Genetic and pathogenic characterizations of newly emerged duck Tembusu virus isolated in Thailand

Abstract

Duck Tembusu virus (DTMUV), a mosquito-borne Flavivirus, caused an emerging disease in ducks in Asia since 2010, resulting in significant economic loss in duck producing industry. After the initial outbreak in 2013, DTUMV cases have been continuously detected in several duck farms in Thailand. Thus, the disease surveillance and the genetic characterization of the Thai DTMUV are necessary. Therefore, the first objective of this dissertation was to investigate the genetic characteristic of DTMUVs circulating in Thailand. Our retrospective study demonstrated the presence of DTMUV in the Thai ducks since 2007. Phylogenetic analysis of the polyprotein gene sequence revealed that the 2007 Thai DTMUV was belonged within DTMUV cluster 1, which was genetically different from the currently circulating Thai and Chinese DTMUVs. In addition, the genetic characteristic of DTMUVs recently circulating in Thailand were investigated. Of the 288 clinical samples obtained from 89 ducks farms in Thailand during 2015-2017, 65 samples (22.57%) of 34 duck farms (38.20%) were DTMUV positive. Our results demonstrated the extensive distribution of DTMUV in duck raising areas of Thailand. Our finding indicated that three clusters of DTMUVs were associated with the current DTMUV outbreaks in Asia and suggested the correlation between virus cluster and geographic location. Phylogenetic analysis demonstrated that DTMUVs circulating in Thailand were divided into 3 clusters (1, 2.1 and 3), in which subcluster 2.1 was a predominant cluster of DTMUV circulating in ducks in Thailand during 2015-2017. A novel cluster of DTMUV (cluster 3) was first identified in this study. Our results highlight the high genetic diversity of DTMUVs. This highlighted the need for well-validated detection assay for broad detection of all DTMUV clusters. Therefore, the second objective of this dissertation was to develop and validate a universal one-step RT-PCR assay for broad detection of all DTMUV clusters. Our newly developed RT-PCR assay targeting conserved region of NS5 gene could specifically detect all clusters of DTMUV without cross-reaction with common duck viruses and other related flaviviruses. The assay was able to detect DTMUV as low as 0.001 ELD50/ml. The performance of the assay was also evaluated by testing with 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 E-specific RT-PCR assay (30%) from field clinical samples. In addition, the third objective of this dissertation was to investigate the pathogenesis of a Thai DTMUV in three different ages of ducks. Our findings indicated that all duck ages were susceptible to Thai DTMUV infection. However, younger ducks were more susceptible to DTMUV infection than in older ducks, suggesting age-related susceptibility to Thai DTMUV in ducks. In summary, this dissertation provided useful information on genetic characteristic of Thai DTMUVs, developed the well-validated universal one-step RT-PCR assay for broad detection of all DTMUV clusters and demonstrated the pathogenesis of Thai DTMUV in different ages of ducks. Overall, this study highlights the necessity of the continued routine surveillance of DTMUV in ducks particularly in adult ducks as well as the importance of continuously validating the performance of currently used diagnostic assays against newly emerging strains for early and effective detection, control and prevention of DTMUV.