BackgroundPublications 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/FindingsWe 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).ConclusionDifferences 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).

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 p epitope 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.

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.

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.

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.

Human Parechovirus (HPeV), a member of the Picornaviridae family, is an infectious agent mostly affecting children. There are 16 recognized genotypes which have globally spread. This study incorporated a total of 2957 nasopharyngeal (NP) swab and 759 fecal samples that were collected from different parts of Thailand. The NP of HPeV was detected in 0.4% of NP swab and 6.1% of fecal samples. The majority of HPeV infections occur in infants below the age of 2 years, while infections were detected in children above the age of 10 years as well. Various genotypes comprising 1A, 1B, 2, 3, 4, 5, 6, 10 and 14 have been characterized. This study revealed recombination events in 16 samples in which HPeV1B was shown as the highest frequency. In conclusion, HPeV can be detected in both the respiratory and GI tract. Moreover, HPeV which circulates in Thailand is highly diverse and subject to recombination.

The neuraminidase inhibitors (NAIs) oseltamivir and zanamivir are commonly used for the treatment and control of influenza A and B virus infection. However, the emergence of new influenza virus strains with reduced susceptibility to NAIs may appear with the use of these antivirals or even naturally. We therefore screened the neuraminidase (NA) sequences of seasonal influenza virus A(H1N1), A(H1N1)pdm09, A(H3N2), and influenza B virus strains identified in Thailand for the presence of substitutions previously reported to reduce susceptibility to NAIs. We initially examined oseltamivir resistance (characterized by the H275Y mutation in the NA gene) in 485 A(H1N1)pdm09 strains circulating in Thailand and found that 0.82% (4/485) had this substitution. To further evaluate the evolution of the NA gene, we also randomly selected 98 A(H1N1)pdm09, 158 A(H3N2), and 69 influenza B virus strains for NA gene amplification and sequencing, which revealed various amino acid mutations in the active site of the NA protein previously shown to be associated with reduced susceptibility to NAIs. Phylogenetic analysis of the influenza virus strains from this study and elsewhere around the world, together with the estimations of nucleotide substitution rates and selection pressure, and the predictions of B-cell epitopes and N-linked glycosylation sites all provided evidence for the ongoing evolution of NA. The overall rates of NA evolution for influenza A viruses were higher than for influenza B virus at the nucleotide level, although influenza B virus possessed more genealogical diversity than that of influenza A viruses. The continual surveillance of the antigenic changes associated with the NA protein will not only contribute to the influenza virus database but may also provide a better understanding of selection pressure exerted by antiviral use.

Antigenic changes in the HA1 domain of the influenza A/H3N2 hemagglutinin (HA) present a challenge in the design of the annual influenza vaccine. We examined the genetic variability in the nucleotide and amino acid of encoding HA1 sequences of the influenza A/H3N2 virus during the 2015 influenza season in Thailand. Toward this, the HA genes of 45 influenza A/H3N2 strains were amplified and sequenced. Although a clade 3C.3a strain (A/Switzerland/9715293/2013) was chosen for the 2015 vaccine, phylogenetic analysis demonstrated that strains belonging to clade 3C.2a (96 %) instead of clade 3C.3a (4 %) were circulating that year. Sequence analysis showed that seven codons were under positive selection, five of which were located inside the antigenic epitopes. The percentages of the perfect match vaccine efficacy (VE) estimated by the P epitope model against circulating strains suggested antigenic drift of the dominant epitopes A and B, which contributed to reduced VE of the 2015 vaccine. However, the 2016 vaccine strain (A/Hong Kong/4801/2014) was closely related and well matched against the circulating strain (mean of VE = 79.3 %). These findings provide data on the antigenic drift of the influenza A/H3N2 virus circulating in Thailand and further support continual monitoring and surveillance of the antigenic changes on HA1.