Rare Influenza A (H3N2) Variants with Reduced Sensitivity to Antiviral Drugs

In 2007 and 2008 in Myanmar, we detected influenza viruses A (H3N2) that exhibited reduced sensitivity to both zanamivir and amantadine. These rare and naturally occurring viruses harbored a novel Q136K mutation in neuraminidase and S31N mutation in M2

Phylogenetic analysis of an off-seasonal influenza virus A (H3N2) in

SUMMARY: The objective of this study was to characterize the off-seasonal influenza virus A subtype H3N2, which caused an outbreak in an elderly hospital in Niigata, Japan. Virus isolates were subtyped by the hemagglutination-inhibition test and screened for antiviral drug sensitivity by real-time PCR using cycling probe technology and the 50z inhibitory concentration (IC 50 ) method. Whole genome sequencing was performed in order to determine the phylogeny of the outbreak virus. Seven virus isolates were analyzed in this study, and the results showed that all belonged to the influenza virus A (H3N2). These viruses exhibited the S31N mutation in M2, which confers resistance to amantadine. The results of the IC 50 analysis showed that these viruses were sensitive to both oseltamivir and zanamivir. Whole genome analysis revealed that the virus was similar to the A/Perth/16/2009 strain and that it is a triple reassortant virus with a 3 ＋ 3 ＋ 2 pattern of segment recombination

Phylogenetic analysis of the A) HA1 fragment of hemagglutinin, HA gene (885nt) and B) neuraminidase, NA gene (1,404nt) of influenza B viruses.

<p>Trees were constructed using the Neighbor-Joining method. Numbers at the nodes indicate confidence levels of bootstrap analysis with 1,000 replicates as percentage value. Amino acid substitutions that characterized a particular branch are indicated on the left side node. Vaccine strains are italicized and in red. Reference strains are boldfaced.</p

Genetic Characterization of Human Influenza Viruses in the Pandemic (2009–2010) and Post-Pandemic (2010–2011) Periods in Japan

<div><h3>Background</h3><p>Pandemic influenza A(H1N1) 2009 virus was first detected in Japan in May 2009 and continued to circulate in the 2010–2011 season. This study aims to characterize human influenza viruses circulating in Japan in the pandemic and post-pandemic periods and to determine the prevalence of antiviral-resistant viruses.</p> <h3>Methods</h3><p>Respiratory specimens were collected from patients with influenza-like illness on their first visit at outpatient clinics during the 2009–2010 and 2010–2011 influenza seasons. Cycling probe real-time PCR assays were performed to screen for antiviral-resistant strains. Sequencing and phylogenetic analysis of the HA and NA genes were done to characterize circulating strains.</p> <h3>Results and Conclusion</h3><p>In the pandemic period (2009–2010), the pandemic influenza A(H1N1) 2009 virus was the only circulating strain isolated. None of the 601 A(H1N1)pdm09 virus isolates had the H275Y substitution in NA (oseltamivir resistance) while 599/601 isolates (99.7%) had the S31N substitution in M2 (amantadine resistance). In the post-pandemic period (2010–2011), cocirculation of different types and subtypes of influenza viruses was observed. Of the 1,278 samples analyzed, 414 (42.6%) were A(H1N1)pdm09, 525 (54.0%) were A(H3N2) and 33 (3.4%) were type-B viruses. Among A(H1N1)pdm09 isolates, 2 (0.5%) were oseltamivir-resistant and all were amantadine-resistant. Among A(H3N2) viruses, 520 (99.0%) were amantadine-resistant. Sequence and phylogenetic analyses of A(H1N1)pdm09 viruses from the post-pandemic period showed further evolution from the pandemic period viruses. For viruses that circulated in 2010–2011, strain predominance varied among prefectures. In Hokkaido, Niigata, Gunma and Nagasaki, A(H3N2) viruses (A/Perth/16/2009-like) were predominant whereas, in Kyoto, Hyogo and Osaka, A(H1N1)pdm09 viruses (A/New_York/10/2009-like) were predominant. Influenza B Victoria(HA)-Yamagata(NA) reassortant viruses (B/Brisbane/60/2008-like) were predominant while a small proportion was in Yamagata lineage. Genetic variants with mutations at antigenic sites were identified in A(H1N1)pdm09, A(H3N2) and type-B viruses in the 2010–2011 season but did not show a change in antigenicity when compared with respective vaccine strains.</p> </div

Phylogenetic analysis of the A) HA1 fragment of hemagglutinin, HA gene (954nt) and B) neuraminidase, NA gene (1,388nt) of influenza A(H3N2) isolates.

<p>Trees were constructed using the Neighbor-Joining method. Numbers at the nodes indicate confidence levels of bootstrap analysis with 1,000 replicates as percentage value. Amino acid substitutions that characterized a particular branch are indicated on the left side node. Vaccine strains are italicized and in red. Reference strains are boldfaced.</p

Detection of influenza virus type, subtype and antiviral-resistance by cycling probe real time PCR.

*<p>oseltamivir-resistant (OsR): H275Y mutation in NA.</p>**<p>amantadine-resistant (AmR): S31N mutation in M2.</p><p>“-” no samples collected.</p>a<p>Percentage of antiviral resistant viruses in each season.</p

Amino acid mutations differences in the NA of influenza virus isolates in Japan, 2009–2010 and 2010–2011.

<p>Three-dimensional structures of monomeric NA were downloaded from the Protein Data Bank (RCSB PDB, <a href="http://www.pdb.org" target="_blank">http://www.pdb.org</a>) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036455#pone.0036455-Berman1" target="_blank">[44]</a> and visualized using PyMol (<a href="http:www.pymol.org" target="_blank">http:www.pymol.org</a>). The top view of the NA is shown. (A) The amino acid differences between A(H1N1)pdm09 isolates in Japan and vaccine strain, A/California/07/2009 were compared. Amino acid substitutions V241I and N369K are shown in yellow. These amino acid changes are located outside the antigenic sites but are phylogenetically relevant. PDB entry: 3NSS. (B) Antigenic site mutations between Japanese A(H3N2) isolates and vaccine strain, A/Perth/16/2009 were compared. L338F mutation is located in antigenic site F’ (olive); K369T is localized in antigenic site I’; and R400K and N402D are located in antigenic site K’. PDB entry: 1IVG (C) Amino acid substitutions in the NA antigenic sites of Japanese influenza B isolates and vaccine strain, B/Brisbane/60/2008 were compared. Mutations at N329D is localized at antigenic site F’ (olive); and D340N/D is located in antigenic site G’ (violet). PDB entry: 1INF.</p

Secretion of Galectin-9 as a DAMP during Dengue Virus Infection in THP-1 Cells

Damage-associated molecular patterns (DAMPs) are endogenous cellular molecules released to the extracellular environment in response to stress conditions such as virus infection. Galectins are β-galactoside-binding proteins that are widely expressed in cells and tissues of the immune system, are localized in the cell cytoplasm, and have roles in inflammatory responses and immune responses against infection. Elevated levels of galectin-9 (Gal-9) in natural human infections have been documented in numerous reports. To investigate the effect of dengue virus (DENV) infection on expression of endogenous Gal-9, monocytic THP-1 cells were infected with varying doses of DENV-3 (multiplicity of infection (MOI) 0.01, 0.03 and 0.1) and incubated at varying time points (Day 1, Day 2, Day 3). Results showed augmentation of Gal-9 levels in the supernatant, reduction of Gal-9 levels in the cells and decreased expression of LGALS9 mRNA, while DENV-3 mRNA copies for all three doses remained stable through time. Dengue virus induced the secretion of Gal-9 as a danger response; in turn, Gal-9 and other inflammatory factors, and stimulated effector responses may have limited further viral replication. The results in this pilot experiment add to the evidence of Gal-9 as a potential DAMP

Observed mutations in the antigenic sites of HA of influenza virus isolates in Japan, 2009–2010 and 2010–2011.

<p>Three-dimensional structures of trimeric HA were downloaded from the Protein Data Bank (RCSB PDB, <a href="http://www.pdb.org" target="_blank">http://www.pdb.org</a>) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036455#pone.0036455-Berman1" target="_blank">[44]</a> and visualized using PyMol (<a href="http:www.pymol.org" target="_blank">http:www.pymol.org</a>). (A) The amino acid differences in the antigenic sites of HA between Japanese A(H1N1)pdm09 isolates and vaccine strain, A/California/07/2009 were compared. Amino acid substitutions at G140E, A141S, I166V, G170E, S203T, R203T, D222E, E235K are located in antigenic site Ca (orange); L70F is located in antigenic site Cb (blue); K153T, K160M, K163T/N in antigenic site Sa (magenta); and S185T, A186T in antigenic site Sb (cyan). Amino acid changes outside the antigenic sites are shown in yellow. PDB entry: 3LZG. (B) HA antigenic site mutations between Japanese A(H3N2) isolates and vaccine strain, A/Perth/16/2009 were compared. N144K mutation is localized in antigenic site A (red); P162S in antigenic site B (orange); G50E/K. T212A, and E280A/S/T are localized in antigenic site C (green); I260M and R261Q are located in antigenic site E (blue). PDB entry: 1MQL (C) Amino acid substitutions in the HA antigenic sites of influenza B isolates in Japan and vaccine strain, B/Brisbane/60/2008 were compared. Mutations at A127T, V146I, and S150I are localized at antigenic site A (red); N165K/Y and K209N are located in antigenic site B (orange); and S229D is located in antigenic site D (violet). PDB entry: 2RFT.</p