关键词: 坦布苏病毒(TMUV); 分支3; 分离鉴定; 鹅; 致病性

Abstract: 【Objective】 This study was aimed to understand the genomic variation of Cluster 3 goose Tembusu virus (TMUV) and its pathogenicity to geese.【Method】 BHK-21 cells were used to isolate the liver tissue samples of geese infected with TMUV,and identified by RT-PCR,indirect immunofluorescence assay (IFA),transmission electron microscope observation,and the growth curve of isolated viruses were detected.After the whole genome amplification of TMUV isolated strains,the genetic evolution was analyzed by ModelFinder,MrBayes and other softwares.The amino acid mutation sites of E protein of TMUV isolated strains were analyzed.After detecting the viral titers of TMUV isolates,the isolates were used to challenge 30-day-old geese,respectively.Then the clinical dissection of each tissue and organ,and the histopathological changes were observed.The viral load in each tissue and organ was detected by Real-time PCR.【Result】 Two TMUV nucleic acid positive samples were successfully identified by RT-PCR.After inoculation into BHK-21 cells,significant lesions were observed within 60 h.Significant red fluorescence could be observed by IFA detection of the third-generation virus solution,and virus particles with a diameter of about 50 nm and a capsule could be observed by transmission electron microscopy.Two strains of TMUV were successfully isolated from diseased goose liver tissue and named JM3 and JM1205,respectively.The one-step growth curve of the virus showed that JM3 and JM1205 strains had the highest virus titers after 60 and 48 h of cultivation,respectively.The whole genome amplification results showed that the full-length genomes of JM3 and JM1205 strains were both 10 994 bp.The genetic evolutionary tree showed that both JM3 and JM1205 strains were members of Cluster 3 TMUV,and had the closest genetic distance to the CTLN isolate of Cluster 3 TMUV chicken source.The analysis of amino acid mutation sites showed that compared with the earliest uploaded TMUV strain MM1775 in GenBank,JM3 and JM1205 strains had multiple amino acid site mutations in the E protein,among which the V157A mutation might be related to the increased virulence of TMUV.After 1 d of infection,geese began to show symptoms of green and sparse feces,and after 7 d of infection,neurological symptoms began to appear.JM3 group continued to detoxify 14 d after challenge,while the JM1205 group continued to detoxify until 11 d after challenge.After 6 d of infection,the geese showed a slowdown and decrease in weight growth,and began to recover slowly and increase by 10 d.Autopsy revealed varying degrees of splenomegaly,pancreatic necrosis,liver whitening,and cerebral congestion in the infected geese.In addition,JM3 strain infected geese showed ovarian bleeding and pericardial effusion.Geese infected with JM1205 strain showed cardiac bleeding.The viral load in the spleen was highest at all time points after infection,reaching its peak at 3 d after infection and gradually decreasing thereafter.【Conclusion】 This study isolated two strains of Cluster 3 TMUV-JM3 and JM1205 from goose farms in Guangdong province.Both isolates were pathogenic to geese and could replicate in multiple organs,causing symptoms such as ataxia and weight loss.

Key words: Tembusu virus (TMUV); Cluster 3; isolation and identification; goose; pathogenicity