Evaluation and comparison of hemagglutination inhibition and indirect immunofluorescence tests for the detection of antibodies against duck Tembusu virus.

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.

Dynamics of cellular and humoral immune responses following duck Tembusu virus infection in ducks.

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+ and CD8+ T, B and non-T and B lymphocytes as well as the levels of neutralizing antibodies were quantified in parallel with DTMUV loads in blood and target organs. Our results demonstrated that DTMUV infection caused severe losses of non-T and B lymphocyte/myeloid cell subpopulation, and reduction in phagocytic activity during 3-5 days after infection. We also found that the numbers of T and B cells were increased during the first week of DTMUV infection. A significant negative correlation between the levels of CD4+ and CD8+ T, B and non-T and B lymphocytes and viral loads in blood and target organ (spleen) was observed during the early phase of infection. Additionally, DTMUV infection induced an early and robust neutralizing antibody response, which was associated with DTMUV-specific IgM and IgG responses. The presence of neutralizing antibody also correlated with reduction of viremia and viral load in the spleen. Overall, DTMUV elicited both cellular and humoral immune responses upon infection, in which the magnitude of these responses was correlated with reduction of viremia and viral loads in the target organ (spleen). The results suggested the critical role of both cellular and humoral immunity against DTMUV infection. This study expands our understanding of the immunological events following DTMUV infection in ducks.

Interactions of duck Tembusu virus with Aedes aegypti and Aedes albopictus mosquitoes: Vector competence and viral mutation.

Duck Tembusu virus (DTMUV) is an emerging flavivirus that causes severe disease in avian hosts, while also affecting mammalian hosts; however, information on viral interaction with mosquito vectors for mammalian hosts is limited. Vector competence of Aedes (Ae.) aegypti and Aedes albopictus mosquitoes for DTMUV were investigated. Both Aedes mosquito species were orally infected with DK/TH/CU-1 strain of Thai DTMUV and isolated DTMUV from BALB/c mouse. Genomes of the viruses isolated from hosts and vectors were analyzed and compared with the positive virus. Findings showed that both Aedes mosquito species could serve as vectors for DTMUV with minimum viral titer in blood meal of 106 TCID50/mL. After taking blood meal with viral titer at 107 TCID50/mL, vector competence of the mosquitoes was significantly different from the lower titer in both species. Both Aedes species did not support development of the isolated viruses from mouse. A point mutation of nucleotide and amino acid was found in all isolated DTMUV from Ae. aegypti saliva, while other viruses were similar to the positive virus. Our findings demonstrated that both Ae. aegypti and Ae. albopictus had potential to transmit the virus and play important roles in the viral transmission cycle in mammalian hosts, while viral mutation occurred in Ae. aegypti mosquitoes.

Patterns of duck Tembusu virus infection in ducks, Thailand: a serological study.

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.

Pathogenesis of Thai duck Tembusu virus in BALB/c mice: Descending infection and neuroinvasive virulence.

Duck Tembusu virus (DTMUV) is an emerging flavivirus that causes systemic disease in an avian host. The predominant cluster of DTMUV circulating in Thailand was recently classified as cluster 2.1. The pathogenesis of this virus has been extensively studied in avian hosts but not in mammalian hosts. Six-week-old BALB/c mice were intracerebrally or subcutaneously inoculated with Thai DTMUV to examine clinical signs, pathological changes, viral load and virus distribution. Results demonstrated that the virus caused disease in BALB/c mice by the intracerebral inoculation route. Infected mice demonstrated both systemic and neurological symptoms. Pathological changes and virus distribution were observed in all tested organs. Viral load in the brain was significantly higher than in other organs (p < .05), and the virus caused acute death in BALB/c mice. The virus was disseminated in all parts of the body, but no virus shedding was recorded in saliva and faeces. Findings highlighted the potential of Thai DTMUV to transmit disease in mammalian hosts.

Evaluation of host systems for efficient isolation and propagation of duck Tembusu virus.

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.

Development and Validation of a Universal One-Step RT-PCR Assay for Broad Detection of Duck Tembusu Virus.

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.

Pathogenesis of Thai duck Tembusu virus in Cherry Valley ducks: The effect of age on susceptibility to infection.

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.

Genetic characterization of duck Tembusu virus in Thailand, 2015-2017: Identification of a novel cluster.

Duck Tembusu virus (DTMUV) infected cases have increasingly been observed in several duck farms in Thailand since its first report in 2013. However, information on the genetic characteristic of DTMUVs recently circulating in ducks in Thailand is limited. In this study, we investigated the geographic distribution and genetic characteristic of DTMUVs recently circulating in ducks in Thailand during 2015-2017. Of the 288 clinical samples obtained from 89 ducks farms located in duck raising areas of Thailand, 65 samples (22.57%) of 34 duck farms (38.20%) were DTMUV positive. Our results demonstrated that DTMUV was extensively distributed in duck raising areas of Thailand. Phylogenetic analysis of the E and NS5 genes revealed that DTMUVs circulating in Thailand were divided into three distinct clusters, including cluster 1, subcluster 2.1 and a novel cluster 3. Among these three clusters, subcluster 2.1 was a predominant cluster of DTMUV circulating in duck populations in Thailand during 2015-2017. It is interesting to note that a novel cluster of DTMUV (cluster 3), which was genetically different from any of the previously reported DTMUV clusters, was first identified in this study. In conclusion, our data demonstrated the circulation of different clusters of DTMUV and the presence of a novel DTMUV cluster in ducks in Thailand. This study highlights the high genetic diversity of DTMUVs in Thailand and the necessity of the routine surveillance of DTMUV for early detection, prevention and control of newly emerging DTMUVs.

Serological evidence of duck Tembusu virus infection in free-grazing ducks, Thailand.

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.

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.