November 15, 2012 - class notes.docx

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University of Toronto St. George
Molecular Genetics and Microbiology
Richard Brown

November 15, 2012 H5N1 Virulence determinants – c-terminal – Zielecki et al paper - Polybasic cleavage site in HA – cleavage by furin-like proteases - NS1 antiviral effects - Polymorphisms o All three above contribute to high virulence of H5N1 - PDZ domain – interact with Ns1 o May disturb function of various cellular protiens – thus disease = hypothesis - Purpose- understand role of N-terminal of NS1 - VN/1203 – isolated from human fatal case - Use reverse genetics o Produce recombinant Vn/1203 virus o NS1 Cterminal either avian ESEV or human RSKV o Three variants: truncated, RSEV, ESEV - Conclusion o PDZ domain ligand sequence (ESEV or RSKV) in NS1 contributes little to the virulence of H5N1 viruses in mice and chickens Figure 1 - A – three viruses with different o RSKV – changed NS2 protein at position 70 – changed amino acid at this position Figure 2 - Presence of PL motif reduces capacity of NS1 protein to support viral replication Figure 3 - Intracellular location of NS1 protein in transfected and infected cell cultures - Little NS1 in nucleus at all time points – RSKV in A549 - More nuclear ESEV - CEF and DEF o WT – little in nucleus; mainly cytosolic o ESEV – nuclear accumulation of NS1 is controlled in avian cells - PL motif modulates intracellular localization of Figure 4 - NS1 affects interferon response – wanted to look at whether the mutants can still interfere with interferon - Renilla luciferase – o NS1 from all three viruses – still inhibit interferon induction - B – western blot confirming proteins - C+D o H3N2 is seasonal strain – little interferon response o All three H5N1 strains give higher response – but slightly lower ESEV o In D, ESEV also doesn’t grow as well  H5N1 results in higher interferon response than H3N2 Figure 5 - Infecting mice - Fifty percent mean lethal dose of the recombinant WT virus is less than 5PFU/mouse Figure 6 - Plaque assays of virus from different organs from different time points - No significant differences - Order of events: lungs: spleen, heart; kidney, liver, brain Figure 7 - Histopathological o Equally severe for all three H5N1 strains (WT and ESEV and RSKV) C terminal NS1 amino acids cannot be considered a major virulence determinant of HPAIV-H5N1 in mice Conflicts with Jackson et al – but used mouse-adapted Figure 8 - In chickens - Dead after day 3 post-infection – similar to Figure 8A o Indistinguishable high virulence for all three viruses - Intranasal – - NS1 PL motif contributes to viral fitness between limit of detection - Or avian influenza viruses propagate in low pathotype form in feral birds, CONCLUSION - No major impact – PL motif at C terminus of NS1 protein has no major impact Injecting egg - Use egg punch to make small hole, insert needle, seal hole with melted paraffin wax; incubate; take off paraffin wax, insert needle to suck up the fluid o Keep sterility - Amniotic sac immediately surrounding embryo OR inject into allantoic sac which surrounds the amniotic sac FOR NEXT WEEK Herfst et al 336 Science 2012 M Imai et al Nature 486 2012 CA Russell et al Science 336 2012 H5N1 – high mortality; high virulence not yet determined why – NS1, polymerase, hemagglutinin important - Virulence is MULTIFACTORIAL Why is transmission from person to person inefficient? 1997 – H5N1 first a problem in people - Isolated – 18 people fell ill, two third died => 60% - Hong Kong 2003 – virus resurfaced - Egypt, Thailand – still an issue 2006 – first publication on transmission - Due to the sialic acid with different linkages – alpha two three (avian), alpha two six (human) - To look at presence of receptor, Lectins that bind to sialic acid of one or the other linkage o Lectin in red binds to sialic acid with alpha two three linkage; lectin in green that binds to alpha two six o Human tissue o Taken tissue from various parts of respiratory tract o Alpha two six all the way through respiratory tract – from upper to lower respiratory tract o But alpha two three linkages only found in lower respiratory tract - Virus binding o Extensive binding of H5N1 from human binding to bronchus and alveolus - Viral replication in alveoli (deep in lower respiratory tract) only for a H5N1 isolate from duck; but isolated from human, replication in bronchus and alveoli - Explai
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