Isolation,Characterization And Pathogencity Of A New Goose Tembusu Virus Variant Identified From Hunan Province

Tembusu virus（TMUV）is a single-stranded,positive-sense RNA arbovirus and it belongs to the genus Flavivirus in the Flaviviridae family.It can cause reproductive diseases and neurological diseases in poultry such as chickens,ducks,geese.At the same time,the incidence and recurrence rate are high.Since 2010,TMUV has become prevalent in several Asian countries,including China,causing serious economic losses to the aquaculture industry.In the past ten years,although the transmission of the virus has been controlled by certain measures,due to the virus variation and improper prevention and control measures,the outbreak of Tembusu virus still appears in various regions.In August 2020,a goose farm in Ningxiang City,Hunan Province,was infected by a suspected Tembuxu virus.TMUV was identified from the tissue samples of diseased geese by testing in our laboratory,at the same time,two strains of TMUV virus were successfully isolated using chicken fibroblast cell line（DF-1）,and the whole genome of one of the viruses（TMUV-NX-2）was determined.Genome sequence analysis found that the virus strain has low homology（<90%）with the reported goose and duck Tembusu virus,and the highest homology（about 97%）with the Tembusu virus from Culex tritaeniorhynchus.Phylogenetic analysis further showed that the strains clustered in a single branch,which indicates that the virus strain may be a new variant of Tembusu virus from the cross-species transmission of Culex tritaenior to domestic geese.The viral envelope protein（E）was successfully expressed and purified by the prokaryotic expression system,and polyclonal antibody against E protein was obtained by immunizing Balb/C mice.Indirect immunofluorescence assay（IFA）showed that the polyclonal antibody could specifically recognize the virus in cell culture.In addition,this experiment also determined the half of the tissue cell infection dose(TCID₅₀)of the virus(TCID₅₀=10^5.88).This study shows that mosquitoes,as an important carrier of Tembusu virus,are potentially a major threat to the healthy breeding of geese and ducks.At the same time,this study shows that Tembusu virus has high genetic divergence,which poses a challenge to the research of targeted vaccines.In addition,this study provide a basis for further research on the pathogenicity and pathogenic mechanism of goose tembusu virus,the mechanism of cross-species transmission of the virus,and the development of targeted specific vaccines.