Evaluation of rapid influenza virus tests in patients with influenza-like illness in Thailand.

BACKGROUND: The influenza virus is responsible for causing major respiratory tract symptoms. A fast, accurate diagnosis is essential for efficient treatment, especially in patients with complications. The Rapid Influenza Diagnostic Tests (RIDTs) for influenza detection have been developed to subtype the influenza virus. The re-evaluation of the rapid test is needed in terms of specificity, sensitivity, and accuracy. METHODS: From August 13, 2010 to September 22, 2011, 1,076 nasal aspirates were obtained from patients, ages ranging from 15 days to 98 years, with symptoms of influenza-like illness (ILI) and evaluated by 2 types of RIDTs, Standard Diagnosis (SD) and QuickVue (QV) Rapid tests followed by real-time RT-PCR. The results from the rapid test diagnoses were compared to those from real-time RT-PCR. RESULTS: During 2010 and 2011, the estimated sensitivity of the SD rapid test for seasonal H3, human pandemic H1N1, and influenza B infection was 49.4%, specificity was 84.1%, positive predictive value was 47.6%, and negative predictive value was 85% while those of the QV rapid test were 63.4%, 96.7%, 94.8%, and 80.3%, respectively. Infant patients (< or = 5 years) yielded less false negatives while adolescents and adults (older than 5 years) showed more false negatives 8.8% and 15.2%, respectively. Using rapid test diagnosis, H3N2 influenza virus was found with more false negative results (11.1%) than the other viruses (1.1 - 3.5%). The SD rapid test appeared to be more sensitive than the QV test during high season activity while the QV test was more sensitive during the period of low influenza virus activity. CONCLUSIONS: Due to persistent genetic drift of the influenza virus, the available RIDTs should be re-evaluated each year. During 2010 - 2011, the QV rapid test showed more reliable results than the SD rapid test. However, the false negative results of H3N2 influenza virus detection during its peak should be cause for concern. Some of the results, e.g. patients with complications, should be compared with real-time RT-PCR as the gold standard method for detecting influenza virus infection.

Influenza surveillance in southern Thailand during 2009-2010.

The aim of this study was to determine the epidemiology of influenza infection among patients with influenza-like illness by real-time RT-PCR in southern Thailand from August 2009 to January 2011. The predominant strain in Thung Song District was influenza A. Sporadic cases of influenza occured year round but the incidence peaked from August to November 2009 and July to November 2010. During August to November 2009, pandemic H1N1 (pH1N1) activity was observed along with a low level of seasonal influenza co-circulation. Subsequently, seasonal influenza (H3) activity increased and became the predominant influenza strain, with co-circulation with pH1N1 and influenza B during the 2010 influenza season. Continual surveillance of influenza activity is useful for public health planning in southern Thailand and plays a major role in future influenza control and prevention measures.

Serological analysis of human pandemic influenza (H1N1) in Thailand.

The study was aimed at determining the prevalence of pandemic influenza (H1N1) 2009 among patients with respiratory tract diseases during July-December 2009 using real-time reverse transcription polymerase chain reaction. Haemagglutination inhibition (HI) assay was performed to detect antibody titres against pandemic influenza in 255 medical personnel, 307 members of the general population during the second week of December 2009 in Khon Kaen province, Thailand, and in 100 stored sera collected from people of different age-groups during 2008. The results showed that the pandemic (H1N1) 2009 had occurred during July-December 2009. The results of the HI test after the wave of this outbreak showed that 123 (48%) of the 255 sera collected from the medical personnel, 109 (36%) of the 307 sera obtained from the general population, and only two of the 100 stored sera from 2008 contained antibodies (HI titres > or = 40) against pandemic influenza. Antibody against the pandemic (H1N1) 2009 was found in at least one-third of the population. In conclusion, the prevalence of virus and serological data obtained from the study can be used as the serological background level of the Thai population after the July-December pandemic. Finally, the serological data might be useful for outbreak-prevention and control strategies and for the management of vaccination for the pandemic (H1N1) 2009 in Thailand.

Determination of antibody response to the human pandemic influenza H1N1 2009 among patients with influenza-like illness and high risk groups.

The global population has been exposed to the novel pandemic H1N1 influenza virus since mid March 2009, causing the expansion of respiratory illness around the world, including Thailand. To evaluate the antibody titers against human pandemic influenza (H1N1) in Thai people with influenza-like illness (ILI), 45 paired serum samples (acute and convalescent) were subjected to hemagglutination inhibition (HI) test and real-time RT-PCR. Most serum samples of ILI patients positive by real-time RT-PCR displayed an at least four-fold antibody increase of HI titers against pandemic influenza (H1N1). In addition, to determine cross-reactivity with human seasonal H1N1 influenza, viral antigen from the seasonal H1N1 was used to detect antibody against seasonal H1N1 influenza and all sera showed negative results. We also studied the single sera samples from the high risk medical personals collected before and after the pandemic influenza (HIN1) outbreaks for antibodies against seasonal H3 influenza virus infection. The results showed lack of cross-reactivity to the human pandemic H1N1 influenza virus. HI antibody testing to pandemic influenza (H1N1) can be used for the diagnosis, preventive and control measures of potential outbreaks.

Hemoptysis in children with pandemic influenza H1N1 2009 infection.

Three patients were admitted to Hat Yai Hospital, Songkhla Thailand with hemoptysis. They were previously healthy children aged 6, 13, and 14 years old who had attended schools in which outbreaks of influenza had occurred. They all had a history of fever, rhinorrhea, and severe cough accompanied by hemoptysis. Two developed hemoptysis on Day 3 and the third on Day 6 of illness, with one of them displaying massive hemoptysis. Chest radiographs were compatible with viral pneumonia in two cases and the third case was unremarkable. Coagulation profiles in the severe case were carried out and were normal. All the patients responded very well to treatment with oseltamivir and did not require intubation. Their subsequent nasopharyngeal swabs were positive for human pandemic influenza A H1N1 by real-time reverse transcription-polymerase chain reaction (RT-PCR), and their sputum for acid-fast bacilli and tuberculin skin tests were negative.

Genetic Variations in XRCC4 (rs1805377) and ATF6 (rs2070150) are not Associated with Hepatocellular Carcinoma in Thai Patients with Hepatitis B Virus Infection.

The liver is one of the most common sites of cancer in the world, hepatocellular carcinoma (HCC) predominating. Chronic hepatitis B virus infection (CHB) is considered as an important potential risk factors for HCC. Different people have diverse responses to HBV infection regarding the likelihood of HCC development, and host factors such as single nucleotide polymorphisms (SNPs) might account for this. The present study was conducted to evaluate any association between SNP frequencies in two genes, XRCC4 (rs1805377) and ATF6 (rs2070150), and the risk of CHB and HCC development in Thai patients. The study covered 369 subjects including 121 HCC patients, 141 with chronic hepatitis B virus infection (CHB) and 107 healthy controls. With TaqMan real-time PCR, the results showed that no significant association between XRCC4 (rs1805377) and ATF6 (rs2070150) and risk of HCC in the Thai population. From this first study of the 2 polymorphisms and HCC in Thailand it can concluded that rs1805377 and rs2070150 polymorphisms may not be applicable as genetic markers in the Thai population for HCC assessment.

Human microRNAs profiling in response to influenza A viruses (subtypes pH1N1, H3N2, and H5N1).

MicroRNAs (miRNAs) play an important role in regulation of gene silencing and are involved in many cellular processes including inhibition of infected viral replication. This study investigated cellular miRNA expression profiles operating in response to influenza virus in early stage of infection which might be useful for understanding and control of viral infection. A549 cells were infected with different subtypes of influenza virus (pH1N1, H3N2 and H5N1). After 24 h post-infection, miRNAs were extracted and then used for DNA library construction. All DNA libraries with different indexes were pooled together with equal concentration, followed by high-throughput sequencing based on MiSeq platform. The miRNAs were identified and counted from sequencing data by using MiSeq reporter software. The miRNAs expressions were classified into up and downregulated miRNAs compared to those found in non-infected cells. Mostly, each subtype of influenza A virus triggered the upregulated responses in miRNA expression profiles. Hsa-miR-101, hsa-miR-193b, hsa-miR-23b, and hsa-miR-30e* were upregulated when infected with all three subtypes of influenza A virus. Target prediction results showed that virus infection can trigger genes in cellular process, metabolic process, developmental process and biological regulation. This study provided some insights into the cellular miRNA profiling in response to various subtypes of influenza A viruses in circulation and which have caused outbreaks in human population. The regulated miRNAs might be involved in virus-host interaction or host defense mechanism, which should be investigated for effective antiviral therapeutic interventions.

Original Research: Analysis of hepatic microRNA alterations in response to hepatitis B virus infection and pegylated interferon alpha-2a treatment.

Interferons play important roles in defense mechanisms against viral infection, and thus interferon therapy has been a standard treatment in chronic hepatitis B patients. Interferons signaling pathways promote interferon-inducible genes including microRNAs. In this research, we aimed to determine microRNAs expression profiles in vitro and in vivo For in vitro model, Huh7 cells were transfected with or without hepatitis B virus plasmid for 6 h, and then treated with 100 ng of pegylated-interferon alpha-2a for 24 h. In vivo, we defined microRNAs expression profiles in pair-liver tissues of chronic hepatitis B patients in comparison between before and after treatment of pegylated-interferon alpha-2a for 48 weeks. Cellular small RNAs were extracted followed by library preparation. To determine microRNAs expression profiles, the next-generation sequencing was carried out on MiSeq platform (Illumina®). In vitro analysis demonstrated that microRNAs can be classified into up-regulated and down-regulated microRNAs in response to hepatitis B virus, interferon, and combination of hepatitis B virus and interferon. Moreover, in vivo analysis revealed microRNAs profiles in non-responders, responders without hepatitis B surface antigen clearance, and responders with hepatitis B surface antigen clearance. The target genes of the candidate microRNAs were determined in terms of roles in cellular pathways and immune response, which might be related to treatment in chronic hepatitis B patients. Results revealed that two down-regulated microRNAs including miR-185-5p and miR-186-5p were correlated in both in vitro and in vivo studies. These two microRNAs might be represented as specific hepatic microRNAs responding to hepatitis B virus and pegylated-interferon alpha-2a treatment, which may remarkable and attractive for further study involving in the association of their target genes and prediction of pegylated-interferon alpha-2a response. Interestingly, microRNAs expression patterns might be useful for understanding the response mechanism and serve as biomarkers for prediction of pegylated-interferon alpha-2a treatment response in patients with chronic hepatitis B.

Human miR-3145 inhibits influenza A viruses replication by targeting and silencing viral PB1 gene.

MicroRNAs (miRNAs) play an important role in the regulation of gene expression and are involved in many cellular processes including inhibition of viral replication in infected cells. In this study, three subtypes of influenza A viruses (pH1N1, H5N1 and H3N2) were analyzed to identify candidate human miRNAs targeting and silencing viral genes expression. Candidate human miRNAs were predicted by miRBase and RNAhybrid based on minimum free energy (MFE) and hybridization patterns between human miRNAs and viral target genes. In silico analysis presented 76 miRNAs targeting influenza A viruses, including 70 miRNAs that targeted specific subtypes (21 for pH1N1, 27 for H5N1 and 22 for H3N2) and 6 miRNAs (miR-216b, miR-3145, miR-3682, miR-4513, miR-4753 and miR-5693) that targeted multiple subtypes of influenza A viruses. Interestingly, miR-3145 is the only candidate miRNA targeting all three subtypes of influenza A viruses. The miR-3145 targets to PB1 encoding polymerase basic protein 1, which is the main component of the viral polymerase complex. The silencing effect of miR-3145 was validated by 3'-UTR reporter assay and inhibition of influenza viral replication in A549 cells. In 3'-UTR reporter assay, results revealed that miR-3145 triggered significant reduction of the luciferase activity. Moreover, expression of viral PB1 genes was also inhibited considerably (P value < 0.05) in viral infected cells expressing mimic miR-3145. In conclusion, this study demonstrated that human miR-3145 triggered silencing of viral PB1 genes and lead to inhibition of multiple subtypes of influenza viral replication. Therefore, hsa-miR-3145 might be useful for alternative treatment of influenza A viruses in the future.

Human miR-5193 Triggers Gene Silencing in Multiple Genotypes of Hepatitis B Virus.

Hepatitis B virus (HBV) infection can lead to various disease states including asymptomatic, acute hepatitis, chronic hepatitis, liver cirrhosis and hepatocellular carcinoma (HCC), which remain a major health problem worldwide. Previous studies demonstrated that microRNA (miRNAs) plays an important role in viral replication. This study aimed to predict and evaluate human miRNAs targeting multiple genotypes of HBV. Candidate human miRNAs were analyzed by data obtained from miRBase and RNAhybrid. Then miRNAs were selected based on hybridization patterns and minimum free energy (MFE). The silencing effect of miRNA was evaluated by real-time PCR, the luciferase reporter assay and the ELISA assay. Five human miRNAs including miR- 142-5p, miR-384, miR-500b, miR-4731-5p and miR-5193 were found to target several HBV genotypes. Interestingly, miR-5193 was found to be the most potent miRNA that could target against all HBV transcripts in almost all HBV genotypes with a highly stable hybridization pattern (5' canonical with MFE lower than -35 kcal/mol). Moreover, miR-5193 caused significant silencing in luciferase activity (53% reduction), luciferase transcript (60% reduction) and HBV surface antigen (HBsAg) production (20-40% reduction depending on genotypes). Therefore, miR-5193 might be useful and have a vital role for inhibition of HBV replication in the future.

Single Nucleotide Polymorphisms in miR-149 (rs2292832) and miR-101-1 (rs7536540) Are Not Associated with Hepatocellular Carcinoma in Thai Patients with Hepatitis B Virus Infection.

MicroRNAs directly and indirectly influence many biological processes such as apoptosis, cell maintenance, and immune responses, impacting on tumor genesis and metastasis. They modulate gene expression at the post- transcriptional level and are associated with progression of liver disease. Hepatocellular carcinoma (HCC) is a cancer which mostly occurs in males. There are many factors affect HCC development, for example, hepatitis B virus (HBV), hepatitis C virus (HCV) and human immunodeficiency virus (HIV), co-infection, environmental factors including alcohol, aflatoxin consumption and host-related factors such as age, gender immune response, microRNA and single nucleotide polymorphisms (SNPs). Chronic infection with the hepatitis B virus is the major factor leading to HCC progression since it causes the liver injury. At present, there are many reports regarding the association of SNPs on miRNAs and the HCC progression. In this research, we investigated the role of miR- 149 (rs2292832) and miR-101-1 (rs7536540) with HCC progression in Thai population. The study included 289 Thai subjects including 104 HCC patients, 90 patients with chronic hepatitis B virus infection (CHB) and 95 healthy control subjects. The allele and genotype of rs2292832 and rs7536540 polymorphisms were determined by TaqMan real-time PCR assay. Our results revealed no significant association between miR-149 (rs2292832) and miR-101-1 (rs7536540) and the risk of HCC in our Thai population. However, this research is the first study of miR-149 (rs2292832) and miR-101-1 (rs7536540) in HCC in Thai populations and the results need to be confirmed with a larger population.

Global alert to avian influenza virus infection: from H5N1 to H7N9.

Outbreak of a novel influenza virus is usually triggered by mutational change due to the process known as 'antigenic shift' or re-assortment process that allows animal-to-human or avian-to-human transmission. Birds are a natural reservoir for the influenza virus, and subtypes H5, H7, and H9 have all caused outbreaks of avian influenza in human populations. An especially notorious strain is the HPAI influenza virus H5N1, which has a mortality rate of approximately 60% and which has resulted in numerous hospitalizations, deaths, and significant economic loss. In March 2013, in Eastern China, there was an outbreak of the novel H7N9 influenza virus, which although less pathogenic in avian species, resulted in 131 confirmed cases and 36 deaths in humans over a two-month span. The rapid outbreak of this virus caused global concern but resulted in international cooperation to control the outbreak. Furthermore, cooperation led to valuable research-sharing including genome sequencing of the virus, the development of rapid and specific diagnosis, specimen sharing for future studies, and vaccine development. Although a H7N9 pandemic in the human population is possible due to its rapid transmissibility and extensive surveillance, the closure of the live-bird market will help mitigate the possibility of another H7N9 outbreak. In addition, further research into the source of the outbreak, pathogenicity of the virus, and the development of specific and sensitive detection assays will be essential for controlling and preparing for future H7N9 outbreaks.

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.

Erythrocyte binding preference of human pandemic influenza virus a and its effect on antibody response detection.

BACKGROUND: Validation of hemagglutination inhibition (HI) assays is important for evaluating antibody responses to influenza virus, and selection of erythrocytes for use in these assays is important. This study aimed to determine the correlation between receptor binding specificity and effectiveness of the HI assay for detecting antibody response to pandemic influenza H1N1 (pH1N1) virus. METHODS: Hemagglutination (HA) tests were performed using erythrocytes from 6 species. Subsequently, 8 hemagglutinating units of pH1N1 from each species were titrated by real-time reverse transcription-PCR. To investigate the effect of erythrocyte binding preference on HI antibody titers, comparisons of HI with microneutralization (MN) assays were performed. RESULTS: Goose erythrocytes showed most specific binding with pH1N1, while HA titers using human erythrocytes were comparable to those using turkey erythrocytes. The erythrocyte binding efficiency was shown to have an impact on antibody detection. Comparing MN titers, HI titers using turkey erythrocytes yielded the most accurate results, while those using goose erythrocytes produced the highest geometric mean titer. Human blood group O erythrocytes lacking a specific antibody yielded results most comparable to those obtained using turkey erythrocytes. Further, pre-existing antibody to pH1N1 and different erythrocyte species can distort HI assay results. CONCLUSIONS: HI assay, using turkey and human erythrocytes, yielded the most comparable and applicable results for pH1N1 than those by MN assay, and using goose erythrocytes may lead to overestimated titers. Selection of appropriate erythrocyte species for HI assay allows construction of a more reliable database, which is essential for further investigations and control of virus epidemics.

Whole genome characterization, phylogenetic and genome signature analysis of human pandemic H1N1 virus in Thailand, 2009-2012.

BACKGROUND: Three waves of human pandemic influenza occurred in Thailand in 2009-2012. The genome signature features and evolution of pH1N1 need to be characterized to elucidate the aspects responsible for the multiple waves of pandemic. METHODOLOGY/FINDINGS: Forty whole genome sequences and 584 partial sequences of pH1N1 circulating in Thailand, divided into 1(st), 2(nd) and 3(rd) wave and post-pandemic were characterized and 77 genome signatures were analyzed. Phylogenetic trees of concatenated whole genome and HA gene sequences were constructed calculating substitution rate and d(N)/d(S) of each gene. Phylogenetic analysis showed a distinct pattern of pH1N1 circulation in Thailand, with the first two isolates from May, 2009 belonging to clade 5 while clades 5, 6 and 7 co-circulated during the first wave of pH1N1 pandemic in Thailand. Clade 8 predominated during the second wave and different proportions of the pH1N1 viruses circulating during the third wave and post pandemic period belonged to clades 8, 11.1 and 11.2. The mutation analysis of pH1N1 revealed many adaptive mutations which have become the signature of each clade and may be responsible for the multiple pandemic waves in Thailand, especially with regard to clades 11.1 and 11.2 as evidenced with V731I, G154D of PB1 gene, PA I330V, HA A214T S160G and S202T. The substitution rate of pH1N1 in Thailand ranged from 2.53×10(-3)±0.02 (M2 genes) to 5.27×10(-3)±0.03 per site per year (NA gene). CONCLUSIONS: All results suggested that this virus is still adaptive, maybe to evade the host's immune response and tends to remain in the human host although the d(N)/d(S) were under purifying selection in all 8 genes. Due to the gradual evolution of pH1N1 in Thailand, continuous monitoring is essential for evaluation and surveillance to be prepared for and able to control future influenza activities.

Detection of oseltamivir sensitive/resistant strains of pandemic influenza A virus (H1N1) from patients admitted to hospitals in Thailand.

Oseltamivir has been used widely for prophylaxis or treatment during outbreaks of the pandemic influenza virus (H1N1) in several countries. The aim of this study was to develop a real-time RT-PCR (reverse transcription-polymerase chain reaction) to be applied for detection and monitoring of the oseltamivir resistant strains of this virus during three outbreaks (May 2009 to October 2010) in Thailand. The real-time RT-PCR assay for detecting H275Y proved highly specific for the pandemic influenza virus (H1N1) as no cross-amplification was detected with other respiratory viruses or human total RNA. The assay was also highly sensitive with a detection limit as low as 100 copies/µL for both wild-type and resistant strains. The performance of the assay was evaluated in terms of amplification efficiency (100%). The results obtained by real-time RT-PCR were in complete agreement with direct nucleotide sequencing. However, real-time RT-PCR provided more detail on the relative quantities of ratios between resistant and sensitive strains in each individual. The results revealed that four of 1288 (0.31%) patients were infected with the oseltamivir resistant strain. The number of patients infected by resistant strains was higher during the third (0.61%) and second (0.24%) waves than during the first (0%) outbreak. In conclusion, the real-time RT-PCR assay for H275Y detection is advantageous because it is specific, sensitive, and provides quantitative data. And it would be useful for large-scale testing and monitoring of oseltamivir resistant strains of the pandemic influenza A virus (H1N1).

Epidemiological and serological surveillance of human pandemic influenza A virus infections during 2009-2010 in Thailand.

Since April 2009, the outbreak of human pandemic influenza A (H1N1) virus (pH1N1) infection has spread from North America to other parts of the world, and currently, pH1N1 is the predominant circulating strain of influenza viruses. Our objectives were to perform a serological survey of medical personnel at the Chumphae Hospital in Thailand and to investigate the prevalence of pH1N1 in randomly selected patients diagnosed with respiratory tract disease. Prevalence of pH1N1 in the patients was determined by performing real-time reverse transcription-polymerase chain reaction. The study was carried out between July 2009 and November 2010. Seroprevalence of hemaglutination inhibition (HI) titers among medical personnel was established in three cross-sectional studies at the end of each wave of the pandemic by performing HI assay to detect antibodies against pH1N1. Infection by the pH1N1 peaked between July and October 2009; the second wave was from January to March 2010 and the third wave from June to November 2010. The HI titers after the first, second, and third waves were 48.2%, 22.4%, and 25.7%, respectively. After the second and third waves, 52.1% and 45.3% of the medical personnel who had received pH1N1 vaccination had HI titers ≥ 40. These findings show that seasonal influenza strain in Chumphae and the predominant influenza strain from each wave was pH1N1. HI assay results also represent the severity of the attack rate in each wave.

Molecular evolution of human H1N1 and H3N2 influenza A virus in Thailand, 2006-2009.

BACKGROUND: Annual seasonal influenza outbreaks are associated with high morbidity and mortality. OBJECTIVE: To index and document evolutionary changes among influenza A H1N1 and H3N2 viruses isolated from Thailand during 2006-2009, using complete genome sequences. METHODS: Nasopharyngeal aspirates were collected from patients diagnosed with respiratory illness in Thailand during 2006-2009. All samples were screened for Influenza A virus. A total of 13 H1N1 and 21 H3N2 were confirmed and whole genome sequenced for the evolutionary analysis using standard phylogenetic approaches. RESULTS: Phylogenetic analysis of HA revealed a clear diversification of seasonal from vaccine strain lineages. H3N2 seasonal clusters were closely related to the WHO recommended vaccine strains in each season. Most H1N1 isolates could be differentiated into 3 lineages. The A/Brisbane/59/2007 lineage, a vaccine strain for H1N1 since 2008, is closely related with the H1N1 subtypes circulating in 2009. HA sequences were conserved at the receptor-binding site. Amino acid variations in the antigenic site resulted in a possible N-linked glycosylation motif. Recent H3N2 isolates had higher genetic variations compared to H1N1 isolates. Most substitutions in the NP protein were clustered in the T-cell recognition domains. CONCLUSION: In this study we performed evolutionary genetic analysis of influenza A viruses in Thailand between 2006-2009. Although the current vaccine strain is efficient for controlling the circulating outbreak subtypes, surveillance is necessary to provide unambiguous information on emergent viruses. In summary, the findings of this study contribute the understanding of evolution in influenza A viruses in humans and is useful for routine surveillance and vaccine strain selection.