Napawan Bunpapong

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.

Genetic characterization of canine influenza A virus (H3N2) in Thailand

In January 2012, several clinical cases of dogs with flu-like symptoms, including coughing, sneezing, nasal discharge, and fever, were reported in a small-animal hospital located in Bangkok, Thailand. One influenza A virus was identified and characterized as an avian-like influenza virus H3N2. The virus was named A/canine/Thailand/CU-DC5299/12. A phylogenetic analysis indicated that the canine virus belonged to an avian Eurasian lineage and was genetically related to the canine influenza viruses H3N2 from China and Korea. This canine virus displays a unique genetic signature with two amino acid insertions in the NA protein, which is similar to the canine influenza viruses from eastern China (Zhejiang and Jiangsu). This study constitutes the first report of H3N2 canine influenza virus infection in a small-animal hospital in Thailand.

Porcine Deltacoronavirus, Thailand, 2015

To the Editor: Porcine deltacoronavirus (PDCoV) was first reported in Hong Kong in 2012 and included the HKU15-44 and HKU15-155 strains (1). In early 2014, PDCoV was reported in pigs with diarrhea on swine farms in Ohio, USA (2), and later in other states (2–5). In April 2014, PDCoV strain KNU14-04 was reported in pigs in South Korea (6). A retrospective study in 2012 reported PDCoV strain S27 in Sichuan, China (7). Recently PDCoV strain CNJXNI2 has been reported in pigs with diarrhea in Jiangxi, China (8). There are currently 28 complete PDCoV genomes from China, South Korea, and the United States available in GenBank. We report emergence of PDCoV infections on a commercial swine farm in Thailand. In June 2015, we investigated reports of acute diarrhea in piglets, gilts, and sows on a swine farm. An outbreak occurred on a commercial swine farm (3,000 sows) located in the eastern province of Thailand. Clinical signs, including acute watery diarrhea, loss of appetite, and agalactia, were observed in gilts and sows in the breeding and gestation houses. Subsequently, piglets in farrowing houses had clinical signs (depression, fever, watery diarrhea, and severe dehydration). Although clinical signs were detected less frequently in fattening pigs in growth-finishing houses, PDCoVs were later detected from blood samples of fattening pigs. The outbreak lasted 6 weeks (June 10–July 20, 2015). The mortality rate was 27.63% (829/3,000) in sows and 64.27% (2,892/4,500) in piglets but was lower than that usually observed for porcine epidemic diarrhea virus (PEDV) infection. A total of 865 (19.22%) piglets died and were culled during 10 production weeks. Postmortem examination of dead piglets showed emaciated animals and yellow pasty feces. Intestines and colons showed thin walls with a watery content and curdled milk. Histopathologic examination showed shortened and fused villi in the jejunum and ileum. An attenuated and vacuolated cytoplasm in enterocytes was also observed (Technical Appendix Figure 1) (9,10). We examined 30 samples from the affected swine farm. Blood (n = 10), intestine (n = 8), lymph node (n = 2), feces (n = 6), and feed (n = 4) samples were collected for 2 day-old piglets and 17-, 19-, and 20-week-old fattening pigs. A total of 26 samples were positive for PDCoV by reverse transcription PCR (2) (Technical Appendix Table 1). Because sick pigs had clinical signs similar to those of pigs with other swine virus diseases, all samples were tested for transmissible gastroenteritis coronavirus; PEDV; rotaviruses A, B, and C; porcine reproductive and respiratory syndrome virus; and circovirus. All test results were negative. We selected 2 PDCoVs (S5011 and S5015L) for whole-genome sequencing and 14 PDCoVs for sequencing of spike (S), envelope (E), membrane (M), and nucleocapsid (N) genes and the 3′-untranslated region (UTR). Nucleotide sequences obtained were submitted to GenBank (Technical Appendix Table 2). Sequence analysis of the 2 PDCoVs from Thailand showed that their whole genomes had 99.98% nt identity (only 4 nt differences) with each other and highest nucleotide identities with PDCoVs from China (98.43% with AH2004). S gene sequences showed greatest diversity (99.97%–100% nt identities and 99.91%–100% aa identities) for PDCoVs from Thailand and 95.93%–96.68% with other reference PDCoVs, which is consistent with findings of previous report (5). In contrast, E, M, and N genes were conserved (100% nt identities for PDCoVs from Thailand and 99.19%–100% for E genes, 98.28%–99.07% for M genes, and 96.88%–97.81% for N genes with reference PDCoVs) (Technical Appendix Table 3). Phylogenetic analysis of the whole genome of PDCoVs from Thailand showed close relatedness with AH-2004, HKU15-44, S27-2012, and HKU15-155 virus strains from China. However, these viruses from Thailand were in a different subcluster than PDCoVs from the United States (Figure; Technical Appendix Figure 2). PDCoVs identified in this study might represent a new variant of PDCoV because these 2 viruses have unique sequence characteristics: 3-nt (TCT) and 1-nt (A) deletions in the 5′-UTR, 6-nt (AGTTTG) and 9-nt (GAGCCAGTC) deletions in open reading frame 1a/b, and 4-nt (CTCT) insertion in the 3′-UTR (Technical Appendix Table 4). Figure Phylogenetic analysis of whole-genome sequences of porcine deltacoronaviruses (PDCoVs), Thailand. Black circles indicate strains isolated in this study. The tree was constructed by using MEGA version 6.06 (http://www.megasoftware.net/) with the neighbor-joining ... We identified PDCoV on a commercial swine farm in Thailand. Affected pigs had clinical signs of acute watery diarrhea, similar to those of pigs infected with PEDV, and had moderate illness and low mortality rates. PDCoVs were detected in symptomatic piglets, sows, and fattening pigs, although clinical signs in fattening pigs were least severe. Swine farmers and veterinarians should be aware of PDCoV as another causative agent of watery diarrhea in pigs. Similar to PEDV, Wang et al. reported that sequence deletions, insertions, and mutations in PDCoVs in pigs might contribute to variant virus virulence (2). Our findings might assist in development of diagnostic assays for differentiating PDCoVs in Thailand from PDCoVs in other countries. Because PDCoVs from Thailand were highly related to each other, PDCoV might have transmitted into Thailand by a single event. However, verification of this possibility would be difficult. Similar to the situation in the United States, PDCoV might be underdiagnosed in Thailand. Technical Appendix: Methods used and additional information for detection of porcine deltacoronavirus, Thailand, 2015. Click here to view.(709K, pdf)

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.

Avian Influenza Viruses in Wild Land Birds in Northern Vietnam

Given a paucity of data on the occurrence of avian influenza viruses (AIVs) in wild passerines and other small terrestrial species in Southeast Asia and the importance of highly pathogenic Asian-strain H5N1 outbreaks in humans and domestic poultry in these areas, we focused on surveillance for influenza A viral nucleic acids and antibodies for AIVs in wildcaught birds in northern Vietnam. Four of 197 serum samples collected in 2007 from Black-crested Bulbul (Pycnonotus melanicterus), Crow-billed Drongo (Dicrurus annectans), Buff-breasted Babbler (Pellorneum tickelli), and Black-browed Fulvetta (Alcippe grotei) were antibody positive for the H5 subtype. Fourteen of 193 samples collected in 2008 were positive for the influenza A viral M gene by real-time reverse transcriptase-polymerase chain reaction. These included samples from 10 Japanese White-eyes (Zosterops japonicus), two Puff-throated Bulbuls (Alophoixus pallidus), one White-tailed Robin (Cinclidium leucurum), and one Striped Titbabbler (Macronous gularis). Almost all positive samples were from bird species that forage in flocks, including Japanese White-eyes with an unusually high prevalence of 14.9%. We collected samples from birds from three habitat types but detected no strong pattern in prevalence. Our results suggest that attention should be given to terrestrial species, particularly flocking passerines, in AIV surveillance and monitoring programs.

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.

Influenza A Virus Surveillance in Live-Bird Markets: First Report of Influenza A Virus Subtype H4N6, H4N9, and H10N3 in Thailand

SUMMARY. A one-year influenza A survey was conducted in 10 live bird markets (LBMs) in H5N1 high-risk areas in Thailand from January to December 2009. The result from the survey showed that the occurrence of influenza A virus (IAV) in LBMs was 0.36% (19/5304). Three influenza A subtypes recovered from LBMs were H4N6 (n  =  2), H4N9 (n  =  1), and H10N3 (n  =  16) from Muscovy ducks housed in one LBM in Bangkok. These influenza subtypes had never been reported in Thailand, and therefore such genetic diversity raises concern about potential genetic reassortment of the viruses in avian species in a particular setting. Two influenza A subtypes (H4N6 and H4N9) were isolated from oropharyngeal and cloacal swabs of the same duck, suggesting co-infection with two influenza subtypes and possible genetic reassortment in the bird. In addition, H10N3 infection in ducks housed in the same LBM was observed. These findings further support that LBMs are a potential source of IAV transmission and genetic reassortment.

Influenza A(H9N2) Virus, Myanmar, 2014–2015

Routine surveillance of influenza A virus was conducted in Myanmar during 2014–2015. Influenza A(H9N2) virus was isolated in Shan State, upper Myanmar. Whole-genome sequencing showed that H9N2 virus from Myanmar was closely related to H9N2 virus of clade 4.2.5 from China.

Sentinel model for influenza A virus monitoring in free-grazing ducks in Thailand.

Influenza A virus (IAV) can cause influenza in birds and mammals. In Thailand, free-grazing ducks are known IAV reservoirs and can spread viruses through frequent movements in habitats they share with wild birds. In this study, the sentinel model for IAV monitoring was conducted over 4 months in two free-grazing duck flocks. IAV subtypes H4N6 (n=1) and H3N8 (n=5) were isolated from sentinel ducks at the ages of 13 and 15 weeks. Clinical signs of depression and ocular discharge were observed in the infected ducks. Phylogenetic analysis and genetic characterization of the isolated IAVs indicated that all Thai IAVs were clustered in the Eurasian lineage and pose low pathogenic avian influenza characteristics. Serological analysis found that antibodies against IAVs could be detected in the ducks since 9-weeks-old. In summary, our results indicate that the sentinel model can be used for IAV monitoring in free-grazing duck flocks. Since free-grazing ducks are potential reservoirs and transmitters of IAVs, routine IAV surveillance in free-grazing duck flocks can be beneficial for influenza prevention and control strategies.

Genetic characterization of influenza A virus subtypes H1N3 and H1N9 isolated from free-grazing ducks in Thailand

Influenza A virus (IAV) subtype H1 has been reported to infect birds, pigs and humans. In this study, we characterized IAVs subtype H1N3 and H1N9 isolated from free-grazing ducks in Thailand. Phylogenetic analysis showed that Thai IAV-H1 isolates cluster with avian Eurasian-lineage but not pandemic H1N1 viruses. Analysis of the viruses indicated low-pathogenic avian influenza (LPAI) characteristics. This study is the first report of avian H1N3 and H1N9 in Thailand. Although Thai IAV-H1 viruses do not pose a risk of a pandemic, routine surveillance and genetic monitoring of IAVs should be conducted.