Molecular Epidemiology and Evolution of Human Enterovirus Serotype 68 in Thailand, 2006–2011

BACKGROUND: Publications worldwide have reported on the re-occurrence of human enterovirus 68 (EV68), a rarely detected pathogen usually causing respiratory illness. However, epidemiological data regarding this virus in particular on the Asian continent has so far been limited. METHODOLOGY/FINDINGS: We investigated the epidemiology and genetic variability of EV68 infection among Thai children with respiratory illnesses from 2006-2011 (n = 1810). Semi-nested PCR using primer sets for amplification of the 5'-untranslated region through VP2 was performed for rhino-enterovirus detection. Altogether, 25 cases were confirmed as EV68 infection indicating a prevalence of 1.4% in the entire study population. Interestingly, the majority of samples were children aged >5 years (64%). Also, co-infection with other viruses was found in 28%, while pandemic H1N1 influenza/2009 virus was the most common co-infection. Of EV68-positive patients, 36% required hospitalizations with the common clinical presentations of fever, cough, dyspnea, and wheezing. The present study has shown that EV68 was extremely rare until 2009 (0.9%). An increasing annual prevalence was found in 2010 (1.6%) with the highest detection frequency in 2011 (4.3%). Based on analysis of the VP1 gene, the evolutionary rate of EV68 was estimated at 4.93 × 10(-3) substitutions/site/year. Major bifurcation of the currently circulating EV68 strains occurred 66 years ago (1945.31 with (1925.95-1960.46)95% HPD). Among the current lineages, 3 clusters of EV68 were categorized based on the different molecular signatures in the BC and DE loops of VP1 combined with high posterior probability values. Each cluster has branched off from their common ancestor at least 36 years ago (1975.78 with (1946.13-1984.97)95% HPD). CONCLUSION: Differences in epidemiological characteristic and seasonal profile of EV68 have been found in this study. Results from Bayesian phylogenetic investigations also revealed that EV68 should be recognized as a genetically diverse virus with a substitution rate identical to that of enterovirus 71 genotype B (4.2 × 10(-3 )s/s/y)

Respiratory syncytial virus genotypes NA1, ON1, and BA9 are prevalent in Thailand, 2012–2015

Respiratory syncytial virus (RSV) causes acute lower respiratory tract infection in infants and young children worldwide. To investigate the RSV burden in Thailand over four consecutive years (January 2012 to December 2015), we screened 3,306 samples obtained from children ≤5 years old with acute respiratory tract infection using semi-nested reverse-transcription polymerase chain reaction (RT-PCR). In all, 8.4% (277/3,306) of the specimens tested positive for RSV, most of which appeared in the rainy months of July to November. We then genotyped RSV by sequencing the G glycoprotein gene and performed phylogenetic analysis to determine the RSV antigenic subgroup. The majority (57.4%, 159/277) of the RSV belonged to subgroup A (RSV-A), of which NA1 genotype was the most common in 2012 while ON1 genotype became prevalent the following year. Among samples tested positive for RSV-B subgroup B (RSV-B) (42.6%, 118/277), most were genotype BA9 (92.6%, 87/94) with some BA10 and BA-C. Predicted amino acid sequence from the partial G region showed highly conserved N-linked glycosylation site at residue N237 among all RSV-A ON1 strains (68/68), and at residues N296 (86/87) and N310 (87/87) among RSV-B BA9 strains. Positive selection of key residues combined with notable sequence variations on the G gene contributed to the continued circulation of this rapidly evolving virus

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

Genetic and antigenic divergence in the influenza A(H3N2) virus circulating between 2016 and 2017 in Thailand

<div><p>Influenza virus evolves rapidly due to the accumulated genetic variations on the viral sequence. Unlike in North America and Europe, influenza season in the tropical Southeast Asia spans both the rainy and cool seasons. Thus, influenza epidemiology and viral evolution sometimes differ from other regions, which affect the ever-changing efficacy of the vaccine. To monitor the current circulating influenza viruses in this region, we determined the predominant influenza virus strains circulating in Thailand between January 2016 and June 2017 by screening 7,228 samples from patients with influenza-like illness. During this time, influenza A(H3N2) virus was the predominant influenza virus detected. We then phylogenetically compared the hemagglutinin (HA) gene from a subset of these A(H3N2) strains (n = 62) to the reference sequences and evaluated amino acid changes in the dominant antigenic epitopes on the HA protein structure. The divergence of the circulating A(H3N2) from the A/Hong Kong/4801/2014 vaccine strain formed five genetic groups (designated I to V) within the 3C.2a clade. Our results suggest a marked drift of the current circulating A(H3N2) strains in Thailand, which collectively contributed to the declining predicted vaccine effectiveness (VE) from 74% in 2016 down to 48% in 2017.</p></div

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

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

Demographic characteristics of patients (N = 14,418).

<p>^a Calculated by (number of patients/number of specimen) x 100.</p><p>^b At least one of the expected values is smaller than 0.05, the <i>P</i> value is calculated by the Chi square test.</p><p>N/A Information not available.</p><p>Demographic characteristics of patients (N = 14,418).</p

Analysis of the numbers and influenza B strains of randomly sampled sequences from January 2010 to February 2014.

<p>Number of B/Victoria and B/Yamagata lineage strains found are displayed in red and yellow bars, respectively (left scale). Total number of influenza B positive samples for each year is indicated by dot-line (right scale). The strains B/Brisbane/60/2008, B/Wisconsin/1/2010, and B/Massachusetts/2/2012 included in the Southern hemisphere vaccines for the given year are indicated by the asterisk. Vic denotes Victoria and Yam denotes Yamagata.</p

Phylogenetic analysis of the nucleotide sequences of the HA coding region of A(H3N2).

<p>Sequences of randomly selected samples from this study (n = 62) (designated A/Thailand/CU and denoted in colored dots) and those identified in Thailand from the databases during 2016–2017 (n = 29) were compared to the A(H3N2) vaccine and reference strains of known clades reported by the WHO and others (squares). The phylogenetic tree was constructed using the maximum likelihood method and HKY+G model with 1,000 bootstrap replicates implemented in MEGA (version 6). Branch values of >70% are indicated at the nodes. Dominant epitope (A-E) determined for each sequence are marked with different colored dots (A = yellow, B = pink, C = purple, D = green, and E = blue). The signature amino acid substitutions (colored) occurring on the antigenic epitopes are also shown. Scale bar represents approximately 0.5% nucleotide difference between close relatives. Residue numbers are specific for HA1 (color-coded by epitope) and HA2 (dark blue). The vaccine strain A/Hong Kong/4801/2014 belonged to 3C.2a clade. Shaded area (Group V) highlights strains of interest.</p