Virology lecture 4

Question 1

Basica influenza virus structure

Answer 1

1 - -ve ssRNA, 8 segments
2 - nucleocapsid has helical symmetry. envelope with 2 prominant spikes. one is haemagglutinin (HA) the other is neuraminidase (NA) = glycoproteins
3 - 3rd viral protein in envelope = M2. an ion channel.
4 - M1 protein under envelope
5 - NP (nucleoprotein) associated with RNA genome within the nucleocapsid
6 - PB1, PB2 and PA polypeptides also in virion core which collectively make the RNA dependent RNA polymerase for -ve to +ve ssRNA.
7 - variable but spherical shape in culture. in fresh clinical isolates however it can be filamentous.
8 - an exception to the -ve ssRNA rule in that it replicates in the nucleus.

Question 2

Influenza classification?

Answer 2

1 - orthomyxovirus family
2 - 3 types (A,B and C). A = most disease in man.
3 - 2 methods of classification - type, country of isolation, isolate number, year. ie A/PR/8/34 = type A, Puerto rico, isolate 8, 1934.
4 - second classification mechanism - spcific HA and NA proteins, H1N1 = H1 HA and N1 NA

Question 3

the 8 influenza RNA segments, the protein encoded and its function

Answer 3

1 - PB2 - RNA pol
2 - PB1 - RNA pol
3 - PA - RNA pol
4 - HA - binds sialic acid
5 - NP (nucleoprotein) - encapsidates the RNA genome
6 - NA - surface glycoprotein - cleaves sialic acid fro infected cell surface and virions.
7 - M1 and M2 (matrix proteins) - M1 interacts with envelpoe and nucleocapsid, M2 is an ion channel
8 - NS1 and NS2 (non-structural) - NS1 = evasion of innate immunity

Question 4

how many types of HA and NA in influenza A? natural reservoirs?

Answer 4

1 - 17 HA types (1-17) and 9 NA types (1-9)
2 - birds are the natural reservoirs for most subtypes (all bar H17 but a few can infect man (H1,2,3,5 and N1,2,8) , pigs (H1 and 3, N1 and 2), horses (H1 and 7, N7 and 8), specific fruit bats (H17) and seals (H4 and 7, N7).

Question 5

influenza patterns of transmission and death

Answer 5

1 - highly seasonal, epidemics during winter due to close proximity as an aerosol with an envelope.
2 - even in an ordinary year it causes 10-15,000 deaths in the UK

Question 6

influenza entry pattern

Answer 6

HA binds sialic acid, endocytosis, acidification of endosome, confromational change of HA, membrane fusion, nucleocapsid released into the cytosol then transported to nucleus for replication.

Question 7

HA structure

Answer 7

1 - trimer. each monomer include a HA1 and a HA2 - cleaved from HA0.
2 - HA1 is the globular head with sialic acid binding site
3 - HA2 is the stalk connecting the head to the envelope

Question 8

mechanism of influenza membrane fusion during entry. drugs?

Answer 8

1 - N terminus of HA2 has a hydrophobic peptide called the fusion peptide. at neutrla pH its buried on acidification HA1 moves to expose it. it inserts in to the endosome membrane, destabilises it and promotes fusion. hence fusion is pH dependent.
- amantadine and rimantidine raise the pH to inhibit entry.

Question 9

define epidemic

Answer 9

επί (epi), meaning “upon or above” and δήμος (demos), meaning “people”) occurs when new cases of a certain disease, in a given human population, and during a given period, substantially exceed what is expected based on recent experience.

Question 10

define pandemic

Answer 10

pandemic comes from the Greek pandemos meaning “pertaining to all people”. The Greek word pan means “all” and the Greek word demos means “people”. Denoting a disease affecting or attacking the population of an extensive region, country, continent, global; extensively epidemic

Question 11

which influenza mRNAs produce more than one protein? how? can they be used to replicate the genome?

Answer 11

1 -segment 7 for M1 and M2, segment 8 for NS1 and NS2. alternative splicing.
viral mRNA from RNA-dep RNA pol are not complete copies so cant make up the genome. viral RNA pol makes +ve from the -ve then -ve from that - go to templates for more mRNA or packaged

Question 12

how does new influenza escape its host cell? drugs?

Answer 12

Neuraminidase cleaves sialic residues from proteins to stop it getting stuck to the outside of the cell. it also removes sialic acid residues from HA and NA on virions to prevent their aggregation.
tamiflue and relenza inhibit NA.

Question 13

how does influenza cause repeated infection?

Answer 13

antigenic drift and shift. also new viruses from animal reservoirs ie H5N1 bird flu and H1N1 swine flu.

Question 14

antigenic drift in influenza?

Answer 14

1 - neutralising antibodies bind HA, causes natural selection for aa mutations to evades these. this gradual accumilation of changes = drift.
H3 found in man in 1968 is very different to the H3 strain circulated in 2003. huge numbers of aa changes.

Question 15

antigenic shift in influenza?

Answer 15

1 - acquisition of a completely new HA from another influenza virus.
2 - more than one influenza type infects a cell, reassortment during packaging of new virions produces a new virus. often this is of no consequence, one combination is dangerous - if 7 genes are fro the huma virus it’ll replicate well, add the avian gene for HA and it’ll evade any existing immunity. this causes pandemics.

Question 16

date of influenza pandemics and genotype.

Answer 16

1 - 1918 - H1N1 - devastating pandemic w/ 40-50 million dead.
2 - 1957 - H2N2
3 - 1968 - H3N2 - HA changes more rapidly as its critical to reinfection, the NA antibody is less important.
4 - 1977 - H1N1 - reemergence from a 1930s circulation
5 - 2009 - H1N1 - derived from mexican swine. low 0.03% mortality rate vs the 2.5% of the 1918 strain

Question 17

why is the H5N1 influenza virus of concern?

Answer 17

1 - highly virulent, severe epidemics in birds, spread to other species inc man. 60% mortality in man. several hundered deaths but no evidence of human to human transmission yet.
2- factors influencing its adaptation to man-
a - better binding
b - better replication in cells
c - better escape from innate immune system
d - better transmission between humans

Question 18

difference between human influenza and avian in HA binding to sialic acid?

Answer 18

1 - human preferentially binds sialic acid with a alpha2’-6’ link to the terminal galactose, avian to a alpha2’-6’ link. one amino acid mutation in the HA structure can change the preference allowing adaptation!

Question 19

what influences the ability of influenza to replicate in human cells?

Answer 19

the aa in the PB2 subunit of the RNA pol. is the aa E627 is present it replicates well in avian but not human, change to K627 and this switches.

Question 20

how does the influenza virus resist host immunity?

Answer 20

NS1 protein confers resistance to interferon mediated inhibition. avian influenza version works well in birds but less efficient in humans but it could adapt to increase its virulence in humans.

Question 21

HIV first investigated and why? year identified? close relative?
no infected in 1990 and 1996

Answer 21

1 - 1981 - CDC spotted unusual cluster of opportunistic infections - pneumonitis carinii, disseminated HCMV, mucosal candida and chronic HSV.
2 - retrovirus identified in paris in 1983
3 - 1990 - 307,000 AIDS cases, 9 million HIV.
4 - 1996 - 28million infected, 5.8million AIDS cases and over 75% of these were dead.

Question 22

outcome of HIV?

death to infection ratio in 2010?

Answer 22

1 - DEATH

2 - for every 2 death, 3 more were infected so the pandemic is spreading. 7000 new infections a day, 42% in 15-24yo.

Question 23

HIV family and general structure

Answer 23

1 - retrovirus, lentivirus subfamily. more complex genome than standard retrovirus so slow infection.
2 - +ve ssRNA, 5’ can 3’ poly A tail. but doesnt act as mRNA.
3 - diploid genome (2 sets)
4 - genome contains a tRNA to prime reverse transcription. dsDNA - provirus.

Question 24

HIV capsid and envelope structure

Answer 24

1 - cone like capsid, composed of gag24 protein.
2 - lipid envelope with gp120 and gp41 proteins from gp160 precursor. gp120 is eavily glycosylated to provide a glycan shield to make it harder for antibodies to target. quite variable env proteins.
3 - p24 gag more conserved than env protein so target of antigen in diagnostic kits.

Question 25

how does the HIV provirus genome differ from the RNA genome?

Answer 25

1 - has LTR (long terminal repeats) at either end. the 5’ one contains the promoter to recruit host DNA dep RNA pol 2.

Question 26

what are the standard genes encoded by all retroviruses? which are distinct to HIV?

Answer 26

1 - gag, pol and env.
2 - early expression genes for regulation and immune evasion , heavily spliced mRNA. tat and rev proteins are regulatory and are needed to switch to capsid and envelope proteins late in infection. Vpu has several functions inc down regulating CD4 and inhibiting NF-kB pathway to block innate immunity.

Question 27

binding receptors for HIV? human resistance?

Answer 27

1 - gp120 to CD4 on T helper cells and macrophages/DCs. co-receptor = CCR5 or CXCR4 = hydrophobic chemokine TMRs.
2 - CCR5 = predom in early infection and = predominant transmission. CXCR4 increasingly so as immunodeficiency developes.
3 - CCR5 polymorphism truncates it causes non-function. present in caucasians, 16% heteozygotes, 1% homo. reduces HIV prevalence in this pop as it’s the predom for HIV transmission. hetero = 16% of caucasians but only 10% of HIV infected hence resistance. homozygotes can still be infected.

Question 28

HIV gene expression?

Answer 28

1 - promoter present in (unique 3 region - U3) left LTR recruits host RNA dep RNA pol 2 .
2 - early - mRNA spliced so small reg proteins predom. as their conc rises the focus shifts to larger structural proteins.
3 - gag expressed like eukaryotic proteins - ribosome binds 5’ cap and scans to the first AUG codon. (mature capsid proteins cleaved from it).
4 - pol translated by ribosomal frameshifting. as it reaches the end of gag a secondary RNA structure causes a slip of one nucleotide (in 1 in 20 instances) to a different reading frame. gag-pol protei produced and later cleaved. provides quantative control of gag vs pol as more gag is needed as its structural not an enzyme.
5 - env - gag and pol mRNA regions spliced out to put the env open reading frame near the 5’ cap. gp160 product cleaved to gp120 and gp41 envelope proteins.

Question 29

how is HIV released?

Answer 29

1 - buds through plasma membrane where the env proteins are located. doesnt kill the cell rapidly so it is prolonged. can evade CD8 CTL or innate etc by going latent, this is why its so hard to get rid of.

Question 30

describe HIV pathogenesis

Answer 30

1 - acute phase - approx 2 months - burst of replication, HIV specific CTL response to clear infected CD4 cells, causing a dramatic drop in cell count. AB removes free viruses, viral load drops off and cell count recovers.
2 - asymptomatic phase - up to 15yrs, low levels of replication controlled by CD8
3 - AIDS - 1 to 3 years, virus escapes immune containment, increasing replication and CD4 destruction. CD4 count drops so less help for CD8 so that drops, positive feedback and the immune system breaks down to immunodeficiency.
4 - in most cases an opportunistic infection kills the patient.
5 - these patients are classed as slow progressors.
6 - other potential outcomes are long term non-progressors and rapid progressors

Question 31

how do long term HIV non progressors and rapid progressors work?

Answer 31

1 - LTNP - retain high CD8 and CD4, low viral load and low infected cell count.
2 - RP - poor prognosis, only a weak CD8 to the initial HIV burst so a rapid progression to immunodeficiency within 1-5 years.

Question 32

why no HIV vaccine?

Answer 32

1 - reverse transcriptase is very error prone so there is rapid antigenic variation. neutralising AB for gp120 but all failed so far. an estimated 10^6 different HIV mutants in one patient in one day… CD8 CTL based vaccines also tried but still antigenic variationof peptides on MHC1 allow escape.

Question 33

anti-HIV drugs?

Answer 33

1 - over 25 approved by the US FDA.
2 - first, 1987, AZT (also called zidovudine and retrovir), a nucleoside analogue, phosphorylated to dNTP and acts as a substrate for reverse transcriptase but functions as a chain terminator to block further addition. better substrate for reverse transcriptase than cell DNA pol hence specific.
- 3 other chain terminating nucleoside analogues - dideoxycytidine, dideoxyinosine, and 3TC (lamivudine). some toxicity.
3 - portease inhibitors - target HIV asp protease that cleaves gag to its mature form.
4 - fusion inhibitors - enfurvitide
5 - integrase inhibitors

Question 34

HIV drug resistance issue?

Answer 34

1 - high mutability allows mutation to give resistance to a drug, change drug and repeate.
2 - HAART now used in developed countries - Highly active anti-retroviral theraphy, multiple drugs at once so resistance less likely. v successful but v expensive so poor provision in developing countries where HIV is most prevalent. this is improving however.
- currently no vaccine and no cure so prevention through education is important.