Совершенствование инновационной деятельности предприятия (на примере ОАО «СтанкоГомель»)

Background Wild waterfowl is the natural reservoir of influenza A virus (IAV); hosted viruses are very variable and provide a source for genetic segments which can reassort with poultry or mammalian adapted IAVs to generate novel species crossing viruses. Additionally, wild waterfowl act as a reservoir for highly pathogenic IAVs. Exposure of wild birds to the antiviral drug oseltamivir may occur in the environment as its active metabolite can be released from sewage treatment plants to river water. Resistance to oseltamivir, or to other neuraminidase inhibitors (NAIs), in IAVs of wild waterfowl has not been extensively studied. Aim and Methods In a previous in vivo Mallard experiment, an influenza A(H6N2) virus developed oseltamivir resistance by the R292K substitution in the neuraminidase (NA), when the birds were exposed to oseltamivir. In this study we tested if the resistance could be maintained in Mallards without drug exposure. Three variants of resistant H6N2/R292K virus were each propagated during 17 days in five successive pairs of naive Mallards, while oseltamivir exposure was decreased and removed. Daily fecal samples were analyzed for viral presence, genotype and phenotype. Results and Conclusion Within three days without drug exposure no resistant viruses could be detected by NA sequencing, which was confirmed by functional NAI sensitivity testing. We conclude that this resistant N2 virus could not compete in fitness with wild type subpopulations without oseltamivir drug pressure, and thus has no potential to circulate among wild birds. The results of this study contrast to previous observations of drug induced resistance in an avian H1N1 virus, which was maintained also without drug exposure in Mallards. Experimental observations on persistence of NAI resistance in avian IAVs resemble NAI resistance seen in human IAVs, in which resistant N2 subtypes do not circulate, while N1 subtypes with permissive mutations can circulate without drug pressure. We speculate that the phylogenetic group N1 NAs may easier compensate for NAI resistance than group N2 NAs, though further studies are needed to confirm such conclusions

Тепловой расходомер

Полезная модель относится к области измерительной техники, а именно к тепловым устройствам для измерения расхода жидких или газообразных сред, и может быть использована в химической, электронной, энергетической и других отраслях промышленности

Influenza A(H7N9) Virus Acquires Resistance-Related Neuraminidase I222T Substitution When Infected Mallards Are Exposed to Low Levels of Oseltamivir in Water

Influenza A virus (IAV) has its natural reservoir in wild waterfowl, and new human IAVs often contain gene segments originating from avian IAVs. Treatment options for severe human influenza are principally restricted to neuraminidase inhibitors (NAIs), among which oseltamivir is stockpiled in preparedness for influenza pandemics. There is evolutionary pressure in the environment for resistance development to oseltamivir in avian IAVs, as the active metabolite oseltamivir carboxylate (OC) passes largely undegraded through sewage treatment to river water where waterfowl reside. In an in vivo mallard (Anas platyrhynchos) model, we tested if low-pathogenic avian influenza A(H7N9) virus might become resistant if the host was exposed to low levels of OC. Ducks were experimentally infected, and OC was added to their water, after which infection and transmission were maintained by successive introductions of uninfected birds. Daily fecal samples were tested for IAV excretion, genotype, and phenotype. Following mallard exposure to 2.5 μg/liter OC, the resistance-related neuraminidase (NA) I222T substitution, was detected within 2 days during the first passage and was found in all viruses sequenced from subsequently introduced ducks. The substitution generated 8-fold and 2.4-fold increases in the 50% inhibitory concentration (IC50) for OC (P < 0.001) and zanamivir (P = 0.016), respectively. We conclude that OC exposure of IAV hosts, in the same concentration magnitude as found in the environment, may result in amino acid substitutions, leading to changed antiviral sensitivity in an IAV subtype that can be highly pathogenic to humans. Prudent use of oseltamivir and resistance surveillance of IAVs in wild birds are warranted

Oseltamivir Resistance in Influenza A(H6N2) Caused by an R292K Substitution in Neuraminidase Is Not Maintained in Mallards without Drug Pressure

Background Wild waterfowl is the natural reservoir of influenza A virus (IAV); hosted viruses are very variable and provide a source for genetic segments which can reassort with poultry or mammalian adapted IAVs to generate novel species crossing viruses. Additionally, wild waterfowl act as a reservoir for highly pathogenic IAVs. Exposure of wild birds to the antiviral drug oseltamivir may occur in the environment as its active metabolite can be released from sewage treatment plants to river water. Resistance to oseltamivir, or to other neuraminidase inhibitors (NAIs), in IAVs of wild waterfowl has not been extensively studied. Aim and Methods In a previous in vivo Mallard experiment, an influenza A(H6N2) virus developed oseltamivir resistance by the R292K substitution in the neuraminidase (NA), when the birds were exposed to oseltamivir. In this study we tested if the resistance could be maintained in Mallards without drug exposure. Three variants of resistant H6N2/R292K virus were each propagated during 17 days in five successive pairs of naive Mallards, while oseltamivir exposure was decreased and removed. Daily fecal samples were analyzed for viral presence, genotype and phenotype. Results and Conclusion Within three days without drug exposure no resistant viruses could be detected by NA sequencing, which was confirmed by functional NAI sensitivity testing. We conclude that this resistant N2 virus could not compete in fitness with wild type subpopulations without oseltamivir drug pressure, and thus has no potential to circulate among wild birds. The results of this study contrast to previous observations of drug induced resistance in an avian H1N1 virus, which was maintained also without drug exposure in Mallards. Experimental observations on persistence of NAI resistance in avian IAVs resemble NAI resistance seen in human IAVs, in which resistant N2 subtypes do not circulate, while N1 subtypes with permissive mutations can circulate without drug pressure. We speculate that the phylogenetic group N1 NAs may easier compensate for NAI resistance than group N2 NAs, though further studies are needed to confirm such conclusions

Viral NA inhibition by oseltamivir carboxylate and zanamivir.

<p>Mean concentrations of drugs ± standard deviations that inhibited 50% (IC₅₀) of the viral NA activity in a functional assay with the MUNANA substrate. 292R = arginine at NA residue 292. 292K = lysine at NA residue 292. Values were calculated on duplicates of six wild type (292R) and five mutated (292K) viral samples.</p><p>Viral NA inhibition by oseltamivir carboxylate and zanamivir.</p

Viral excretion.

<p>IAV was detected by RRT-PCR of the matrix gene from daily fecal samples. The Y-axis displays cycle threshold (CT) values as a quantitative measure of viral excretion. Samples with CT values ≥ 45 were considered negative. The X-axis displays which days of the experiment samples were collected from the Mallards. G1 = two birds in each of three experiments (n = 6), etc. G1 and G2 were OC exposed, G3 was OC exposed day 0–2, and G4 and G5 were unexposed. Value points display mean CT values of 6 samples and error bars display standard errors of the mean (SEM). No significant difference in excretion was detected between drug-exposed (G1 and G2 with resistant genotype NA-292K) and unexposed (G4 and G5 with wild type genotype NA-292R) birds day 1, 4 or 5, while drug-exposed birds had lower virus excretion levels day 2 and 3(<i>P</i> = 0.034).</p

Experimental design and NA sequence results of residue 292.

<p>The experiment was repeated three times with three H6N2/R292K resistant IAV variants, each consisting of five overlapping generations of Mallards, two birds in each.</p><p>*Oseltamivir carboxylate (OC) concentration in the experimental water with range of relative standard deviations (%RSD) for each of the three experiments at respective exposure level (n = 4 at 10 μg/L, n = 3 at 3 μg/L and n = 4 at 1 μg/L). The three experiments had the same OC concentration in the water. Dashed horizontal lines indicate change in OC concentrations.</p><p>G1 = generation one, G2 = generation two etc.</p><p>E1 = experiment with NA substitutions R292K + D113N. E2 = experiment with NA substitutions R292K + D141N. E3 = experiment with NA substitutions R292K. K displays lysine, R displays arginine, and <i>k</i> displays a mixed proportion of R and K at NA residue 292, as determined by Sanger sequencing of daily fecal samples of each bird. Wild type genotype correlated to unexposed Mallards (<i>P</i> = 0.000002)</p><p>Experimental design and NA sequence results of residue 292.</p

Primers for NA amplification and sequencing.

<p>Primers for NA amplification and sequencing.</p