Nutthawan Nonthabenjawan

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)

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.

Tembusu-Related Flavivirus in Ducks, Thailand.

Since 2013, outbreaks of disease caused by duck Tembusu virus (DTMUV) have been observed in layer and broiler duck farms in Thailand. The virus is closely related to Chinese DTMUVs and belongs to the Ntaya group of mosquitoborne flaviviruses. These findings represent the emergence of DTMUV in ducks 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.

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.

The presence of duck Tembusu virus in Thailand since 2007: A retrospective study

Duck Tembusu virus (DTMUV), a newly emerging virus in ducks, was first reported in China in 2010. However, an unknown severe contagious disease associated with severe neurological signs and egg production losses in ducks, resembling to DTMUV infection, was observed in Thailand since 2007. To determine the presence of DTMUV in 2007, the clinical samples from affected ducks collected in 2007 were tested for DTMUV using pathological and virological analyses. Gross and histopathological lesions of affected ducks were mostly restricted to the ovary, brain and spinal cord, and correlated with the presence of flavivirus antigen in the brain and spinal cord samples. Subsequently, DTMUV was identified by RT-PCR and nucleotide sequencing of the polyprotein gene. Phylogenetic analysis of the polyprotein gene sequence revealed that the 2007 Thai DTMUV was a unique virus, belonged within DTMUV cluster 1, but distinctively separated from the Malaysian DTMUV, which was the most closely related DTMUV. It is interesting to note that the 2007 Thai DTMUV was genetically different from the currently circulating Thai and Chinese DTMUVs, which belonged to cluster 2. Our findings indicated that the 2007 Thai DTMUV emerged earlier from a common ancestor with the recently reported DTMUVs; however, it was genetically distinctive to any of the currently circulating DTMUVs. In conclusion, our data demonstrated the presence of DTMUV in the Thai ducks since 2007, prior to the first report of DTMUV in China in 2010. This study indicates that DTMUV may have circulated in the region long before 2010 and highlights high genetic diversity of DTMUVs in Asia.

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.