Isolation and identification of Mangshi virus in sentinel cattle from border areas of Yunnan Province

Objective  To analyze the diversity, dissemination risk of animal arboviruses in border areas of Yunnan Province.

Method  Three sentinel cattles were placed in Jinghong City, Yunnan Province and blood samples were weekly collected for arboviruses monitoring and virus isolation. The isolated virus was identified through agar-gel electrophoresis, electron microscopy, RT-PCR amplification, cloning and sequencing. The infection characteristics of the virus in infected cattle were retrospectively analyzed by qRT-PCR and serum neutralizing test (SNT).

Result  In 2019, a virus strain (V301/YNJH/2019) which caused cytopathic effect on C6/36 cells, was isolated from blood sample collected from one of the cattle at the 13^th week of monitoring period. Electron microscope observation revealed that the virions were icosahedral symmetry with a diameter of about 70 nm. The result of agar-gel electrophoresis showed that the viral genome was composed of double stranded RNA. The isolated virus was identified as Mangshi virus (MSV) by RT-PCR identification. Sequence analysis exhibited the lengths of Seg-4 and Seg-7 from the isolated virus were 2 055 and 1 122 bp respectively, encoding viral out-most shell VP4 (628 aa) and VP7 (298 aa) proteins, while Seg-2 and Seg-9 were 3 055 and 1 076 bp respectively in length encoding viral inner-core VP2 (956 aa) and VP9 (283 aa) proteins. Phylogenetic analysis showed that the isolated virus had the closest relationship with MSV/DH13M041 isolated from Mangshi of Yunnan Province, with their nucleic acid sequence similarities ranging from 97.4% (Seg-9) to 98.5% (Seg-2) and amino acid sequence similarities ranging from 96.4% (VP9) to 98.4% (VP2). Retrospective investigation results of the virus infection indicated that virus nucleic acid was first detected in the blood of the infected cattle at the 11^th week of monitoring period. The virus nucleic acid in the blood reached the peak at the 13^th week and dropped rapidly, becoming not detectable within 5 weeks. The results of SNT showed that neutralization antibody against V301/YNJH/2019 was first detected at the 13^th week (antibody titer 1∶14), peaked from the 16^thto 18^thweek (antibody titer 1∶226) and decreased to 1∶57 when monitoring terminated at the 24^thweek.

Conclusion  The MSV isolation from cattle indicating cattle is one of the susceptible animals of MSV. In naturally infected cattle, the virus is characterized by “transient infection” with no clinical symptoms. Our study imparts a foundation for study of epidemiology, pathogenicity, and diagnostic reagents of MSV.