Evolutionary and Genetic Recombination Analyses of Coxsackievirus A6 Variants Associated with Hand, Foot, and Mouth Disease Outbreaks in Thailand between 2019 and 2022.

Coxsackievirus (CV)-A6 infections cause hand, foot, and mouth disease (HFMD) in children and adults. Despite the serious public health threat presented by CV-A6 infections, our understanding of the mechanisms by which new CV-A6 strains emerge remains limited. This study investigated the molecular epidemiological trends, evolutionary dynamics, and recombination characteristics of CV-A6-associated HFMD in Thailand between 2019 and 2022. In the HFMD patient samples collected during the 4-year study period, we identified enterovirus (EV) RNA in 368 samples (48.7%), of which CV-A6 (23.7%) was the predominant genotype, followed by CV-A4 (6%), EV-A71 (3.7%), and CV-A16 (3.4%). According to the partial viral protein (VP) 1 sequences, all these CV-A6 strains belonged to the D3 clade. Based on the viral-RNA-dependent RNA polymerase (RdRp) gene, four recombinant forms (RFs), RF-A (147, 84.5%), RF-N (11, 6.3%), RF-H (1, 0.6%), and newly RF-Y (15, 8.6%), were identified throughout the study period. Results from the similarity plot and bootscan analyses revealed that the 3D polymerase (3Dpol) region of the D3/RF-Y subclade consists of sequences highly similar to CV-A10. We envisage that the epidemiological and evolutionarily insights presented in this manuscript will contribute to the development of vaccines to prevent the spread of CV-A6 infection.

Human norovirus GII.4 Hong Kong variant shares common ancestry with GII.4 Osaka and emerged in Thailand in 2016.

Human norovirus is a leading cause of non-bacterial acute gastroenteritis, which affects all age groups and are found globally. Infections are highly contagious and often occur as outbreaks. Periodic emergence of new strains are not uncommon and novel variants are named after the place of first reported nucleotide sequence. Here, we identified human norovirus GII.4 Hong Kong variant in stool samples from Thai patients presented with acute gastroenteritis. Comparison of amino acid residues deduced from the viral nucleotide sequence with those of historical and contemporary norovirus GII.4 strains revealed notable differences, which mapped to the defined antigenic sites of the viral major capsid protein. Time-scaled phylogenetic analysis suggests that GII.4 Hong Kong shared common ancestry with GII.4 Osaka first reported in 2007, and more importantly, did not evolve from the now-prevalent GII.4 Sydney lineage. As circulation of norovirus minor variants can lead to eventual widespread transmission in susceptible population, this study underscores the potential emergence of the GII.4 Hong Kong variant, which warrants vigilant molecular epidemiological surveillance.

Prevalence of antibodies against seasonal influenza A and B viruses among older adults in rural Thailand: A cross-sectional study.

Assessing the seroprevalence of the high-risk individuals against the influenza virus is essential to evaluate the progress of vaccine implementation programs and establish influenza virus interventions. Herein, we identified the pre-existing cross-protection of the circulating seasonal influenza viruses among the older-aged population. A cross-sectional study was performed base on the 176 residual sera samples collected from older adults aged 60 to 95 years without a history of vaccination in rural Thailand in 2015. Sera antibody titers against influenza A and B viruses circulating between 2016 and 2019 were determined by hemagglutination inhibition assay. These findings indicated the low titers of pre-existing antibodies to circulating influenza subtypes and showed age-independent antibody titers among the old adults. Moderate seropositive rates (HAI ≥ 1:40) were observed in influenza A viruses (65.9%A(H3N2), 50.0% for A(H1N1) pdm09), and found comparatively lower rates in influenza B viruses (14% B/Yam2, 21% B/Yam3 and 25% B/Vic). Only 5% of individuals possessed broadly protective antibodies against both seasonal influenza A and B virus in this region. Our findings highlighted the low pre-existing antibodies to circulating influenza strains in the following season observed in older adults. The serological study will help inform policy-makers for health care planning and guide control measures concerning vaccination programs.

Characterizing genetic and antigenic divergence from vaccine strain of influenza A and B viruses circulating in Thailand, 2017-2020.

We monitored the circulating strains and genetic variation among seasonal influenza A and B viruses in Thailand between July 2017 and March 2020. The hemagglutinin gene was amplified and sequenced. We identified amino acid (AA) changes and computed antigenic relatedness using the Pepitope model. Phylogenetic analyses revealed multiple clades/subclades of influenza A(H1N1)pdm09 and A(H3N2) were circulating simultaneously and evolved away from their vaccine strain, but not the influenza B virus. The predominant circulating strains of A(H1N1)pdm09 belonged to 6B.1A1 (2017-2018) and 6B.1A5 (2019-2020) with additional AA substitutions. Clade 3C.2a1b and 3C.2a2 viruses co-circulated in A(H3N2) and clade 3C.3a virus was found in 2020. The B/Victoria-like lineage predominated since 2019 with an additional three AA deletions. Antigenic drift was dominantly facilitated at epitopes Sa and Sb of A(H1N1)pdm09, epitopes A, B, D and E of A(H3N2), and the 120 loop and 190 helix of influenza B virus. Moderate computed antigenic relatedness was observed in A(H1N1)pdm09. The computed antigenic relatedness of A(H3N2) indicated a significant decline in 2019 (9.17%) and 2020 (- 18.94%) whereas the circulating influenza B virus was antigenically similar (94.81%) with its vaccine strain. Our findings offer insights into the genetic divergence from vaccine strains, which could aid vaccine updating.

Climate factors influence seasonal influenza activity in Bangkok, Thailand.

Yearly increase in influenza activity is associated with cold and dry winter in the temperate regions, while influenza patterns in tropical countries vary significantly by regional climates and geographic locations. To examine the association between influenza activity in Thailand and local climate factors including temperature, relative humidity, and rainfall, we analyzed the influenza surveillance data from January 2010 to December 2018 obtained from a large private hospital in Bangkok. We found that approximately one in five influenza-like illness samples (21.6% or 6,678/30,852) tested positive for influenza virus. Influenza virus typing showed that 34.2% were influenza A(H1N1)pdm09, 46.0% were influenza A(H3N2), and 19.8% were influenza B virus. There were two seasonal waves of increased influenza activity. Peak influenza A(H1N1)pdm09 activity occurred in February and again in August, while influenza A(H3N2) and influenza B viruses were primarily detected in August and September. Time series analysis suggests that increased relative humidity was significantly associated with increased influenza activity in Bangkok. Months with peak influenza activity generally followed the most humid months of the year. We performed the seasonal autoregressive integrated moving average (SARIMA) multivariate analysis of all influenza activity on the 2011 to 2017 data to predict the influenza activity for 2018. The resulting model closely resembled the actual observed overall influenza detected that year. Consequently, the ability to predict seasonal pattern of influenza in a large tropical city such as Bangkok may enable better public health planning and underscores the importance of annual influenza vaccination prior to the rainy season.

Enterovirus A71 Infection, Thailand, 2017.

An outbreak of hand, foot and mouth disease among children in Thailand peaked in August 2017. Enterovirus A71 subgenogroup B5 caused most (33.8%, 163/482) cases. Severe disease (myocarditis and encephalitis) was observed in 1 patient. Coxsackievirus A6 was detected in 6.0% (29/482) of patients, and coxsackievirus A16 was detected in 2.7% (13/482) of patients.

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

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.

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.

Evolution of the neuraminidase gene of seasonal influenza A and B viruses in Thailand between 2010 and 2015.

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.

Evidence for influenza B virus lineage shifts and reassortants circulating in Thailand in 2014-2016.

Towards the surveillance of seasonal influenza viruses between August 2015 and June 2016, respiratory samples (n=3390) were collected from Thai patients with influenza-like illness. One-hundred fifty-seven (4.6%) samples tested positive for influenza B virus by real-time reverse-transcription polymerase chain reaction (RT-PCR). While the influenza B virus Yamagata lineage strains were more prevalent than the Victoria lineage strains in 2015 (77.5% vs. 22.5%), the Victoria lineage strains appeared to dominate the first half of 2016 (62.3%). To better assess possible lineage shift in this transition period, 73 influenza B virus strains circulating between March 2014 and May 2016 were randomly selected for hemagglutinin (HA) and neuraminidase (NA) gene sequencing. Phylogenetic analysis of the HA gene showed clustering in Yamagata clade 3 (61.6%), Victoria clade 1 (20.6%), and Yamagata clade 2 (17.8%). Analyses of both the HA and NA segments together, however, demonstrated that 5 influenza B strains (6.8%) were of mixed lineages. Our findings suggest that the circulating strains of the Victoria and Yamagata lineages underwent another lineage shift in 2016. The identification of mutations and reassortment of influenza B virus underscores the importance of careful surveillance and the selection of optimal vaccine strains.

Lineage-specific detection of influenza B virus using real-time polymerase chain reaction with melting curve analysis.

Influenza B viruses comprise two lineages, Victoria (B/Vic) and Yamagata (B/Yam), which co-circulate globally. The surveillance data on influenza B virus lineages in many countries often underestimate the true prevalence due to the lack of a rapid, accurate, and cost-effective method for virus detection. We have developed a real-time PCR with melting curve analysis for lineage-specific differential detection of influenza B virus. By amplifying a region of the hemagglutinin gene using real-time PCR with SYBR Green I dye, B/Vic and B/Yam could be differentiated based on their melting temperature peaks. This method was efficient (B/Vic = 93.2 %; B/Yam 97.7 %), sensitive (B/Vic, 94.6 %; B/Yam, 96.3 %), and specific (B/Vic, 97.7 %; B/Yam, 97.1 %). The lower detection limit was 10(2) copies per microliter. The assay was evaluated using 756 respiratory specimens that were positive for influenza B virus, obtained between 2010 and 2015. The incidence of influenza B virus was approximately 18.9 % of all influenza cases, and the percentage was highest among children aged 6-17 years (7.57 %). The overall percentage of mismatched influenza B vaccine was 21.1 %. Our findings suggest that real-time PCR with melting curve analysis can provide a rapid, simple, and sensitive lineage-specific influenza B virus screening method to facilitate influenza surveillance.

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.

Complete genome sequence analysis of enterovirus 71 isolated from children with hand, foot, and mouth disease in Thailand, 2012-2014.

The complete genomic sequences of 14 enterovirus 71 (EV71) strains isolated from children with hand, foot, and mouth disease in Thailand from 2012 to 2014 were determined and compared to enterovirus group A prototypes. Phylogenetic analysis revealed that 13 strains resembled the B5 subgroup, while one strain from a fatal case designated THA_1219 belonged to the C4 subgroup. Similarity plot and bootscan analyses suggested that THA_1219 underwent recombination in the P2 and P3 regions. Full-genome data from this work will contribute to the study of evolution dynamics of EV71.

Influenza activity in Thailand and occurrence in different climates.

This study observed influenza activity between June 2009 and July 2014 in Thailand, a country in the Northern hemisphere with a tropical climate, and compared the results to activity in the United States (US) and Australia, which represent temperate climates in the Northern and Southern hemispheres, respectively. From Thailand, a total of 17,416 specimens were collected from patients exhibiting influenza-like illnesses and subjected to real-time PCR for the detection of influenza viruses. For comparison, laboratory confirmations of influenza originating from the US and Australia were obtained from the US CDC's FluView surveillance reports and the Australian Government's Department of Health and Ageing websites. We found that, generally, the influenza season in Thailand starts with the rainy season. This observation of influenza's annual incidence pattern provides a better understanding of its occurrence, suggesting that vaccination campaigns should be started before the influenza season begins in order to reduce transmission.

Molecular epidemiology and phylogenetic analyses of influenza B virus in Thailand during 2010 to 2014.

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.

PREVALENCE OF HUMAN ENTEROVIRUS AMONG PATIENTS WITH HAND, FOOT, AND MOUTH DISEASE AND HERPANGINA IN THAILAND, 2013.

Human enterovirus (EV) infection causes hand, foot, and mouth disease (HFMD) and herpangina (HA). We studied the prevalence of enterovirus (EV) among patients with HFMD and HA in Thailand during 2013. We conducted a study in archived specimens of patients sent for screening for enterovirus. A total of 203 clinical specimens from 184 individuals with painful blister in the oropharynx and on the palms, soles, knees, elbows or buttock were examined by semi-nested polymerase chain reaction (PCR) for the 5'UTR and VP1 genes of EV. Eighty-six samples were positive: EV71 was detected in 14 (30%), CV-A8 in 12 (26%) and CV-A16 in 10 (21%). Classification of EV species detected revealed that 46 specimens were EV-A, 14 specimens were EV-B, 1 specimen was EV-D, and 16 specimens were positive for unclassified enterovirus. The majority of individuals with EV infection were aged 2-6 years. Multiple EV-A serotypes were detected among HFMD and HA patients in our study.

Infantile osteoarticular tuberculosis misdiagnosed as Bacillus Calmette-Guerin related osteomyelitis.

Tuberculosis, a re-emerging public health problem, is uncommon in infancy. Two healthy completely immunized infants presenting with manifestations compatible with osteoarticular infection required surgical debridement. The cultures of the specimens were positive for M. tuberculosis (MTB) complex comprised multiple subspecies. One case was misdiagnosed as a Bacillus Calmette-Guerin (BCG) related osteomyelitis by a polymerase chain reaction (PCR) based on detection of genes at the region of difference 1. Genome extraction and PCR using the rimM gene and sequences analysis against MTB and BCG control samples confirmed that both specimens were infected by M. tuberculosis. The lesions were successfully healed within one year. Surgical debridement of suspected lesions is warranted in infants as a definitive treatment and to obtain tissues for further evaluation. Microbiological cultures only confirm nonspecific MTB complex infection. PCR kits may yield a false positive result. Identification of the pathogen by DNA extraction and sequence analysis should be recommended.

Prevalence and characterization of enterovirus infections among pediatric patients with hand foot mouth disease, herpangina and influenza like illness in Thailand, 2012.

Hand, foot, and mouth disease (HFMD) and herpangina are common infectious diseases caused by several genotypes of human enterovirus species A and frequently occurring in young children. This study was aimed at analyzing enteroviruses from patients with these diseases in Thailand in 2012. Detection and genotype determination of enteroviruses were accomplished by reverse transcription-polymerase chain reaction and sequencing of the VP1 region. Enterovirus-positive samples were differentiated into 17 genotypes (coxsackievirus A4 (CAV4), A5, A6, A8, A9, A10, A12, A16, A21, B1, B2, B4, B5, echovirus 7, 16, 25 and Enterovirus 71). The result showed CAV6 (33.5%), followed by CAV16 (9.4%) and EV71 (8.8%) as the most frequent genotypes in HFMD, CAV8 (19.3%) in herpangina and CAV6 (1.5%) in influenza like illness. Enterovirus infections were most prevalent during July with 34.4% in HFMD, 39.8% in herpangina and 1.6% in ILI. The higher enterovirus infection associated with HFMD and herpangina occurred in infants over one year-old. This represents the first report describing the circulation of multiple enteroviruses in Thailand.

Prevalence and genetic characterization of human coronaviruses in southern Thailand from July 2009 to January 2011.

Emergence of viruses belonging to the coronavirus family has been widespread in the past, causing respiratory infections in humans, such as severe acute respiratory syndrome (SARS). This study investigated the prevalence of human coronavirus (HCoV) and characterized the molecular viral genetics. We collected 1,254 samples from patients diagnosed with respiratory infection in southern Thailand from July 2009 to January 2011 and screened for HCoV by RT-PCR and genotyped by BLAST analysis of nsp12 gene. Phylogenetic analysis was performed based on S gene sequences. Thirty-five of 1,254 samples were positive for HCoV. Viral genotyping revealed 4 genotypes with HCoV-OC43 being the predominant genotype. Viral prevalence and genotype distribution were not in accordance with seasonal distribution. Phylogenetic analysis and deduced amino acid sequences of the S gene showed amino acid variations in each genotype. The S gene sequence of HCoV-OC43 genotype indicated that it resulted from recombination between subgenotypes B and C. Viral genetics analysis disclosed genetic variations of HCoV and additionally, it can provide information suitable for monitoring and prevention of the emergence and re-emergence of various types of coronavirus.

Epidemiology of seasonal influenza in Bangkok between 2009 and 2012.

INTRODUCTION: This study investigated influenza activity in Bangkok, Thailand between June 2009 and July 2012. METHODOLOGY: Real-time reverse transcription polymerase chain reaction (RT-PCR) was performed to detect influenza viruses among patients with influenza-like illnesses. RESULTS: Of the 6417 patients tested, influenza virus infection was detected in 42% (n = 2697) of the specimens. Influenza A pH1N1 viruses comprised the predominant strain between 2009 and 2010, and seasonal influenza (H3) had a high prevalence in 2011. Laboratory data showed a prevalence and seasonal pattern of influenza viruses. In 2009, influenza activity peaked in July, the rainy season. In 2010, influenza activity happened in two phases, with the initial one at the beginning of the year and another peak between June and August 2010, which again corresponded to the rainy period. Influenza activity was low for several consecutive weeks at the beginning of 2011, and high H3N2 activity was recorded during the rainy season between July and September 2011. However, from the beginning of 2012 through July 2012, pH1N1, influenza H3N2, and influenza B viruses continuously circulated at a very low level. CONCLUSION: The seasonal pattern of influenza activity in Thailand tended to peak during rainy season between July and September.