Comparable fitness and transmissibility between Oseltamivir-Resistant Pandemic 2009 and seasonal H1N1 Influenza Viruses with the H275Y Neuraminidase Mutation

Conference Theme: Translating Health Research into Policy and Practice for Health of the PopulationPoster Presentations - Emerging / Infectious Diseases: no. P65-Ab0010INTRODUCTION: Neuraminidase (NA) inhibitors are one of the limited options for the control of influenza. The H275Y NA mutation, which confers resistance to oseltamivir carboxylate, was initially considered to be of little clinical consequence due to limited detection of this mutation in field isolates prior to 2007-2008, when a globally spreading H275Y variant emerged. Oseltamivir-resistant A(H1N1)pdm09 viruses with the H275Y NA mutation has been reported since 2009 but have not replaced the …published_or_final_versio

Hemagglutinin-neuraminidase balance confers respiratory-droplet transmissibility of the Pandemic H1N1 Influenza virus in ferrets

Conference Theme: Translating Health Research into Policy and Practice for Health of the PopulationPoster Presentations: Emerging / Infectious Diseases: no. P66-Ab0011published_or_final_versio

Comparative mutational analyses of influenza A viruses

The error-prone RNA-dependent RNA polymerase (RdRP) and external selective pressures are the driving forces for RNA viral diversity. When confounded by selective pressures, it is difficult to assess if influenza A viruses (IAV) that have a wide host range possess comparable or distinct spontaneous mutational frequency in their RdRPs. We used in-depth bioinformatics analyses to assess the spontaneous mutational frequencies of two RdRPs derived from human seasonal (A/Wuhan/359/95; Wuhan) and H5N1 (A/Vietnam/1203/04; VN1203) viruses using the mini-genome system with a common firefly luciferase reporter serving as the template. High-fidelity reverse transcriptase was applied to generate high-quality mutational spectra which allowed us to assess and compare the mutational frequencies and mutable motifs along a target sequence of the two RdRPs of two different subtypes. We observed correlated mutational spectra (τ correlation P < 0.0001), comparable mutational frequencies (H3N2:5.8 ± 0.9; H5N1:6.0 ± 0.5), and discovered a highly mutable motif "(A)AAG" for both Wuhan and VN1203 RdRPs. Results were then confirmed with two recombinant A/Puerto Rico/8/34 (PR8) viruses that possess RdRP derived from Wuhan or VN1203 (RG-PR8×Wuhan(PB2, PB1, PA, NP) and RG-PR8×VN1203(PB2, PB1, PA, NP)). Applying novel bioinformatics analysis on influenza mutational spectra, we provide a platform for a comprehensive analysis of the spontaneous mutation spectra for an RNA virus

Toll-like receptor (TLR)3 signaling contributes to enhanced disease severity in H5N1 but not pandemic H1N1 infection in mice

Session - Pathogenesis and Transmission - Short TalkConference Theme: Pathogenesis of Influenza: Virus-host Interaction (E3

Generation and characterization of influenza A viruses with altered polymerase fidelity

Genetic diversity of influenza A viruses (IAV) acquired through the error-prone RNA-dependent RNA polymerase (RdRP) or through genetic reassortment enables perpetuation of IAV in humans through epidemics or pandemics. Here, to assess the biological significance of genetic diversity acquired through RdRP, we characterize an IAV fidelity variant derived from passaging a seasonal H3N2 virus in the presence of ​ribavirin, a purine analogue that increases ​guanosine-to-​adenosine mutations. We demonstrate that a single ​PB1-V43I mutation increases selectivity to ​guanosine in A/Wuhan/359/95 (H3N2) and A/Vietnam/1203/04 (H5N1) viruses. The H5N1 ​PB1-V43I-recombinant virus replicates to comparable titres as the wild-type virus in vitro or in the mouse lungs. However, a decrease in viral population diversity at day 3 post inoculation is associated with a tenfold reduced lethality and neurotropism in mice. Applying a fidelity variant with reduced mutational frequency, we provide direct experimental evidence for the role of genetic diversity in IAV pathogenesis

Resistance to Neuraminidase Inhibitors Conferred by an R292K Mutation in a Human Influenza Virus H7N9 Isolate Can Be Masked by a Mixed R/K Viral Population

We characterized the A/Shanghai/1/2013 virus isolated from the first confirmed human case of A/H7N9 disease in China. The A/Shanghai/1/2013 isolate contained a mixed population of R (65%; 15/23 clones) and K (35%; 8/23 clones) at neuraminidase (NA) residue 292, as determined by clonal sequencing. A/Shanghai/1/2013 with mixed R/K at residue 292 exhibited a phenotype that is sensitive to zanamivir and oseltamivir carboxylate by the enzyme-based NA inhibition assay. The plaque-purified A/Shanghai/1/2013 with dominant K292 (94%; 15/16 clones) showed sensitivity to zanamivir that had decreased by >30-fold and to oseltamivir carboxylate that had decreased by >100-fold compared to its plaque-purified wild-type counterpart possessing dominant R292 (93%, 14/15 clones). In Madin-Darby canine kidney (MDCK) cells, the plaque-purified A/Shanghai/1/2013-NAK292 virus exhibited no reduction in viral titer under conditions of increasing concentrations of oseltamivir carboxylate (range, 0 to 1,000 microM) whereas the replication of the plaque-purified A/Shanghai/1/2013-NAR292 and the A/Shanghai/2/2013 viruses was completely inhibited at 250 microM and 31.25 microM of oseltamivir carboxylate, respectively. Although the plaque-purified A/Shanghai/1/2013-NAK292 virus exhibited lower NA enzyme activity and a higher Km for 2'-(4-methylumbelliferryl)-alpha-d-N-acetylneuraminic acid than the wild-type A/Shanghai/1/2013-NAR292 virus, the A/Shanghai/1/2013-NAK292 virus formed large plaques and replicated efficiently in vitro. Our results confirmed that the NA R292K mutation confers resistance to oseltamivir, peramivir, and zanamivir in the novel human H7N9 viruses. Importantly, detection of the resistance phenotype may be masked in the clinical samples containing a mixed population of R/K at NA residue 292 in the enzyme-based NA inhibition assay. IMPORTANCE: The neuraminidase (NA) inhibitors oseltamivir and zanamivir are currently the front-line therapeutic options against the novel H7N9 influenza viruses, which possess an S31N mutation that confers resistance to the M2 ion channel blockers. It is therefore important to evaluate the sensitivity of the clinical isolates to NA inhibitors and to monitor for the emergence of resistant variants. We characterized the A/Shanghai/1/2013 (H7N9) isolate which contained a mixed population of R/K at NA residue 292. While the clinical isolate exhibited a phenotype of sensitivity to NA inhibitors using the enzyme-based NA inhibition assay, the plaque-purified A/Shanghai/1/2013 virus with dominant K292 was resistant to zanamivir, peramivir, and oseltamivir. Resistance to NA inhibitors conferred by the R292K mutation in a human influenza virus H7N9 isolate can be masked by a mixed R/K viral population, and this should be taken into consideration while monitoring antiviral resistance in patients with H7N9 infection.link_to_OA_fulltex

Characterization of the R292K mutation that confers resistance to the neuraminidase inhibitors in a novel H7N9 human isolate

Session 5. PathogenesisWe characterized the A/Shanghai/1/2013 virus isolated from the first confirmed human case of A/H7N9 disease in China. The A/Shanghai/1/2013 isolate contained a mixed population of R (65%; 15/23 clones)/K (35%; 8/23 clones) at the neuraminidase (NA) residue 292, as determined by clonal sequencing. A/Shanghai/1/2013 with mixed R/K at residue 292 exhibited a sensitive phenotype to zanamivir and oseltamivir carboxylate by the enzyme based NA inhibition assay and to zanamivir in vitro. The plaque purified A/Shanghai/1/2013 with dominant K292 (94%; 15/16 clones) showed decreased sensitivity to zanamivir by 30-fold and to oseltamivir carboxylate by >100-fold compared to its plaque purified wild-type counterpart possessing dominant R292 (93%, 14/15 clones). In MDCK cells, the plaque purified A/Shanghai/1/2013-NAK292 virus exhibited no reduction in viral titer under increasing concentrations of oseltamivir carboxylate (range 0-1000 μM), whereas the replication of the plaque purified A/Shanghai/1/2013-NAR292 and the A/Shanghai/2/2013 viruses were completely inhibited at 250 μM and 31.25 μM of oseltamivir carboxylate, respectively. Although the plaque purified A/Shanghai/1/2013-NAK292 virus exhibited lower NA enzyme activity and a higher Km for 2’-(4-methylumbelliferryl)-α-D-N-acetylneuraminic acid than that of the wild-type A/Shanghai/1/2013-NAR292 virus, the A/Shanghai/1/2013-NAK292 virus formed large plaques and replicated efficiently in vitro. Our results confirmed that the NA R292K mutation confers resistance to oseltamivir and zanamivir in the novel human H7N9 viruses. Importantly, detection of the resistance phenotype may be masked in the clinical samples containing a mixed population of R/K at NA residue 292 while applying the in vitro based assay or the fluorescence based NA inhibition assay

Influenza viruses in wild birds in Hong Kong, 2003-2010

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