A novel goose-origin Tembusu virus exhibits pathogenicity in day-old chicks with evidence of direct contact transmission

In late 2020, an outbreak of Tembusu virus (TMUV)-associated disease occurred in a 45 -day-old white Roman geese flock in Taiwan. Here, we present the identification and isolation of a novel goose -origin TMUV strain designated as NTU/C225/2020. The virus was successfully isolated using minimal -path-ogen-free duck embryos. Phylogenetic analysis of the polyprotein gene showed that NTU/C225/2020 clus-tered together with the earliest isolates from Malaysia and was most closely related to the first Taiwanese TMUV strain, TP1906. Genomic analysis revealed sig-nificant amino acid variations among TMUV isolates in NS1 and NS2A protein regions. In the present study, we characterized the NTU/C225/2020 culture in duck embryos, chicken embryos, primary duck embryonated fibroblasts, and DF-1 cells. All host systems were sus-ceptible to NTU/C225/2020 infection, with observable lesions. In addition, animal experiments showed that the intramuscular inoculation of NTU/C225/2020 resulted in growth retardation and hyperthermia in day-old chicks. Gross lesions in the infected chicks included hepatomegaly, hyperemic thymus, and splenomegaly. Viral loads and histopathological damage were displayed in various tissues of both inoculated and naive co-housed chicks, confirming the direct chick-to -chick contact transmission of TMUV. This is the first in vivo study of a local TMUV strain in Taiwan. Our findings provide essential information for TMUV propaga-tion and suggest a potential risk of disease outbreak in chicken populations.