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Marek's disease (MD) is a highly contagious lymphoproliferative disease of chickens caused by Gallid alphaherpesvirus 2, commonly known as serotype 1 Marek's disease virus (MDV-1). Despite widespread vaccination, MD-related cases have been frequently observed worldwide, including in Thailand. However, no information is available on the genetic characteristics of MDV-1 field strains circulating in chickens in Thailand. This study investigated the geographic distribution and genetic characteristics of MDV-1 field strains circulating in chickens in Thailand between 2013 and 2021 by analysing the Meq and pp38 genes. Out of a total of the 286 clinical samples obtained from 70 chicken farms located in major chicken raising areas of Thailand, 138 samples (48.25%) from 46 chicken farms (65.71%) tested positive for MDV-1 field strains. Results demonstrated that MDV-1 field strains were extensively distributed in major chicken raising areas. Phylogenetic analyses based on the Meq gene revealed that four clusters of MDV-1 circulated in chickens in Thailand between 2013 and 2021. Among these clusters, cluster 1 was the predominant cluster circulating in chickens in Thailand. Additionally, our findings based on molecular characteristics of Meq and pp38 gene/protein suggested that most of the Thai MDV-1 field strains were potentially highly virulent. In conclusion, our data demonstrated the circulation of different clusters of MDV-1 with virulence characteristics in chickens in Thailand, indicating high genetic diversity of MDV-1 in Thailand. This study highlights the importance of more effective vaccine development and routine MDV-1 surveillance for early detection and control of highly virulent MDV-1.

Duck Tembusu virus (DTMUV), a mosquito-borne flavivirus, has been identified as a causative agent of an emerging viral disease in ducks, causing significant economic losses to the duck-producing industry. In Thailand, DTMUV has been detected sporadically in ducks since the first report in 2013. However, information on the patterns of DTMUV infection in ducks in Thailand is limited. In this study, a serological survey of DTMUV on ducks raised in farming and free-grazing systems was conducted during 2015-2016. Blood samples of farm ducks (n = 160) and free-grazing ducks (n = 240) were collected in the summer, rainy, and winter seasons during 2015-2016 and tested for DTMUV infection. Our results showed that DTMUV infection in ducks in Thailand occurred all year-round; however, the patterns of DTMUV infection varied between 2 duck-raising systems. Significant seasonal pattern was found in free-grazing ducks, whereas no seasonality was observed in farm ducks. Notably, DTMUV infection in ducks in Thailand was highest in the winter season. In conclusion, our data indicate distinct patterns of DTMUV infection between farm and free-grazing ducks, and the year-round circulation of DTMUV in ducks in Thailand, with peaks in the winter season. This information will help reduce the risk of DTMUV transmission through prevention and control strategies focusing on the peak period. Routine surveillance of DTMUV in ducks is essential for early detection of DTMUV allowing the implementation of control measures in a timely manner.

Currently, duck Tembusu virus (DTMUV), an emerging avian pathogenic flavivirus, is widely spread and becomes endemic in duck populations in Asia, causing significant economic losses in the duck producing industry. To early detection and control of DTMUV, the well-validated diagnostic tests for efficient detection of DTMUV infection in ducks are needed. In this study, we validated and compared hemagglutination inhibition (HI) and indirect immunofluorescence (IFA) tests for identifying antibodies against DTMUV in duck serum samples. Our results demonstrated that HI and IFA tests can both be used to detect antibodies against DTMUV in duck serum samples with high sensitivity (100%), specificity (87%) and overall agreement with the gold standard serum neutralization (SN) test (90%). Additionally, DTMUV-specific antibody titres determined by HI and IFA tests correlated well with the neutralizing antibody titres obtained by SN test. No cross-reactivity against common duck viruses and other flaviviruses was observed in both tests. It is interesting to note that HI test had higher diagnostic specificity and exhibited a stronger positive correlation with SN test than IFA test. Evaluating the performance of HI and IFA tests with experimental and field serum samples revealed that both tests showed comparable performance with SN test in terms of antibody kinetic and detection rate. Collectively, these findings support the use of both tests, particularly HI test, as the alternative to SN test for measuring the antibody responses against DTMUV in ducks. These tests could be the suitable choices for DTMUV diagnosis, epidemiological study and vaccine efficacy evaluation.

Duck Tembusu virus (DTMUV), an emerging avian pathogenic flavivirus, causes severe neurological disorders and acute egg drop syndrome in ducks. However, the effects of DTMUV on duck immunological components and functions remain largely unknown. In this study, the dynamics of cellular and humoral immune responses of DTMUV-infected ducks were investigated. The numbers of CD4

Several phylogenetic clusters of duck Tembusu virus (DTMUV) that caused outbreaks in ducks in Asia have been identified since its emergence in 2010, highlighting the need for an efficient host system that can support isolation of all circulating phylogenetic clusters of DTMUV. In this study, various host systems, including different avian embryonated eggs (duck and chicken) and cell cultures (primary duck embryo fibroblast (DEF), primary chicken embryo fibroblast (CEF), baby hamster kidney (BHK-21), African green monkey kidney (Vero) and Aedes albopictus clone C6/36 (C6/36) cells), were evaluated and compared for their ability to support DTMUV isolation and propagation. Our results showed that all host systems were susceptible to DTMUV infection; however, BHK-21 and primary DEF cells supported more efficient replication of DTMUV compared to the other host systems. BHK-21 cells had the highest DTMUV isolation rate when tested with experimental and field clinical samples. All circulating phylogenetic clusters of DTMUV, including clusters 1, 2 and 3, were successfully isolated from duck clinical samples using BHK-21 cells. In conclusion, our findings supported the use of BHK-21 cells as a host system for primary isolation of all circulating phylogenetic clusters of DTMUV from duck clinical samples. This study highlights the importance of selecting the most appropriate host system for efficient isolation and propagation of DTMUV from duck clinical samples.RESEARCH HIGHLIGHTS DTMUV replicated more efficiently in BHK-21 and primary DEF cells than in other host systems tested.BHK-21 cells had the highest DTMUV isolation rate.All DTMUV phylogenetic clusters were successfully isolated from the samples using BHK-21 cells.BHK-21 cells were the most efficient host system for DTMUV isolation.

Duck Tembusu virus (DTMUV), a mosquito-borne flavivirus, has been identified as a causative agent of an emerging disease in ducks. Since its first report in 2010, several clusters of DTMUV have increasingly been identified and caused outbreaks in many Asian countries. This highlights the need for improved and novel broad detection assays in order to detect all circulating clusters of DTMUV. In this study, a universal one-step reverse-transcription PCR (RT-PCR) assay targeting a highly conserved region of the NS5 gene was developed and validated for broad detection of all DTMUV clusters. The newly developed universal RT-PCR assay could specifically detect all clusters of DTMUV without cross-reactions with common duck viruses and other related flaviviruses. The assay was able to detect DTMUV as low as a 0.001 50% embryo lethal dose/milliliter. The performance of the assay was evaluated by using experimental and field clinical samples. The assay could successfully detect DTMUV in all experimentally DTMUV-infected samples and gave a higher DTMUV detection rate (36%) than the previously reported envelope-specific RT-PCR assay (30%) in field clinical samples. All the positive samples were confirmed DTMUV-positive by DNA sequencing. In conclusion, the newly developed universal RT-PCR assay exhibited high accuracy, specificity, and sensitivity in broad DTMUV detection, thus providing an improved screening assay for routine detection and epidemiologic surveillance of DTMUV.

Several duck Tembusu virus (DTMUV) clusters have been identified since its first emergence in 2010. However, the pathogenesis evaluation of DTMUV has been restricted to cluster 2.2 Chinese DTMUVs. In this study, the pathogenesis of a cluster 2.1 Thai DTMUV was investigated in three ages of Cherry Valley ducks (1-, 4- and 27-week-old). In each age, 35 ducks were inoculated with a cluster 2.1 Thai DTMUV and evaluated for clinical signs, virus distribution and shedding, pathology and serological response. Our results demonstrated that all duck ages were susceptible to Thai DTMUV; however, Thai DTMUV induced greater disease severity in younger ducks (1- and 4-week-old) when compared to older ducks (27-week-old) reflected by higher morbidity and mortality rates, and higher degree of pathological severity. Corresponding to these results, longer-term viremia, higher levels of viral loads in tissues and lower neutralizing antibody titers were also observed in younger ducks compared to those in older ducks. However, it should be noted that a significant drop in egg production was found in older ducks, which also indicates the susceptibility to Thai DTMUV in older ducks. Interestingly, prolonged shedding period with high viral loads was observed in older ducks even without showing clinical signs, suggesting the potential role of the older ducks as the carriers of Thai DTMUV. This finding highlights the importance of monitoring DTMUV and preventing the transmission of DTMUV in adult ducks. Overall, this study provides insights into the pathogenesis and infection dynamics of a cluster 2.1 Thai DTMUV in ducks.

Duck Tembusu virus (DTMUV) has been reported in ducks raised in farming system since its emergence in 2010. No information is available on DTMUV infection in free-grazing ducks, which are commonly raised and widespread in several Asian countries. To determine the presence of DTMUV infection in free-grazing ducks in Thailand, retrospective serum samples collected from 1,000 free-grazing ducks during 2008-2015 were tested for DTMUV infection. Our result showed that 91 (9.10%) were positive for DTMUV neutralizing antibodies and DTMUV seropositive ducks have been detected in Thailand since 2008. To further investigate the seroprevalence and geographic distribution of DTMUV infection in free-grazing ducks in Thailand, a cross-sectional serological survey of DTMUV was conducted in 2016. Of 1,200 free-grazing ducks in the 60 flocks from 20 provinces located in the major free-grazing duck raising areas of Thailand, 365 (30.42%) were positive for DTMUV neutralizing antibodies and 56 flocks (93.33%) had at least one DTMUV seropositive duck. Additionally, DTMUV seropositive ducks were observed in all provinces tested. In conclusion, our data demonstrated the presence of DTMUV infection in free-grazing ducks since 2008 and widespread DTMUV infection in free-grazing ducks in Thailand with a relatively high seroprevalence. These findings suggest the potential role of free-grazing ducks in the dissemination of DTMUV and highlight the necessity of systemic DTMUV surveillance in free-grazing ducks in addition to farm ducks for early detection, prevention, and control of this emerging disease.

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.

Quail is considered as an intermediate host for generation of the novel reassortant influenza A viruses (IAVs). In this study, we evaluated the replication ability of the three novel H3N1 reassortant viruses recovered from pandemic H1N1 2009 (pH1N1) and duck H3N2 (dkH3N2) co-infected quail generated from our previous study in embryonated chicken eggs, mammalian (MDCK) and human lung derived (A549) cells. Our study demonstrated that all of the reassortant viruses replicated efficiently in avian and mammalian cells, albeit with slightly lower titers than the parental viruses. Of note, all of the reassortant viruses showed enhanced replication in human lung derived A549 cells compared to their parental viruses. Interestingly, among the reassortant viruses tested, a reassortant virus (P(NA,NS)-DK) containing NA and NS genes derived from pH1N1 and the other genes from dkH3N2 exhibited the highest replication ability in all in vitro models, indicating a high level of gene compatibility of this reassortant virus. Our results highlight the potential role of quail as intermediate hosts for the generation of the viable reassortant viruses with ability to replicate efficiently in avian, mammalian, and particularly human lung derived cells. These findings emphasize the need for the continuous IAV surveillance in quail to prevent the risk of the emergence of the novel viable reassortant viruses.

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.