October 26, 2012.rtf

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

October 26, 2012 Kuss et al Science paper - enteric microbiota - two strains of mice, PVR and PVR IFNAR - mixture of four antibiotics - different mechanisms, target different groups of bacteria - Figure 1 C - same effect regardless of whether treated with antibiotics or not - expected because intraperitoneal infection D - antibiotic-treated mice had higher titres in feces - dashed line is not expected - WHY? - antibiotic treated mice is delayed - for the antibiotic treated mice, took longer to pass the dye - gut movement is slower, hence the viral titre peak is delayed E - use light-sensitive virus - pre-treat virus with neutral red dye, which bind to RNA - expose dye to light, dye crosslinks, virus cannot replicate any more - percent replication = (light insensitive = progeny only)/(UNexposed = total titre) times 100% - DOUBLE CHECK THIS - antibiotic treated mice, little replication in feces F - surprising - untreated mice - almost same amount of replication as PVR transgenic, IFNAR mice - more susceptible to infection - when assayed virus replication, it's normal - interferon not a large impact on viral replication - but the virus replication only assessed to three days - but see part B, where it shows that after day five, six, etc. where mice start dying - perhaps only need interferon to prevent dissemination from gut to the CNS - in the gut, not as important to have interferon Figure 2. - antibiotic acting on virus directly? Or it is acting through depletion of microbiota? Part C - protection lost because have antibiotic RESISTENT bacteria in gut Part D - virus replication is higher than antibiotic-treated mice for mice given antibiotic resistent BACTERIA Figure 1 and 2 - total virus titre in feces not accurate representation of virus replication antibiotic depletion of gut negatively impact Reovirus - morphological differences in feces and intestines - so can assay whether infection present or not Figure 3 - Peyers patches - the reason for looking at the ileum - part D - the difference in white bars due to presence/absence of bacteria use T3SA strain because it is systemic - part E - inhibition of replication, NOT inhibition of spread of reovirus Figure 4 - microbiota affecting cells or poliovirus or both? - no increase in infectiousness of virus - thirty seven degrees = physiological temperature, and because using bacteria, do not want to induce any stress responses - forty-two degrees = degrade poliovirus - part D - both gramp ositive and negative bacteria enhance replication - dose-dependent - not the feces that is enhancing replication part E - bacterial component - LPS had affect according to part C - looked at other components also found in gut - all four are polysaccharides and have N-aceytlgluocsamine in their sturcture - but hyaluronic acid did not improve virus replication despite having the two requirements - suggesting that the two are requirements but not necessarily SUFFICIENT for replication Part F - increasing compound concentration - higher PFU formed - Part G - LPS binding assay with avidin column - - poliovirus infectivity is enhanced by presence of bacteria, by bacterial components, which are polysacchardies and contain N-acetylglucosamine - LPS is potent enahncer - modification to virion appears to enhance thermostability ______________________________________________________________________________ ____ TEST UPCOMING - will include questions from the paper that should have been but is not presented today ______________________________________________________________________________ ____ Influenza Papers for next week: - R W Chan et al PLoS One 5 2010 Many things can cause influenza-like illnesses - flu-like symptoms does not definitively indicate influenza infection Hemagluttination - hemaglutinin binds sialic acids in cells of the respiratory system - avian strain binds better to sialic acid with alpha two three linkages - human strains bind to alpha two six - in humans, alpha two three linkages are deeper in the lungs - postulated to be reason why H1N1 infects humans with difficulty - this binding is first step in infection Neuriminidase - cleaves sialic acids - to allow release of progeny virus and thus spread - cleaving non-productive reactions - - Tamiflu inhibits neuriminidase - blocks the active site of enzyme - designer inhibitor - so virus stays stuck to cell that produced it Enveloped cells - picks up cell's plasma membrane upon budding from infected cell Undergo antigenic shift - eight segments of the genome - reassortments of the genome - reassortment can give antigenic shift - 1918 flu - avian virus that adapted to humans - antigenic shift not from reassortment - antigenic shift depends on H and N proteins - ANTIGENic shift - after this process, virus cannot be recognized by antibodies that recognize the virus before the shift - hence, no pre-existing protection - hence, pandemic = worldwide epidemic - higher rate of influenza than expected in given year - does not necessarily translate to fatalities - Antigenic drift - Vaccine - contains the H and N subuni
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