Isolation and characterization of a novel goose ovaritis-associated cluster 3 Tembusu virus

Tembusu virus (TMUV) is a member of the genus Flavivirus in the family Flaviviridae. Cur-rently, TMUV was classified into 4 distinct clusters, with cluster 2 strains widely distributed in duck and goose populations in Asia, causing significant economic losses to the producing industries. In this study, a novel TMUV strain TMUV/goose/CHN/2019/HNU-NX2 (HNU-NX2-2019) was isolated and characterized from geese with ovaritis from Hunan province, China. Phylo-genetic analyses of genome and the E gene indicated the present TMUV could be grouped into the newly defined TMUV cluster 3. The genome of HNU-NX2-2019 showed the highest identities of 98.1% to 98.2% to the cluster 3 TMUVs newly identified in 2020 and 2021 from chickens with a severe egg-drop syndrome from Guang-dong, Guangxi and Shandong provinces of China, which were all showing a close relation to a mosquito-origin TMUV (KT607936) identified in 2012. Further experiments confirmed HNU-NX2-2019 could grow well in chicken fibroblast cell line DF-1 and in SPF chicken embryos, with titers varied from 107.3 to 108.8 viral geno-mic copies per mL in the culture solutions. A pilot virus challenge study in 3-day-old chicks demonstrated that this virus could efficiently infect chicks with virus dis-tributed in the brains, small intestines and other visceral organs, with titers varied from 105.4 to 106.7viral geno-mic copies per gram of the tissues. Furthermore, HNU-NX2-2019 can induce specific antibody in ducklings but with no obvious disease and virus shedding, and on nec-ropsy no TMUV was detected in the tissues in the present study. This is the first report to identify a novel cluster 3 TUMV from goose, and further demonstrated this goose TMUV strain could infect chicken efficiently but not in ducklings under the present experimental con-ditions, which highlighted intensive attentions may be paid to this novel mosquito-origin cluster 3 TMUV.