kong Flu: 1967 (H3N2)

Front 79

What are the steps of pathogenicity of the virus?

Back 79

1) Virus infects epithelial cells of the upper respiratory tract
2) Desquamation of the ciliated epithelium and loss of protective musco-ciliary escalator
3) Virus replicatin causes induction of IFN, cytokines and soluble response factors
-> Flu symptoms, headache, myalgias (fatigue), malaise...
4)secondary bacterial pneumonia as a result of desquamation of ciliated epithelium

Front 80

Which viral protein is responsible for mb fusion with the cell?

Back 80

Hemaglutinnin (HA)

Front 81

How does HA0 become fusion competent? What is the function of HA1? HA2?

Back 81

-HA0 is proteolitically cleaved into 2 subunits
-HA1: surface subunit, responsible for recognizing and binding to the sialic acid receptors on the host
-HA2: transmembrane subunit, contains fusion peptide region at its amino terminus and remains anchored to the viral ptn

Front 82

How do the viral particles fuse?

Back 82

-HA1 binds receptor (sialic acid)
-Enters cell by endocytosis
--pH of endosome drops, HA-> conformational change, exposes fusion active peptide and allows juxtaposition of vral and host cell membranes
-Fusion btw endosomal mbs and entry of viral RNPs into the cell cytoplasm

Front 83

What are the domains of the M2?

Back 83

-N-term extracellular domain
-Transmb domain (only 1)
-Large cytoplasmic domain

Front 84

What is M2 a component of? Why is it so important?

Back 84

-M2 is component of the viral envelope
-Imp cuz of its ability to form a highly selective, pH-regulated H+ conducting transmb ion-channel
-M2 H+ channel facilitates viral uncoating (in the endosome)
-Allows H+ to enter inside of virion where it can disrupt interaction between M1 and RNP core of virus
-Also modulat pH of trans-Golgi network

Front 85

What is amantadine? What does it do?

Back 85

-Amantadine is an anti-influenza drug
-It block the M2 H+ channel (viral uncoating is .: incomplete and RNP can't initiate transcription)

Front 86

What is the most abundant protein in the virus infected cells?

Back 86

-NS1

Front 87

What are the two functional domains of NS1?

Back 87

-Amino-terminal RNA-binding domain: essential for dimerization of the protein
-Effector domain (in C-terminal): Mediates inhibition of poly(A)-containing RNAs
->Interacts with CPSF (cleavage and polyadenylation specific factor) and PABII (poly(A)-binding ptn II)

Front 88

What happenes when NS1 binds CPSF?

Back 88

Inhibits cleavage of 3' cleavage and polyadenylations of CELLUAR pre-mRNAs .: prevents nuclear export, .: pre-mRNAs are degraded
-**viral mRNAs are synthesized/polyadenylated by virus transcriptase, .: it is unaffected

Front 89

What happens when NS1 binds to U6 snRNA?

Back 89

-Inhibits pre-mRNA splicing
(not much known about this

Front 90

Why does NS1 bind ds RNA?

Back 90

-inhibits host cell IFN response by inhibiting IFN-specific paths
-without this, mutant viruses can only replicate in cells where interferon response is deficient

Front 91

What does NA do?

Back 91

-catalyzes cleavage of sialic acid (receptor of HA) from carb side gps of glycoptns
-->this prevents aggregation of budding virions at ell surface
-->this may help virus to penetrate the surface mucin layer of resp. epithelium so that the virus can bind to the epithelium cells

Front 92

What are the seq/domains of the glycoptn HA?

Back 92

-amino-terminal signal seq
-amino-terminal mb anchor domain
(these are cleaved by a signal peptidase)

Front 93

What is the domain of glycoptn NA?

Back 93

-combine amino-terminal signal/anchor domain

Front 94

How is M2 different from NA and HA?

Back 94

-M2 is not glycosylated or cleaved
-anchored to the mb through its hydrphobic domain in its core
-forms an ion channel in mbs

Front 95

What arethe functions of M2 late in infection?

Back 95

-regualte intracompartmental pH in trans-Golgi network (TGN)
-keep pH in TGN above HA fusion conf. threshold (don't want HA-mediated fusion at this point)

Front 96

How do virus particles exit the cell?

Back 96

-precursor to budding virus particle= region of outer mb containing viral envelope ptns
-RNP-M1-NS2 complex ass't with the env ptn cytoplasmic tails
-Don't know exact mechanisms on influenza's budding process or how its genome RNA segments are packaged into equal amount of virions

Front 97

What is antigenic drift? Antigenic shift?

Back 97

-Drift:Slow but continuous change of antigenic domains of the virus surface glycoptns (continuous point mutations in the surface glycoprotein genes)
-Shift: recombination between dif human and animal strains of influenza. Occurs suddenly and episodically

Front 98

How are vaccines made for this virus?

Back 98

-Reflect the influenza A and B strains of the previous year
-Inactivated vaccines so that they cannot cause disease (roduced in hen eggs)

Front 99

What do amantidine and rimantadine do?

Back 99

-Originally anti-parkinsons drug abut can be used against Influenza A (NOT B) to bind M2 channel and block passage of H+ ions

Front 100

What are Zanamivir and oseltamivir?

Back 100

-synthetic NA inhibitor for influenza A and B

Front 101

Why are NA inhibitors better then M2 channel inhibitors?

Back 101

1) Works against influenza B also
2)Rare dev'l of resistance
3) Fewer side effects