African Horse Sickness Virus Serotype 1 on Horse Farm, Thailand, 2020.

To investigate an outbreak of African horse sickness (AHS) on a horse farm in northeastern Thailand, we used whole-genome sequencing to detect and characterize the virus. The viruses belonged to serotype 1 and contained unique amino acids (95V,166S, 660I in virus capsid protein 2), suggesting a single virus introduction to Thailand.

Human Norovirus Infection in Dogs, Thailand.

In July 2018, recombinant norovirus GII.Pe-GII.4 Sydney was detected in dogs who had diarrhea in a kennel and in children living on the same premises in Thailand. Whole-genome sequencing and phylogenetic analysis of 4 noroviruses from Thailand showed that the canine norovirus was closely related to human norovirus GII.Pe-GII.4 Sydney, suggesting human-to-canine transmission.

First detection and genetic characterization of canine Kobuvirus in domestic dogs in Thailand.

BACKGROUND: Canine Kobuvirus (CaKoV) has been detected both in healthy and diarrheic dogs and in asymptomatic wild carnivores. In this study, we conducted a survey of CaKoV at small animal hospitals in Bangkok and vicinity of Thailand during September 2016 to September 2018. RESULTS: Three hundred and seven rectal swab samples were collected from healthy dogs (n = 55) and dogs with gastroenteritis symptoms (n = 252). Of 307 swab samples tested by using one-step RT-PCR specific to 3D gene, we found CaKoV positivity at 17.59% (54/307). CaKoVs could be detected in both sick (19.44%) and healthy (9.09%) animals. In relation to age group, CaKoV could be frequently detected in younger dogs (25.45%). Our result showed no seasonal pattern of CaKoV infection in domestic dogs. In this study, we characterized CaKoVs by whole genome sequencing (n = 4) or 3D and VP1 gene sequencing (n = 8). Genetic and phylogenetic analyses showed that whole genomes of Thai CaKoVs were closely related to Chinese CaKoVs with highest 99.5% amino acid identity suggesting possible origin of CaKoVs in Thailand. CONCLUSIONS: In conclusion, this study was the first to report the detection and genetic characteristics of CaKoVs in domestic dogs in Thailand. CaKoVs could be detected in both sick and healthy dogs. The virus is frequently detected in younger dogs. Thai CaKoVs were genetically closely related and grouped with Chinese CaKoVs. Our result raises the concerns to vet practitioners that diarrhea in dogs due to canine Kobuvirus infection should not be ignored.

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.

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.

Genetic characterization of influenza A (H7N6) virus isolated from a live-bird market in Thailand.

A one-year influenza A virus (IAV) monitoring program was conducted in a live-bird market (LBM) in Thailand. Using one-step real-time RT-PCR (rRT-PCR), 2.39 % of live birds were found to be IAV positive. Twenty viruses could be identified as IAV subtype H7N6. Eight IAV-H7N6 viruses were subjected to whole-genome sequencing and genetic characterization. Phylogenetic analysis showed that the HA gene of Thai H7N6 is grouped with those of the H7 Eurasian viruses. The NA gene is closely related to those of the N6 Eurasian viruses. This is the first report of IAV subtype H7N6 in Thailand.

Genetic diversity of swine influenza viruses in Thai swine farms, 2011-2014.

The pig is known as a "mixing vessel" for influenza A viruses. The co-circulation of multiple influenza A subtypes in pig populations can lead to novel reassortant strains. For this study, swine influenza surveillance was conducted from September 2011 to February 2014 on 46 swine farms in Thailand. In total, 78 swine influenza viruses were isolated from 2,821 nasal swabs, and 12 were selected for characterization by whole genome sequencing. Our results showed that the co-circulation of swine influenza subtypes H1N1, H3N2, and H1N2 in Thai swine farms was observable throughout the 3 years of surveillance. Furthermore, we repeatedly found reassortant viruses between endemic swine influenza viruses and pandemic H1N1 2009. This observation suggests that there is significant and rapid evolution of swine influenza viruses in swine. Thus, continuous surveillance is critical for monitoring novel reassortant influenza A viruses in Thai swine populations.

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 1.18 % 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.

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.

Genetic characterization of influenza A virus subtypes H11N6, H11N7, and H11N9 isolated from free-grazing ducks, Thailand.

Influenza A viruses (IAVs) infect avian species and several mammalian species including humans. Anseriformes water birds are an important reservoir of IAVs. In this study, we identified and characterized IAV subtypes H11N6 (n = 5), H11N7 (n = 3), and H11N9 (n = 3) isolated during the influenza surveillance program in free-grazing ducks from 2012 to 2015 in Thailand. Eleven IAV-H11 viruses were characterized by either whole genome sequencing (n = 5) or HA and NA gene sequencing (n = 6) for phylogenetic and amino acid analyses. Phylogenetic analysis showed that Thai IAV-H11 were grouped into Avian Eurasian lineage. Amino acid analysis showed that all Thai IAV-H11 viruses have low pathogenic avian influenza (LPAI) characteristics and sensitive to Oseltamivir and Amantadine. Novel reassortant viruses (IAV-H11N7 and IAV-H11N9) have been observed. The reassortant viruses contained NP, M, and NS gene segments which originate from intercontinental sources which never been reported in Thai IAVs. In summary, this study demonstrated high genetic diversity of IAV-H11 circulating in free-grazing ducks. Free-grazing ducks infected with IAVs generated novel reassortant IAV-H11. Thus, surveillance of IAVs in free-grazing ducks should be routinely conducted to monitor novel reassortant viruses and subsequently potential virulence viruses.

Serological survey of antibodies against SARS-CoV-2 in dogs and cats, Thailand.

Coronavirus disease of 2019 (COVID-19) caused by severe acute respiratory syndrome virus type 2 (SARS-CoV-2) is an emerging severe acute respiratory disease affecting global human health. In this study, a large-scale serological survey of antibodies against SARS-CoV-2 in dogs and cats was conducted during the first and second waves of COVID-19 outbreaks in Thailand, from April to December 2020. A total of 3215 serum samples were collected from dogs (n = 2102) and cats (n = 1113) living in Bangkok and in the vicinities. Serum samples were tested for SARS-CoV-2 antibodies by using an indirect multispecies enzyme-linked immunosorbent assay (ELISA). Positive and suspected samples were additionally tested for neutralizing antibodies by the surrogate virus neutralization test (sVNT). The indirect ELISA results showed that 1.66% (35 out of 2103) of dogs and 0.36% (four out of 1112) of cats were positive for SARS-CoV-2 antibodies. The sVNT results showed that all ELISA-positive and suspected samples were negative for neutralizing antibodies. Positive serum samples (35 dogs and four cats) were obtained from clinically healthy animals and animals with mild respiratory signs aged <1-13 years living in Bangkok and Samutprakarn Provinces. In summary, a serological survey revealed evidence of anti-N-IgG antibodies suggesting SARS-CoV-2 exposure in both dogs and cats during the first and second COVID-19 outbreaks in Thailand.

Genetic characterization of hypervirulent Klebsiella pneumoniae responsible for acute death in captive marmosets.

Klebsiella pneumoniae is a Gram-negative bacterium implicated as the causative pathogen in several medical health issues with different strains causing different pathologies including pneumonia, bloodstream infections, meningitis and infections from wounds or surgery. In this study, four captive African marmosets housed in Thailand were found dead. Necropsy and histology revealed congestion of hearts, kidneys and adrenal glands. Twenty-four bacterial isolates were obtained from these four animals with all isolates yielding identical phenotypes indicative of K. pneumoniae based on classical identification schema. All the isolates show the susceptibility to amikacin, cephalexin, doxycycline, gentamicin, and enrofloxacin with intermediate susceptibility to amoxycillin/clavulanic acid. One isolate (20P167W) was chosen for genome analysis and determined to belong to sequence type 65 (ST65). The genome of 20P167W possessed multiple virulence genes including mrk gene cluster and iro and iuc gene cluster (salmochelin and aerobactin, respectively) as well as multiple antibiotic resistance genes including bla SHV-67, bla SHV-11, oqxA, oqxB, and fosA genes resembling those found in human isolates; this isolate has a close genetic relationship with isolates from humans in Ireland, but not from Thailand and California sea lions. Phylogenetic studies using SNP show that there was no relation between genetic and geographic distributions of all known strains typing ST65, suggesting that ST65 strains may spread worldwide through multiple international transmission events rather than by local expansions in humans and/or animals. We also predict that K. pneumoniae ST65 has an ability to acquire genetic mobile element from other bacteria, which would allow Klebsiella to become an even greater public health concern.

First cases of SARS-CoV-2 infection in dogs and cats in Thailand.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused the coronavirus disease 2019 (COVID-19) pandemic in humans since late 2019. Here, we investigated SARS-CoV-2 infection in dogs and cats during COVID-19 quarantine at private veterinary hospitals in Thailand. From April to May 2021, we detected SARS-CoV-2 in three out of 35 dogs and one out of nine cats from four out of 17 households with confirmed COVID-19 patients. SARS-CoV-2 RNA was detected from one of the nasal, oral, rectal and environmental swabs of dog-A (15 years old, mixed breed, male dog), cat-B (1 year old, domestic shorthair, male cat), dog-C (2 years old, mixed breed, female dog) and dog-D (4 years old, Pomeranian, female dog). The animals tested positive for SARS-CoV-2 RNA from 4 to 30 days after pet owners were confirmed to be COVID-19 positive. The animals consecutively tested positive for SARS-CoV-2 RNA for 4 to 10 days. One dog (dog-A) showed mild clinical signs, while the other dogs and a cat remained asymptomatic during quarantine at the hospitals. SARS-CoV-2 specific neutralizing antibodies were detected in both the dogs and cat by surrogate virus neutralization tests. Phylogenetic and genomic mutation analyses of whole genome sequences of three SARS-CoV-2 strains from the dogs and cat revealed SARS-CoV-2 of the Alpha variant (B.1.1.7 lineage). Our findings are suggestive of human-to-animal transmission of SARS-CoV-2 in COVID-19-positive households and contamination of viral RNA in the environment. Public awareness of SARS-CoV-2 infection in pet dogs and cats in close contact with COVID-19 patients should be raised.

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.

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

For the past 10 years, 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.

Influenza A virus surveillance in live-bird markets: first report of influenza A virus subtype H4N6, H4N9, and H10N3 in Thailand.

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 coinfection 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.

Molecular detection and whole genome characterization of Canine Parainfluenza type 5 in Thailand.

Parainfluenza virus type 5 (PIV-5) causes respiratory infection in several animal species and humans. Canine parainfluenza virus type 5 (CPIV-5) causes respiratory disease in domestic dogs worldwide. In this study, we conducted a cross-sectional survey of CPIV-5 in dogs with respiratory symptoms from small animal hospitals in Thailand from November 2015 to December 2018. Our results showed that 32 out of 571 nasal swab samples (5.6%) were positive for CPIV-5 by RT-PCR specific to the NP gene. To characterize the viruses, three representative CPIV-5 were subjected to whole genome sequencing, and an additional ten CPIV-5 were subjected to HN, F, SH and V/P gene sequencing. Pairwise sequence comparison and phylogenetic analysis showed that Thai CPIV-5 was closely related to the CPIV-5 isolated from China and Korea. In conclusion, this study constitutes a whole genome characterization of CPIV-5 from dogs in Thailand. The surveillance of CPIV-5 should be further investigated at a larger scale to determine the dynamics, distribution and potential zoonotic transmission of CPIV-5.

Molecular characterization identifies intra-host recombination and zoonotic potential of canine rotavirus among dogs from Thailand.

From September 2016 to January 2019, we collected 710 rectal swabs from both healthy and sick dogs from small animal hospitals in 5 provinces of Thailand. The samples were tested for canine rotavirus group A (CRV) by using one-step RT-PCR specific to the VP6 gene. Our results showed that 0.70% (5/710) were positive for CRV. The five CRVs were then characterized by whole-genome sequencing. Our results showed that the genotype of Thai CRVs is G3P[3], which is the predominant genotype reported in dogs. The Thai CRVs posed a novel genetic constellation 'G3-P[3]-I3-R3-C3-M3-A9-N2-T3-E3-H6', which has never been reported in CRVs from dogs but has been reported in rotaviruses from humans. Based on phylogenetic analysis, the Thai CRVs are the result of multiple reassortments in which gene segments might have originated from human and bat rotaviruses and suggests the zoonotic potential of the virus.

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.