Objective To investigate the status of arboviruses carried by midges in Mangshi, Dehong Dai and Jingpo autonomous prefecture (Dehong prefecture), Yunnan province, China, and to provide a scientific basis for the research and control on arboviruses. Methods The light-trap method was used to collect specimens of midges from cattle and sheep farms in suburbs of Mangshi, Dehong prefecture in July 2013. The collected Culicoides midges were ground and centrifuged, and the supernatants were inoculated with baby hamster kidney-21 (BHK-21) and Aedes albopictus C6/36 cells separately for virus isolation. The specific primers for Alphavirus, Flavivirus, and Bunya virus were used to preliminarily identify the positive isolates, and the specific primers for Sindbis virus (SINV) NS1 gene were used to perform the reverse transcription polymerase chain reaction (RT-PCR), sequencing, and sequence analysis. Results A total of 4 500 midges were collected, which were divided into 44 batches for virus isolation. One positive isolate (serial number: YN222) was obtained, which produced obvious cytopathic effect in 24 hours after inoculation of BHK-21 cells, and in 72 hours after inoculation of C6/36 cells. The RT-PCR amplification showed a positive result by using the primers for Alphavirus, whereas showed negative results by using those for Flavivirus and Bunya virus, suggesting that the isolate YN222 was from the genus Alphavirus. The BLAST sequence analysis after the amplification of positive products using the primers for Alphavirus showed that the isolate YN222 had a nucleotide homology of up to 93.7% with SINV MRE16 strain. The RT-PCR amplification showed a positive result by using the specific primers for NS1 gene of MRE16 strain, and a 1 605-nt sequence was obtained via sequencing. The phylogenetic analysis showed that the newly isolated virus YN222 from midges was in the same evolutionary branch as the Oriental-Australian type of SINV, with a high homology in nucleotides (93.7%-99.6%) and amino acids (99.4%). Conclusion The virus YN222 isolated from midges collected in Dehong prefecture of Yunnan province is Oriental-Australian type of SINV. This is the first time that SINV has been isolated from Ceratopogonidae.
HE Yu-wen, LI Nan, MENG Jin-xin, WANG Jing-lin
. Isolation and identification of Sindbis virus from midges collected in Mangshi, Yunnan province, China[J]. Chinese Journal of Vector Biology and Control, 2021
, 32(4)
: 498
-502
.
DOI: 10.11853/j.issn.1003.8280.2021.04.023
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