Distribution and genetic diversity of Enterovirus G (EV-G) on pig farms in Thailand.

BACKGROUND: Enterovirus G (EV-G) causes subclinical infections and is occasionally associated with diarrhea in pigs. In this study, we conducted a cross-sectional survey of EV-G in pigs from 73 pig farms in 20 provinces of Thailand from December 2014 to January 2018. RESULTS: Our results showed a high occurrence of EV-Gs which 71.6 % of fecal and intestinal samples (556/777) and 71.2 % of pig farms (52/73) were positive for EV-G by RT-PCR specific to the 5'UTR. EV-Gs could be detected in all age pig groups, and the percentage positivity was highest in the fattening group (89.7 %), followed by the nursery group (89.4 %). To characterize the viruses, 34 EV-G representatives were characterized by VP1 gene sequencing. Pairwise sequence comparison and phylogenetic analysis showed that Thai-EV-Gs belonged to the EV-G1, EV-G3, EV-G4, EV-G8, EV-G9 and EV-G10 genotypes, among which the EV-G3 was the predominant genotype in Thailand. Co-infection with different EV-G genotypes or with EV-Gs and porcine epidemic diarrhea virus (PEDV) or porcine deltacoronavirus (PDCoV) on the same pig farms was observed. CONCLUSIONS: Our results confirmed that EV-G infection is endemic in Thailand, with a high genetic diversity of different genotypes. This study constitutes the first report of the genetic characterization of EV-GS in pigs in 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.

Outbreaks of Tilapia Lake Virus Infection, Thailand, 2015-2016.

During 2015-2016, several outbreaks of tilapia lake virus infection occurred among tilapia in Thailand. Phylogenetic analysis showed that the virus from Thailand grouped with a tilapia virus (family Orthomyxoviridae) from Israel. This emerging virus is a threat to tilapia aquaculture in Asia and worldwide.

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.

Emergence of novel porcine circovirus 2d strains in Thailand, 2019-2020.

Porcine circovirus 2 (PCV2) has been recognized as a causative agent of porcine circovirus diseases (PCVDs) affecting the global swine industry. In this study, the genetic diversity of PCV2 strains circulating in Thailand between 2019 and 2020 was investigated using 742 swine clinical samples from 145 farms. The results showed PCV2-positive rates of 54.2% (402/742) and 81.4% (118/145) at the sample and farm levels, respectively. Genetic analysis of 51 Thai PCV2 genomic sequences showed that 84.3% (43/51) was PCV2d, 13.7% (7/51) was PCV2b and 1.9% (1/51) was PCV2b/2d recombinant virus. Surprisingly, the majority of the Thai PCV2d sequences from this study (69.77%, 30/43) formed a novel cluster on a phylogenetic tree and contained a unique 133HDAM136 on the ORF2 deduced amino acid sequence, which is in one of the previously identified immunoreactive domains strongly involved in virus neutralization. The PCV2b/2d recombinant virus also carried 133HDAM136. The emergence of the novel PCV2d strains predominating in Thailand was discussed. This study highlights the need for further investigations on the spreading of these PCV2d strains in other regions and the efficacy of current commercial vaccines.

Coronavirus seroprevalence among villagers exposed to bats in Thailand.

A serological survey of human coronavirus antibodies among villagers in 10 provinces of Thailand was conducted during 2016-2018. Serum samples (n = 364) were collected from participants from the villages and tested for coronavirus antibodies using a human coronavirus IgG ELISA kit. Our results showed that 10.44% (38/364; 21 males and 17 females) of the villagers had antibodies against human coronaviruses. The odds ratio for coronavirus positivity in the villagers in the central region who were exposed to bats was 4.75, 95% CI 1.04-21.70, when compared to that in the non-exposed villagers. The sociodemographics, knowledge, attitudes and practices (KAP) of the villagers were also recorded and analysed by using a quantitative structured questionnaire. Our results showed that 62.36% (227/364) of the villagers had been exposed to bats at least once in the past six months. Low monthly family income was statistically significant in increasing the risk for coronavirus seropositivity among the villagers (OR 2.91, 95% CI 1.13-7.49). In-depth interviews among the coronavirus-positive participants (n = 30) showed that cultural context, local norms and beliefs could influence to bat exposure activities. In conclusion, our results provide baseline information on human coronavirus antibodies and KAP regarding to bat exposure among villagers in 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.

Elucidating the Regulon of a Fur-like Protein in Mycobacterium avium subsp. paratuberculosis (MAP).

Intracellular iron concentration is tightly regulated to maintain cell viability. Iron plays important roles in electron transport, nucleic acid synthesis, and oxidative stress. A Mycobacterium avium subsp. paratuberculosis (MAP)-specific genomic island carries a putative metal transport operon that includes MAP3773c, which encodes a Fur-like protein. Although well characterized as a global regulator of iron homeostasis in multiple bacteria, the function of Fur (ferric uptake regulator) in MAP is unknown as this organism also carries IdeR (iron dependent regulator), a native iron regulatory protein specific to mycobacteria. Computational analysis using PRODORIC identified 23 different pathways involved in respiration, metabolism, and virulence that were likely regulated by MAP3773c. Thus, chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) was performed to confirm the putative regulon of MAP3773c (Fur-like protein) in MAP. ChIP-Seq revealed enriched binding to 58 regions by Fur under iron-replete and -deplete conditions, located mostly within open reading frames (ORFs). Three ChIP peaks were identified in genes that are directly related to iron regulation: MAP3638c (hemophore-like protein), MAP3736c (Fur box), and MAP3776c (ABC transporter). Fur box consensus sequence was identified, and binding specificity and dependence on Mn2+ availability was confirmed by a chemiluminescent electrophoresis mobility shift assay (EMSA). The results confirmed that MAP3773c is a Fur ortholog that recognizes a 19 bp DNA sequence motif (Fur box) and it is involved in metal homeostasis. This work provides a regulatory network of MAP Fur binding sites during iron-replete and -deplete conditions, highlighting unique properties of Fur regulon in MAP.

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.

Swine influenza viruses and pandemic H1N1-2009 infection in pigs, Myanmar.

Swine influenza virus (SIV) causes respiratory diseases in pigs and has impacts on both animal and human health. In this study, we conducted swine influenza surveillance in pig farms in the Yangon and Bago regions, Myanmar, during 2017-2019. Nasal swabs (n = 500) were collected from pigs in 10 swine farms. Our results showed that 11 out of 100 pooled samples (11%) were positive for influenza A virus (IAV) by real-time RT-PCR. Five SIVs could be isolated and could be subtyped as SIV-H1N1 (n = 4) or SIV-H3N2 (n = 1). The viruses were further characterized by whole-genome sequencing and classified as pdmH1N1-2009 (n = 3), reassortant H1N1 (n = 1) or reassortant H3N2 (n = 1). Phylogenetic analysis of Myanmar SIVs showed that all genes of the three SIV-H1N1 (pdmH1N1-2009) were clustered with viruses of the pdm/09 lineage. For one SIV-H1N1 (rH1N1), the HA1 gene was clustered with those of endemic SIVs of the classical swine lineage, and seven genes were clustered with those of viruses of the pdm/09 lineage. For SIV-H3N2 (rH3N2), the HA3 and NA2 genes were clustered with those of endemic SIVs of the human-like swine lineage, while six internal genes were clustered with those of viruses of the pdm/09 lineage. Genetic analysis indicated that all the Myanmar SIVs possessed amino acids that favour binding to the human receptor. All the Myanmar SIVs contained amino acids related to amantadine resistance but not oseltamivir resistance. Notably, the pdmH1N1-2009 virus might have been circulating in the Myanmar pig population for a period of time after pdmH1N1-2009 outbreaks in humans. Then, reassortment between endemic SIV-H1N1 or SIV-H3N2 and pdmH1N1-2009 in pig farms in Myanmar could have occurred. Our findings ascertained the genetic diversity of SIVs, especially pdmH1N1-2009, in the pig population in Myanmar, with zoonotic and reverse zoonotic potentials.

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.