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Background: Influenza A(H3N2) virus evolves continuously. Its hemagglutinin (HA) and neuraminidase (NA) genes have high genetic variation due to the antigenic drift. This study aimed to investigate the characteristics and evolution of HA and NA genes of the influenza A(H3N2) virus in Thailand. Methods: Influenza A positive respiratory samples from 2015 to 2018 were subtyped by multiplex real-time RT-PCR. Full-length HA and NA genes from the positive samples of influenza A(H3N2) were amplified and sequenced. Phylogenetic analysis with the maximum likelihood method was used to investigate the evolution of the virus compared with the WHO-recommended influenza vaccine strain. Homology modeling and N-glycosylation site prediction were also performed. Results: Out of 443 samples, 147 (33.18%) were A(H1N1)pdm09 and 296 (66.82%) were A(H3N2). The A(H3N2) viruses circulating in 2015 were clade 3C.2a whereas sub-clade 3C.2a1 and 3C.2a2 dominated in 2016–2017 and 2018, respectively. Amino acid substitutions were found in all antigenic sites A, B, C, D, and E of HA but the majority of the substitutions were located at antigenic sites A and B. The S245N and N329S substitutions in the NA gene affect the N-glycosylation. None of the mutations associated with resistance to NA inhibitors were observed. Mean evolutionary rates of the HA and NA genes were 3.47 × 10−3 and 2.98 × 10−3 substitutions per site per year. Conclusion: The influenza A(H3N2) virus is very genetically diverse and is always evolving to evade host defenses. The HA and NA gene features including the evolutionary rate of the influenza A(H3N2) viruses that were circulating in Thailand between 2015 and 2018 are described. This information is useful for monitoring the genetic characteristics and evolution in HA and NA genes of influenza A(H3N2) virus in Thailand which is crucial for predicting the influenza vaccine strains resulting in high vaccine effectiveness. [ABSTRACT FROM AUTHOR]

Background. Ongoing outbreaks of H5N1 highly pathogenic avian influenza (HPAI) viruses and the emergence of the genetic-related hemagglutinin (HA) gene of reassortant H5Nx viruses currently circulating in wild birds and poultries pose a great global public health concern. In this study, we comprehensively analyzed the genetic evolution of Thai H5N1 HA and neuraminidase (NA) genes between 2003 and 2010. The H5N1 Thailand virus clade 2.3.4 was also genetically compared to the currently circulating clade 2.3.4.4 of H5Nx viruses. Methods. Full-length nucleotide sequences of 178 HA and 143 NA genes of H5N1 viruses circulating between 2003 and 2010 were phylogenetically analyzed using maximum likelihood (ML) phylogenetic construction. Bayesian phylogenetic trees were reconstructed using BEAST analysis with a Bayesian Markov chain Monte Carlo (MCMC) approach. The maximum clade credibility (MCC) tree was determined, and the time of the most recent common ancestor (tMRCA) was estimated. The H5N1 HA nucleotide sequences of clade 2.3.4 Thailand viruses were phylogenetically analyzed using ML phylogenetic tree construction and analyzed for nucleotide similarities with various subtypes of reassortant H5Nx HA clade 2.3.4.4. Results. ML phylogenetic analysis revealed two distinct HA clades, clade 1 and clade 2.3.4, and two distinctNAgroups within the corresponding H5 clade 1 viruses. Bayesian phylogenetic reconstruction for molecular clock suggested that the Thai H5N1 HA and NAemerged in 2001.87 (95% HPD: 2001.34-2002.49) and 2002.38 (95% HPD: 2001.99-2002.82), respectively, suggesting that the virus existed before it was first reported in 2004. The Thai H5N1 HA clade 2.3.4 was grouped into corresponding clades 2.3.4, 2.3.4.1, 2.3.4.2, and 2.3.4.3, and shared nucleotide similarities to reassortant H5Nx clade 2.3.4.4 ranged from 92.4-96.8%. Phylogenetic analysis revealed monophyletic H5Nx clade 2.3.4.4 evolved from H5N1 clade 2.3.4. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract: A total of 300 nasal swabs were collected from 5 pig farms in two provinces in the Eastern part of Thailand in February 2011 and were subjected to viral isolation of influenza A viruses. Two H3N2 and 6 H1N1 influenza A viruses were isolated from swabs collected from clinically healthy weaning pigs on farms in Chonburi and Chachoengsao provinces, respectively. The H3N2 isolates consisted of the hemagglutinin (HA) and neuraminidase (NA) genes closely related to Thai SIVs and derived from a cluster of human seasonal H3N2 strains circulating around 1996–1997. The remaining gene segments of the isolates originated from the Pandemic (H1N1) 2009 (A (H1N1) pdm09) virus. Antigenicity of the H3N2 isolates was distinguishable from a human seasonal vaccine strain in the 1996–1998 seasons that represented antigenicity of the seasonal strains around 1996–1998. Nasal swabs from a Chachoengsao farm yielded A (H1N1) pdm09 viruses in chicken embryonated eggs and MDCK cells. A (H1N1) pdm09 viruses isolated in this study grew poorly in MDCK cells. Deduced amino acid sequences of the HA1 region of the HA protein of egg isolated viruses were identical to the sequences directly amplified from original swab samples. Our result demonstrated that the A (H1N1) pdm09 virus has been established in the Thai pig population and this has resulted in genetic reassortment with Thai SIV that previously circulated among pigs. [Copyright &y& Elsevier]

Abstract: Human infections with highly pathogenic H5N1 avian influenza viruses continue to occur in many parts of the world and pose a considerable public health threat. With the use of animal models, the identification of virulence determinants has been instrumental in improving our understanding of how these viruses cause severe disease in humans. Two genetically similar H5N1 viruses (A/Thailand/16/2004 and A/Thailand/SP83/2004) exhibit high or low virulence phenotypes, respectively, in multiple animal models. Reassortant viruses were generated from this virus pair and evaluated in ferrets. Each of the polymerase genes of A/Thailand/16/2004 virus individually conferred increased virulence to A/Thailand/SP83/2004 virus while the neuraminidase of the low virulence virus reduced virulence and replication efficiency of the virulent virus in ferrets unless the homologous HA was present. Our results demonstrate that H5N1 virus virulence determinants are polygenic and that there is an important correlation between polymerase adaptation, efficient replication in the host, and virulence. [Copyright &y& Elsevier]

Abstract: Amantadine and oseltamivir are used to treat influenza A virus infections; however, resistance to these drugs has been widely reported throughout the world. In this study, the frequency and genetic characteristics of the drug-resistant influenza A viruses that circulated in Thailand from 2006 to 2008 were investigated. The nucleotide sequences of the NA and M2 genes were elucidated in order to identify mutations that confer oseltamivir- and amantadine-resistant phenotypes, respectively. A total of 66 influenza A viruses including 44 H1N1 and 22 H3N2 subtypes isolated in Bangkok and 13 provinces of Thailand from 2006 to 2008 were analyzed. Our results demonstrated that seven out of 32 (22%) of the H1N1 viruses isolated in 2006 in Thailand carried the amino acid S31N substitution, which confers amantadine-resistance, although no isolates in 2007 or 2008 possessed the mutation. In the cases of oseltamivir-resistance, four of 10 (40%) of the H1N1 viruses isolated in 2008 were predicted to be resistant to the drug, although none of the 34 viruses isolated in 2006 or 2007 were predicted to be resistant. Surprisingly, all 9 H3N2 viruses isolated in 2008 appeared to be resistant to the amantadine and none were resistant in 2006 or 2007. Phylogenetic analysis based on the HA, M, and NA genes demonstrated that the amantadine-resistant H1N1 isolates had been produced by genetic reassortment. All of the amantadine-resistant H3N2 viruses were clustered in one of these three genes and possessed double mutations of S193F and D225N in the HA gene. [Copyright &y& Elsevier]

Swine have been known to be a suitable host for influenza A virus. In Thailand, phylogenetic analysis on swine influenza virus (SIV) has as yet not been attempted. The present report presents molecular and phylogenetic analysis performed on SIV in Thailand. In this study, 12 SIV isolates from the central and eastern part of Thailand were subtyped and the molecular genetics of hemagglutinin and neuraminidase were elucidated. Three subtypes, H1N1, H1N2 and H3N2, are described. Phylogenetic analysis of the SIV hemagglutinin and neuraminidase genes shows individual clusters with swine, human or avian influenza virus at various global locations. Furthermore, amino acid substitutions were detected either at the receptor binding site or the antigenic sites of the hemagglutinin gene. [ABSTRACT FROM AUTHOR]

In December 2003, the largest outbreak of highly pathogenic avian influenza H5N1 occurred among poultry in 8 Asian countries. A limited number of human H5N1 infections have been reported from Vietnam and Thailand, with a mortality rate approaching 70%. Deaths have occurred in otherwise healthy young individuals, which is reminiscent of the 1918 Spanish influenza pandemic. The main presenting features were fever, pneumonitis, lymphopenia, and diarrhea. Notably, sore throat, conjunctivitis, and coryza were absent. The H5N1 strains are resistant to amantadine and rimantadine but are susceptible to neuraminidase inhibitors, which can be used for treatment and prophylaxis. The widespread epidemic of avian influenza in domestic birds increases the likelihood for mutational events and genetic reassortment. The threat of a future pandemic from avian influenza is real. Adequate surveillance, development of vaccines, outbreak preparedness, and pandemic influenza planning are important. This article summarizes the current knowledge on avian influenza, including the virology, epidemiology, diagnosis, and management of this emerging disease. [ABSTRACT FROM AUTHOR]

• This is the first report on the antiviral activity of ethanolic extracts of L.strychnifolium stems and leaves against Influezna A subtype H5N1. • L.strychnifolium is a potential neuraminidase inhibitor. • Ethanolic and aqueous extracts of L.strychnifolium stems and leaves showed broad-antibacterial activity against both Gram negative and Gram positive bacteria. • Inositol; Olefins; Palmitic acid; Diterpene; Unsaturated fatty acid; Methyl-p-hydrozybenzoate and 1,2,3-Benzenetriol were identified by GC-MSD scan. The leaves and stems of Lysiphyllum strychnifolium (Craib) A. Schmitz (Fabaceae family) have been traditionally used in Thailand for detoxification and to treat pesticide poisoning in humans. To uncover novel uses of L.strychnifolium , the possible antiviral properties against avian influenza virus A, strain H5N1, were explored in this study. The ethanolic extracts of L.strychnifolium leaves and stems showed good inhibitory activities against H5N1 neuraminidase. Such activities have not been previously reported for this plant. Aqueous extracts did not show any inhibition. Thereafter, the anti-neuraminidase activities of ethanolic extracts were evaluated using fluorometric determination via a MUNANA-based enzyme inhibition assay. Ethanolic extracts of both stems and leaves showed good inhibitory activities against neuraminidase from Influenza A H5N1 with IC 50 values of 55.3 and 70.0 μg/mL, respectively. Moreover, anti-bacterial activities of aqueous and ethanolic extracts of L.strychnifolium leaves and stems were tested using the disc diffusion method. All extracts showed broad antibacterial activities against both Gram positive and Gram negative bacterial strains. Phytochemical constituents of all extracts were identified through Gas Chromatography - Mass Spectrometry (GC-MS) and revealed some compounds such as Methyl-p-hydrozybenzoate; Mome inositol; n-Hexadecanoic acid; Tetradecanamide; (Z)-9-Octadecanamide; 1,2,3,-Benzenetriol; Methylparaben; 4-(4-Hydroxyphenyl)-2-butanone; n-Hexadecanoic acid; Ethyl hexadecanoate; Phytol; (Z)-9-Octadecanoic acid, and Octadecanoic acid. This is the first report of neuraminidase inhibitor against Influenza A H5N1 and antibacterial activities derived from L.strychnifolium extracts. The authors suggest that this plant is an alternative source for treatment against influenza viruses and potential antibiotic agent. [ABSTRACT FROM AUTHOR]

The article reports that Roche AG's Tamiflu anti-influenza drug has been proven to work against the H5N1 avian virus, which the World Health Organization says could become the source of influenza pandemic. In a study, researchers from the Queen Mary Hospital in London said the neuraminidase inhibitor Tamiflu is effective against avian and human forms of the virus. According to researchers, these are the first data to show oseltamivir to be effective against this highly pathogenic strain, which is currently circulating in Vietnam and Thailand.

Influenza A viruses are a threat to poultry and human health. We investigated evolution of influenza A virus H7 and N9 subtypes in wild and domestic birds. Influenza A(H7N9) virus probably emerged after a long silent circulation in live poultry markets in eastern Asia.

A lectin from Thai marine carb (Scylla serrata) hemolymph has been isolated and purified by affinity column chromatography and preparative electrophoresis. The amino acid composition and 10 amino-terminal residues have been deduced, and its reactivities have been studied using a biotin labeling technique. A method for the determination of sialoglycoconjugates in human serum is described using this lectin. The principle is based on the reaction between the sialoglycoconjugates and biotinylated lectin. The bovine submaxillary mucin (BSM) is immobilized on polystyrene microplate. The unknown sample or sialoglycoconjugate (BSM equivalent) standards, together with excess biotinylated purified lectin (B-lectin), are then added. The B-lectin that binds to the immobilized BSM is then incubated with the peroxidase-conjugated monoclonal antibiotin antibody, and the color that develops after the addition of enzyme substrate is determined by light absorption using a microplate reader. The assay is not only convenient and reliable, but also capable of measuring sialoglycoconjugates in solution at the submicrogram level. It was used in determining the sialoglycoconjugates in human serum from normal subjects and samples positive for carcinoembryonic antigen.