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.

Genetic and antigenic characterization of hemagglutinin of influenza A/H3N2 virus from the 2015 season in Thailand.

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.

Characterization of Vibrio parahaemolyticus isolated from clinical specimens and oysters in Thailand.

INTRODUCTION: Vibrio parahaemolyticus is a common pathogen that can cause seafood-borne gastroenteritis in humans. We determined the prevalence and characteristics of V. parahaemolyticus isolated from clinical specimens and oysters in Thailand. METHODOLOGY: Isolates of V. parahaemolyticus from clinical specimens (n = 77) and oysters (n = 224) were identified by biochemical testing, polymerase chain reaction (PCR) assays, and serotyping. The toxin genes, antimicrobial resistance, and ß-lactamase production were determined. RESULTS: A total of 301 isolates were confirmed as V. parahaemolyticus by PCR using specific primers for the toxR gene. The majority of clinical isolates carried the tdh+/trh- genotype (82.1%), and one of each isolate was tdh-/trh+ and tdh+/trh+ genotypes. One isolate from oyster contained the tdh gene and another had the trh gene. Twenty-six serotypes were characterized among these isolates, and O3:K6 was the most common (37.7%), followed by OUT:KUT, and O4:K9. In 2010, most clinical and oyster isolates were susceptible to antibiotics, with the exception of ampicillin. In 2012, clinical isolates were not susceptible to cephalothin (52.4%), streptomycin (95.2%), amikacin (66.6%), kanamycin (61.9%), and erythromycin (95.2%), significantly more frequently than in 2010. More than 95% of isolates that were not susceptible to ampicillin produced ß-lactamase enzymes. CONCLUSIONS: We found toxin genes in two oyster isolates, and the clinical isolates that were initially determined to be resistant to several antibiotics. Toxin genes and antimicrobial susceptibility profiles of V. parahaemolyticus from seafood and environment should be continually monitored to determine the spread of toxin and antimicrobial resistance genes.

Mimotope identification using phage displayed random peptide libraries against monoclonal antibodies specific to house dust mite.

Random heptapeptide T7 and random 12mer M13 phage libraries were employed to identify mimotopes binding to monoclonal antibodies (MAb) specific to house dust mite. After selection of bound phage by bio-panning and determination of binding specificity, DNA of selected bound phages was amplified, sequenced and aligned for peptide similarity. Eight mimotopes which were partially matched with Der f 15 allergen were predominant. The amino acid regions, 411-429 and 480-503 of Der f 15 allergen, appeared to be the main epitope clusters. Five mimotopes of MAb B2 and one mimotope of MAb B1 matched with Der p 1 and Der f 2 precursor, respectively.

Distribution of phylogenetic groups, adhesin genes, biofilm formation, and antimicrobial resistance of uropathogenic Escherichia coli isolated from hospitalized patients in Thailand.

BACKGROUND: Urinary tract infections (UTIs) are the most common bacterial infections and are often caused by uropathogenic Escherichia coli (UPEC). We investigated the distribution of phylogenetic groups, adhesin genes, antimicrobial resistance, and biofilm formation in E. coli isolated from patients with UTIs. METHODS: In the present study, 208 UPEC isolated from Thai patients were classified into phylogenetic groups and adhesin genes were detected using multiplex PCR. Antimicrobial susceptibility testing was performed using agar disk diffusion. The Congo red agar method was used to determine the ability of the UPEC to form biofilm. RESULTS: The most prevalent UPEC strains in this study belonged to phylogenetic group B2 (58.7%), followed by group C (12.5%), group E (12.0%), and the other groups (16.8%). Among adhesin genes, the prevalence of fimH (91.8%) was highest, followed by pap (79.3%), sfa (12.0%), and afa (7.7%). The rates of resistance to fluoroquinolones, trimethoprim-sulfamethoxazole, and amoxicillin-clavulanate were  65%, 54.3%, and 36.5%, respectively. The presence of adhesin genes and antibiotic resistance were more frequent in groups B2 and C compared to the other groups. Of the 129 multidrug-resistant UPEC strains, 54% were biofilm producers. Our findings further indicated that biofilm production was significantly correlated with the pap adhesin gene (p ≤ 0.05). CONCLUSION: These findings provide molecular epidemiologic data, antibiotic resistance profiles, and the potential for biofilm formation among UPEC strains that can inform further development of the appropriate prevention and control strategies for UTIs in this region.

Neuraminidase inhibitor susceptibility and neuraminidase enzyme kinetics of human influenza A and B viruses circulating in Thailand in 2010-2015.

Amino acid substitutions within or near the active site of the viral neuraminidase (NA) may affect influenza virus fitness. In influenza A(H3N2) and B viruses circulating in Thailand between 2010 and 2015, we identified several NA substitutions that were previously reported to be associated with reduced inhibition by NA inhibitors (NAIs). To study the effect of these substitutions on the enzymatic properties of NA and on virus characteristics, we generated recombinant influenza viruses possessing either a wild type (WT) NA or an NA with a single I222V, S331G, or S331R substitution [in influenza A(H3N2) viruses] or a single D342S, A395T, A395V, or A395D NA substitution (in influenza B viruses). We generated recombinant (7:1) influenza A and B viruses on the genetic background of A/Puerto Rico/8/1934 (A/PR/8, H1N1) or B/Yamanashi/166/1998 (B/YAM) viruses, respectively. In contrast to the expected phenotypes, all the recombinant influenza A(H3N2) and B viruses carrying putative NA resistance substitutions were susceptible to NAIs. The Km and Vmax for the NAs of A/PR8-S331G and A/PR8-S331R viruses were higher than for the NA of WT virus, and the corresponding values for the B/YAM-D342S virus were lower than for the NA of WT virus. Although there was initial variation in the kinetics of influenza A and B viruses' replication in MDCK cells, their titers were comparable to each other and to WT viruses at later time points. All introduced substitutions were stable except for B/YAM-D342S and B/YAM-A395V which reverted to WT sequences after three passages. Our data suggest that inferring susceptibility to NAIs based on sequence information alone should be cautioned. The impact of NA substitution on NAI resistance, viral growth, and enzymatic properties is viral context dependent and should be empirically determined.

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.

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.

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.

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.