Neuraminidase inhibitor susceptibility and neuraminidase enzyme kinetics of human influenza A and B viruses circulating in Thailand in 2010-2015.

Amino acid substitutions within or near the active site of the viral neuraminidase (NA) may affect influenza virus fitness. In influenza A(H3N2) and B viruses circulating in Thailand between 2010 and 2015, we identified several NA substitutions that were previously reported to be associated with reduced inhibition by NA inhibitors (NAIs). To study the effect of these substitutions on the enzymatic properties of NA and on virus characteristics, we generated recombinant influenza viruses possessing either a wild type (WT) NA or an NA with a single I222V, S331G, or S331R substitution [in influenza A(H3N2) viruses] or a single D342S, A395T, A395V, or A395D NA substitution (in influenza B viruses). We generated recombinant (7:1) influenza A and B viruses on the genetic background of A/Puerto Rico/8/1934 (A/PR/8, H1N1) or B/Yamanashi/166/1998 (B/YAM) viruses, respectively. In contrast to the expected phenotypes, all the recombinant influenza A(H3N2) and B viruses carrying putative NA resistance substitutions were susceptible to NAIs. The Km and Vmax for the NAs of A/PR8-S331G and A/PR8-S331R viruses were higher than for the NA of WT virus, and the corresponding values for the B/YAM-D342S virus were lower than for the NA of WT virus. Although there was initial variation in the kinetics of influenza A and B viruses' replication in MDCK cells, their titers were comparable to each other and to WT viruses at later time points. All introduced substitutions were stable except for B/YAM-D342S and B/YAM-A395V which reverted to WT sequences after three passages. Our data suggest that inferring susceptibility to NAIs based on sequence information alone should be cautioned. The impact of NA substitution on NAI resistance, viral growth, and enzymatic properties is viral context dependent and should be empirically determined

Assessing Antigenic Drift of Seasonal Influenza A(H3N2) and A(H1N1)pdm09 Viruses.

Under selective pressure from the host immune system, antigenic epitopes of influenza virus hemagglutinin (HA) have continually evolved to escape antibody recognition, termed antigenic drift. We analyzed the genomes of influenza A(H3N2) and A(H1N1)pdm09 virus strains circulating in Thailand between 2010 and 2014 and assessed how well the yearly vaccine strains recommended for the southern hemisphere matched them. We amplified and sequenced the HA gene of 120 A(H3N2) and 81 A(H1N1)pdm09 influenza virus samples obtained from respiratory specimens and calculated the perfect-match vaccine efficacy using the pepitope model, which quantitated the antigenic drift in the dominant epitope of HA. Phylogenetic analysis of the A(H3N2) HA1 genes classified most strains into genetic clades 1, 3A, 3B, and 3C. The A(H3N2) strains from the 2013 and 2014 seasons showed very low to moderate vaccine efficacy and demonstrated antigenic drift from epitopes C and A to epitope B. Meanwhile, most A(H1N1)pdm09 strains from the 2012-2014 seasons belonged to genetic clades 6A, 6B, and 6C and displayed the dominant epitope mutations at epitopes B and E. Finally, the vaccine efficacy for A(H1N1)pdm09 (79.6-93.4%) was generally higher than that of A(H3N2). These findings further confirmed the accelerating antigenic drift of the circulating influenza A(H3N2) in recent years

Molecular Epidemiology and Phylogenetic Analyses of Influenza B Virus in Thailand during 2010 to 2014

<div><p>Influenza B virus remains a major contributor to the seasonal influenza outbreak and its prevalence has increased worldwide. We investigated the epidemiology and analyzed the full genome sequences of influenza B virus strains in Thailand between 2010 and 2014. Samples from the upper respiratory tract were collected from patients diagnosed with influenza like-illness. All samples were screened for influenza A/B viruses by one-step multiplex real-time RT-PCR. The whole genome of 53 influenza B isolates were amplified, sequenced, and analyzed. From 14,418 respiratory samples collected during 2010 to 2014, a total of 3,050 tested positive for influenza virus. Approximately 3.27% (471/14,418) were influenza B virus samples. Fifty three isolates of influenza B virus were randomly chosen for detailed whole genome analysis. Phylogenetic analysis of the HA gene showed clusters in Victoria clades 1A, 1B, 3, 5 and Yamagata clades 2 and 3. Both B/Victoria and B/Yamagata lineages were found to co-circulate during this time. The NA sequences of all isolates belonged to lineage II and consisted of viruses from both HA Victoria and Yamagata lineages, reflecting possible reassortment of the HA and NA genes. No significant changes were seen in the NA protein. The phylogenetic trees generated through the analysis of the PB1 and PB2 genes closely resembled that of the HA gene, while trees generated from the analysis of the PA, NP, and M genes showed similar topology. The NS gene exhibited the pattern of genetic reassortment distinct from those of the PA, NP or M genes. Thus, antigenic drift and genetic reassortment among the influenza B virus strains were observed in the isolates examined. Our findings indicate that the co-circulation of two distinct lineages of influenza B viruses and the limitation of cross-protection of the current vaccine formulation provide support for quadrivalent influenza vaccine in this region.</p></div

Incidence of influenza A and B viruses identified from clinical samples between 2010 and 2014.

<p>(A) The distribution of the influenza isolates for each month, including influenza B (light blue color), influenza A(H1N1)pdm09 (red color), and influenza A(H3N2) (yellow color) shown as bars (left scale). From the total number of specimens collected every month, the percent of influenza-positive cases are shown as grey color area under the curve (right scale). (B) Bar graph illustrating only the total number of influenza B infection monthly (left scale) relative to the number of total specimens collected each month are shown in gray (right scale).</p

Genetic stability of NA substitutions among recombinant influenza A and B viruses after three subsequent passages in MDCK cells.

<p>Genetic stability of NA substitutions among recombinant influenza A and B viruses after three subsequent passages in MDCK cells.</p

NAI susceptibility profiles for recombinant influenza A and B viruses carrying a single NA substitution.

<p>NAI susceptibility profiles for recombinant influenza A and B viruses carrying a single NA substitution.</p

Replication kinetics in MDCK cells of recombinant influenza A and B viruses carrying a single NA amino acid substitution.

<p>MDCK cells were infected with recombinant influenza A and B viruses at an MOI of 1 (A, C) or 0.001 (B, D). Virus titers were determined by TCID₅₀ assays in MDCK cells after incubation for 72 h at 35°C (for influenza A viruses) or 33°C (for influenza B viruses). Each data point represents the mean virus titer (log₁₀TCID₅₀/mL) ± the SD from three independent experiments. *<i>P</i> < 0.05.</p

Influenza B virus clinical isolates sequenced in this study.

<p>Influenza B virus clinical isolates sequenced in this study.</p

Amino acid substitutions found in the HA protein of influenza B virus clinical isolates in this study^a.

<p>^a The defined residue positions on the antigenic epitope according to Ni et al., 2013 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116302#pone.0116302.ref024" target="_blank">24</a>].</p><p>^b The residues are numbered according to that of B/HK/73 HA [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116302#pone.0116302.ref023" target="_blank">23</a>].</p><p>Amino acid substitutions found in the HA protein of influenza B virus clinical isolates in this study<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116302#t003fn001" target="_blank">^a</a>.</p

The NA thermostability of the recombinant influenza A and B viruses carrying a single NA substitution.

<p>Influenza A/PR8 (A, B) and B/YAM (C, D) viruses were incubated for 15 or 30 min at the indicated temperatures. Residual NA enzyme activity was assayed and is presented as a percentage of the NA activity at 4°C (unheated). The bars represent the mean of the relative NA activity ± SD for different temperatures from three experiments. *<i>P</i> < 0.05.</p