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.

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.

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.

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.

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.

Persistence of pdm2009-H1N1 internal genes of swine influenza in pigs, Thailand.

Swine influenza is one of the important zoonotic diseases of pigs. We conducted a longitudinal survey of swine influenza A viruses (S-IAV) circulating in a pig farm with history of endemic S-IAV infection from 2017 to 2018. The samples were collected from 436 pigs including nasal swab samples (n = 436) and blood samples (n = 436). Our result showed that 18.81% (82/436) were positive for influenza A virus and subsequently 57 S-IAV could be isolated. Then 24 out of 57 S-IAVs were selected for whole genome sequencing and could be subtyped as S-IAV-H1N1 (n = 18) and S-IAV-H3N2 (n = 6). Of 24 S-IAVs, we observed 3 genotypes of S-IAVs including rH1N1 (pdm + 1), rH1N1 (pdm + 2), and rH3N2 (pdm + 2). Since all genotypes of S-IAVs in this study contained internal genes from pdmH1N1-2009, it could be speculated that pdmH1N1-2009 was introduced in a pig farm and then multiple reassorted with endemic S-IAVs to generate diversify S-IAV genotypes. Our study supported and added the evidences that pdmH1N1-2009 and it reassortant have predominately persisted in pig population in Thailand. Thus, monitoring of S-IAVs in pigs, farm workers and veterinarians in pig farms is important and should be routinely conducted.

Serological evidence of avian influenza virus subtype H5 and H9 in live bird market, Myanmar.

Avian Influenza (AI), caused by Alphainfluenzaviruses (AIVs), is a contagious respiratory disease in birds and mammals. AIVs have been reported in poultry worldwide and the impact of AIVs on human health is immense. In this study, a serological survey of AIV subtype H5 and H9 was conducted in a live bird market (LBM) in Yangon, Myanmar during February 2016 to September 2016. A total of 621 serum samples were collected from chickens (n = 489) and ducks (n = 132) from 48 vendors in the LBM. The samples were examined for antibodies against influenza viruses by using NP-ELISA and specific antibodies against AIV-H5N1 (Clade 2.3.4) and AIV-H9N2 (Clade 9.4.2) by using Hemagglutination Inhibition (HI) assay. The result of NP-ELISA assay showed that 12.88 % (80/621) of poultry in LBM was positive for AIV antibodies. In detail, 38.06 % (51/134) of layers, 7.08 % (8/113) of backyard chicken, 2.07 % (5/242) of broilers and 12.12 % (16/132) of ducks were AIV positive. The HI test for specific antibodies against AIV-H5N1 and AIV-H9N2 were 1.77 % (11/621) and 4.51 % (28/621), respectively. Our findings revealed the evidence of AIV-H5N1 and AIV-H9N2 exposure in both chicken and ducks in the LBM in Yangon, Myanmar. Risks of influenza infections and transmission among poultry and humans in the LBMs could not be ignored.

Surveillance of influenza A virus subtype H5N1 in a live bird market in Yangon, Myanmar: 2017-2018.

A survey of influenza A viruses (IAVs) in the Mingalar Taung Nyunt live bird market (MTN-LBM), Yangon, Myanmar, was conducted from December 2017 to December 2018. During the survey, 455 swab samples were collected from broilers, layers, backyard chickens and ducks from the MTN-LBM. Ninety-one pooled samples were screened for IAVs by real-time RT-PCR specific to the M gene. Positive pooled samples were individually retested for IAVs. In total, 2.63% of individual samples (12/455) were positive for IAVs. Out of 12 samples, seven samples from layer chickens and the environment were identified as IAV subtype H5N1. In this study, four IAVs were successfully isolated and further characterized by whole genome sequencing. Whole genome sequence analysis revealed that the viruses were characterized as highly pathogenic avian influenza virus subtype H5N1 (HPAIV-H5N1) of clade 2.3.2.1c. Phylogenetic and genetic analyses showed that Myanmar HPAIV-H5N1 was closely related to HPAIV-H5N1 clade 2.3.2.1c isolated from China and Vietnam in 2014. Our results suggested that the live bird market in Myanmar represents a significant risk of HPAIV-H5N1 transmission in poultry and humans. Moreover, HPAIV-H5N1 clade 2.3.2.1c is widely distributed in South-East Asia including Myanmar.

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.

Emergence of canine parvovirus type 2c in domestic dogs and cats from Thailand.

Canine parvovirus type 2 (CPV-2) is an important pathogen causing haemorrhagic enteritis in domestic dogs and wildlife worldwide. In early 2000, canine parvovirus type 2c (CPV-2c) was first reported and subsequently became a predominant subtype circulating in Europe and the Americas. CPV-2c has also been reported in Asia, including cases in China, India, Taiwan and Vietnam. However, CPV-2c has never been reported in Thailand. In this study, we conducted viral enteric disease surveillance in dogs and cats in Thailand during 2016-2018. During 20 months of surveillance, 507 rectal swab samples were collected from dogs (n = 444) and cats (n = 63) with and without clinical signs. The samples were examined for parvovirus by using VP2 gene-specific PCR for parvovirus. Our results showed that the positivity of canine parvovirus (CPV) was 29.95% and that of feline parvovirus (FPV) was 58.73%. In this study, we characterized 34 parvoviruses by VP2 gene sequencing. Moreover, two Thai-CPV-2 (Dog/CU-24 and Cat/CU-21) were characterized by whole genome sequencing. The phylogenetic results showed that Thai-CPV-2 had the highest nucleotide identities and clustered with Asian-CPV-2c but were in separate subclusters from the North American and European CPV-2c. Similarly, whole genome analyses showed that Thai-CPVs are closely related to Asian-CPV-2c, with unique amino acids at positions 297A, 324I, 370R and 426E. In summary, our results demonstrated the emergence of Asian-CPV-2c in dogs and cats in Thailand. Thus, the surveillance of CPV-2 in domestic dogs and cats should be further conducted on a larger scale to determine the dynamics of predominant variants and their distributions in the country and in the Southeast Asia region.

Evidence of pandemic H1N1 influenza exposure in dogs and cats, Thailand: A serological survey.

Influenza A virus causes respiratory disease in both humans and animals. In this study, a survey of influenza A antibodies in domestic dogs and cats was conducted in 47 animal shelters in 19 provinces of Thailand from September 2011 to September 2014. One thousand and eleven serum samples were collected from 932 dogs and 79 cats. Serum samples were tested for influenza A antibodies using a multi-species competitive NP-ELISA and haemagglutination inhibition (HI) assay. The NP-ELISA results showed that 0.97% (9/932) of dogs were positive, but all cat samples were negative. The HI test against pandemic H1N1, human H3N2 and canine H3N2 showed that 0.64% (6/932) and 1.20% (1/79) of dogs and cats were positive, respectively. It is noted that all six serum samples (5 dogs and 1 cat) had antibodies against pandemic H1N1. In summary, a serological survey revealed the evidence of pandemic H1N1 influenza exposure in both dogs and cats in the shelters in Thailand.

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.

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 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 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.