Manoosak Wongphatcharachai

Pandemic (H1N1) 2009 virus on commercial swine farm, Thailand.

A swine influenza outbreak occurred on a commercial pig farm in Thailand. Outbreak investigation indicated that pigs were co-infected with pandemic (H1N1) 2009 virus and seasonal influenza (H1N1) viruses. No evidence of gene reassortment or pig-to-human transmission of pandemic (H1N1) 2009 virus was found during the outbreak.

Brief report: molecular characterization of a novel reassorted pandemic H1N1 2009 in Thai pigs

For the past 10xa0years, endemic swine influenza H1 viruses in Thailand have been characterized as reassortants of swine virus genes from swine influenza viruses (SIV) in US and European pigs. Here the authors report the emergence of a novel reassorted H1N1 (rH1N1) virus consisted of human, avian, and swine virus genes from the pandemic H1N1 2009 (pH1N1) virus with a neuraminidase (NA) gene from a Thai swine H1N1 (ThH1N1) isolate. The rH1N1 virus was detected in nursery pigs during a respiratory disease outbreak in central Thailand in early 2010. The rH1N1 virus was repeatedly isolated from infected pigs, suggesting that it can transmit efficiently among the pig population. The appearance of rH1N1 virus in the field occurred within months of the introduction of pH1N1 virus into the Thai swine population in late 2009. The finding highlights the role of pig in generating newly reassorted influenza A viruses and also the significance of continuing disease surveillance and genetic characterization of SIV in pigs.

Neutralizing DNA Aptamers against Swine Influenza H3N2 Viruses

ABSTRACT Triple reassortant influenza A viruses (IAVs) of swine, particularly the North American H3N2 subtype, circulate in swine herds and may reassort and result in the emergence of novel zoonotic strains. Current diagnostic tools rely on isolation of the viruses, followed by serotyping by hemagglutination or genome sequencing, both of which can be expensive and time-consuming. Thus, novel subtype-specific ligands and methods are needed for rapid testing and subtyping of IAVs in the field. To address this need, we selected DNA aptamers against the recombinant HA protein from swine IAV H3 cluster IV using systematic evolution of ligands by exponential enrichment (SELEX). Four candidate aptamers (HA68, HA7, HA2a, and HA2b) were identified and characterized. The dissociation constants (K d ) of aptamers HA68, HA7, HA2a, and HA2b against recombinant H3 protein were 7.1, 22.3, 16.0, and 3.7 nM, respectively. The binding site of HA68 to H3 was identified to be between nucleotide residues 8 and 40. All aptamers inhibited H3 hemagglutination. HA68 was highly specific to all four lineages within the North American H3N2 subtype. Further, the other three aptamers specifically identified live viruses belonging to the phylogenetic clusters I, II/III, and IV especially the virus that closely related to the recent H3N2 variant (H3N2v). Aptamer HA68 was also able to bind and detect H3N2v isolated from recent human cases. In conclusion, we provide subtype-specific aptamers against H3N2 IAVs of swine that can now be used in rapid detection and typing protocols for field applications.

Genetic characterization of avian influenza subtype H4N6 and H4N9 from live bird market, Thailand

A one year active surveillance program for influenza A viruses among avian species in a live-bird market (LBM) in Bangkok, Thailand was conducted in 2009. Out of 970 samples collected, influenza A virus subtypes H4N6 (n = 2) and H4N9 (n = 1) were isolated from healthy Muscovy ducks. All three viruses were characterized by whole genome sequencing with subsequent phylogenetic analysis and genetic comparison. Phylogenetic analysis of all eight viral genes showed that the viruses clustered in the Eurasian lineage of influenza A viruses. Genetic analysis showed that H4N6 and H4N9 viruses display low pathogenic avian influenza characteristics. The HA cleavage site and receptor binding sites were conserved and resembled to LPAI viruses. This study is the first to report isolation of H4N6 and H4N9 viruses from birds in LBM in Thailand and shows the genetic diversity of the viruses circulating in the LBM. In addition, co-infection of H4N6 and H4N9 in the same Muscovy duck was observed.

Genetic characterization of 2008 reassortant influenza A virus (H5N1), Thailand

In January and November 2008, outbreaks of avian influenza have been reported in 4 provinces of Thailand. Eight Influenza A H5N1 viruses were recovered from these 2008 AI outbreaks and comprehensively characterized and analyzed for nucleotide identity, genetic relatedness, virulence determinants, and possible sites of reassortment. The results show that the 2008 H5N1 viruses displayed genetic drift characteristics (less than 3% genetic differences), as commonly found in influenza A viruses. Based on phylogenetic analysis, clade 1 viruses in Thailand were divided into 3 distinct branches (subclades 1, 1.1 and 1.2). Six out of 8 H5N1 isolates have been identified as reassorted H5N1 viruses, while other isolates belong to an original H5N1 clade. These viruses have undergone inter-lineage reassortment between subclades 1.1 and 1.2 and thus represent new reassorted 2008 H5N1 viruses. The reassorted viruses have acquired gene segments from H5N1, subclade 1.1 (PA, HA, NP and M) and subclade 1.2 (PB2, PB1, NA and NS) in Thailand. Bootscan analysis of concatenated whole genome sequences of the 2008 H5N1 viruses supported the reassortment sites between subclade 1.1 and 1.2 viruses. Based on estimating of the time of the most recent common ancestors of the 2008 H5N1 viruses, the potential point of genetic reassortment of the viruses could be traced back to 2006. Genetic analysis of the 2008 H5N1 viruses has shown that most virulence determinants in all 8 genes of the viruses have remained unchanged. In summary, two predominant H5N1 lineages were circulating in 2008. The original CUK2-like lineage mainly circulated in central Thailand and the reassorted lineage (subclades 1.1 and 1.2) predominantly circulated in lower-north Thailand. To prevent new reassortment, emphasis should be put on prevention of H5N1 viruses circulating in high risk areas. In addition, surveillance and whole genome sequencing of H5N1 viruses should be routinely performed for monitoring the genetic drift of the virus and new reassorted strains, especially in light of potential reassortment between avian and mammalian H5N1 viruses.

Predominant populations of indigenous soybean‐nodulating Bradyrhizobium japonicum strains obtained from organic farming systems in Minnesota

Bradyrhizobium from organic fields in Minnesota were isolated and genotyped to assess diversity of soybean‐bradyrhizobia in organic farming systems that can be used to improve soybean productivity.

Genetic characterization of influenza A virus subtype H12N1 isolated from a watercock and lesser whistling ducks in Thailand

Monitoring of influenza A virus (IAV) was conducted in wild bird species in central Thailand. Four IAV subtype H12N1 strains were isolated from a watercock (order Gruiformes, family Rallidae) (nxa0=xa01) and lesser whistling ducks (order Anseriformes, family Anatidae) (nxa0=xa03). All H12N1 viruses were characterized by whole-genome sequencing. Phylogenetic analysis of all eight genes of the Thai H12N1 viruses indicated that they are most closely related to the Eurasian strains. Analysis of the HA gene revealed the strains to be of low pathogenicity. This study is the first to report the circulation of IAV subtype H12N1 in Thailand and to describe the genetic characteristics of H12N1 in Eurasia. Moreover, the genetic information obtained on H12N1 has contributed a new Eurasian strain of H12N1 to the GenBank database.

A novel method for detection of H9N2 influenza viruses by an aptamer-real time-PCR

H9N2 Influenza subtype has emerged in Tunisia causing epidemics in poultry and resulting in major economic losses. New mutations in their hemagglutinin and neuraminidase proteins were acquired, suggesting their potential to directly infect humans. Effective surveillance tools should be implemented to help prevent potential spillover of the virus across species. We have developed a highly sensitive real time immuno-polymerase chain reaction (RT-I-PCR) method for detecting H9N2 virus. The assay applies aptamers as ligands to capture and detect the virus. First, a panel of specific ssDNA aptamers was selected via a one step high stringency protocol. Next, the panel of selected aptamers was characterized for their affinities and their specificity to H9N2 virus. The aptamer showing the highest binding affinity to the virus was used as ligand to develop a highly sensitive sandwich Aptamer I-PCR. A 3-log increase in analytical sensitivity was achieved as compared to a routinely used ELISA antigen test, highlighting the potential of this approach to detect very low levels of virus particles. The test was validated using clinical samples and constitutes a rapid and a label-free platform, opening a new venue for the development of aptamer -based viability sensing for a variety of microorganisms of economic importance in Tunisia and surrounding regions.

Site-specific distribution and competitive ability of indigenous bean-nodulating rhizobia isolated from organic fields in Minnesota

Organic dry bean production systems have received increasing interest in many regions of the US, including Minnesota. Thus, improving biological N2 fixation would be highly beneficial for organic crop production. To date, only limited work has been done to select efficient N2-fixing rhizobia for organic dry bean production. In this study, soil samples from 25 organic fields in Minnesota, with a previous cropping history of dry beans, soybeans or both, were collected during May to July 2012. Genetic diversity of indigenous dry bean-rhizobia (511 isolates) was determined by using horizontal, fluorophore-enhanced, repetitive, extragenic, and palindromic-PCR (HFERP) DNA fingerprinting and isolates were classified as belonging to 58 different genotypes. The more abundant rhizobia isolated from bean nodules comprised 35.6% of the population. None of the isolates were identical to commonly-used commercial strains used in the U.S., including Rhizobium tropici CIAT899. Seventeen predominant genotypes were shown to represent two main species, Rhizobium leguminosarum bv. phaseoli (67.1%) and Rhizobium etli (30.2%). One of the indigenous strains, orgK9, displayed efficient N2-fixation and competitive ability relative to the commercial strains tested. The lack of large numbers of indigenous dry bean-rhizobia at most study sites will be useful to avoid competition problems between inoculant strains and indigenous rhizobia. This will allow inoculation with highly effective N2-fixing rhizobia, thus resulting in improved crop productivity. Our results highlight the existence of site-specific rhizobial genotypes in different organic fields and identify strains that may prove useful as novel inoculants for organic dry bean production systems.

Genetic characterization of influenza A virus subtype H7N1 isolated from quail, Thailand.

In Thailand, surveillance for the highly pathogenic avian influenza virus H5N1 (HPAI-H5N1) has revealed high prevalence of the virus in quail in live-bird markets. This study monitored avian influenza viruses (AIVs) in quail farms in an area at high risk for HPAI-H5N1 over a 12-month period from 2009 to 2010. One-step real-time RT-PCR (rRT-PCR) results showed that 1.18xa0% of swab samples (24/2,040) were AIV positive. Among the rRT-PCR positive samples, three samples were identified as subtype H7N1. One Thai H7N1 virus designated “A/quail/Thailand/CU-J2882/2009 (H7N1)” was subjected to whole genome sequencing and genetic characterization. Phylogenetic analysis showed that the HA gene of the Thai H7N1 virus groups with those of the H7 Eurasian viruses. Interestingly, the NA gene of the virus was found to be closely related to those of the HPAI-H5N1 viruses from Vietnam and Thailand. This study constitutes the first report on AIV H7N1 in Thailand. Our results suggest the possibility of genetic reassortment between AIV-H7NX and HPAI-H5N1 in quail. The HA cleavage site of the Thai H7N1 virus contains no multiple amino acid insertions, suggesting low pathogenic characteristics for this virus.