November 22, 2012 class notes.docx

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Molecular Genetics and Microbiology
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Richard Brown

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November 22, 2012 – class notes Herst et al. paper in Science - What mutations needed for efficient airborne transmission? - Is genetic shift required? Or is genetic drift sufficient? - A/Indonesia/5/2005 - Animal model – ferret o Mammals tend to have lower body temperatures than birds o But single known mutation that changes this o Ferrets and humans – similar distribution of sialic acid receptors - Inoculated ferret set next to naïve ferret o No fomite transfer o Air flow - Starting virus o Two known mutations that allow to bind to human sialic acid linkages o N182K – also changes viral sepcficity to 2-6 linkages - Initial experiment - Viral shedding figure o Wt virus has highest titre, then double mutation (part C) o Tissues similar for all viruses - PB2 mutation – to allow virus to replicate at human temperature o No higher shedding o No air transmission o Naïve ferrets – no virus given ever o Not enough to cause airborne transmission - Serial passage through ferrets experiment o 10 serial passages since flu mutates quickly o Result in two mutant forms of virus - Figure of virus titers in P1 to P10 o Mutant titres showed improved ability to replicate o Wildtype seems unaffected - Sequencing after the tenth passage o Two mutations in HA, one or other, found in all of samples – serve to remove N-linked glycosylation site in HA – may help in binding to mammalian host receptor o Another mutation in hemagglutinin – forms part of HA trimer interface - Figure 5 o Replicating virus in A o In B, no detectable virus in recipient ferrets infected with the 10 passage WT virus o Replicating virus detected in ¾ recipients ferrets paired to H5N1 passage infected donors o The heterogenous virus population evolved fro the modified strain through 10 passage while the population from wt virus does not - Figure 6 o Three naïve ferrets infected in previous experiment – inoculate two more donor ferrets, which can infect more ferrets o Compare airborne transmission of experimentally passaged A/H5N1 and 2009 pandemic A/H1N1 viruses in individual ferrets  The H1N1 known to be highly efficient at airborne transmission o H5N1 transmission less robust - Supplementary figure 1 – experiment 5 o To test lethality of wild-type o All ferrets infected with any airborne virus able to clear the infection  Lethal if intratrachael infection with high dose –lethal but otherwise, can clear - Supplementary figure 10 - Experiment 6 o Antiviral drug resistance  Tamiflu  Airborne transmitted virus similar to wildtype in Tamiflu resistence - Experiment 7, 8 o Recognition of airborne transmissible virus - Supplementary table 6 o No antibody from any volunteer able to bind to H5N1 - Table 2 o Evaluable impact of HA mutations that emerged during passaging in ferrets in a modified turkey red blood cell assay o Q222L, G224S – changed receptor binding preference of HA – increased affinity of alpha 23 and 2-6 - CONCLUSION o H5N1 with alpha two-six SA affinity and the ability to replicate in URT was able to become airborne transmissible through repeated passages in a mammalian host o All ferrets infected via air did clear the infection F5 ferret five’s virus – had all five substitutions and had least number of other substitutions Reassortment – H1N1 2009 (triple reassortment); H3N2 (segments came in from avian population) Pandemics have arisen due to reassortment Pandemic-causing influenza: must be efficiently transmissible from person to person; and have population where most or all does not have antibodies - All are potential hosts – not immune H5N1 – satisfies the criterion that most of population lacks antibodies Transmission experiments – key studies looking at H5N1 transmissible – mixing and matching segments from human influenza virus and putting in H5N1 – get into making targeted mutations, targeted amino acid substitutions – but regardless of what they did, nothing transmitted efficiently Kawaoka group paper - Focus on receptor-binding part of HA – random mutagenesis by PCR – - Started with Vietnam 1203 isolate – took out bit that encodes amino acids that make up r
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