›› 2018, Vol. 45 ›› Issue (11): 3192-3202.doi: 10.16431/j.cnki.1671-7236.2018.11.026

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Isolation and Identification of Bluetongue Virus Type 16 Strain from Jiangcheng County in Yunnan Province and Sequence Analysis of Its L2 Gene

KOU Meiling, MIAO Haisheng, LI Le, LI Nan, LIAO Defang, LI Huachun   

  1. Yunnan Tropical and Subtropical Animal Virus Disease Laboratory, Yunnan Animal Science and Veterinary Institute, Kunming 650224, China
  • Received:2018-04-13 Online:2018-11-20 Published:2018-11-20


In order to understand the prevalence of Bluetongue virus type 16 (BTV-16) strains from Jiangcheng county in Yunnan province in recent years,and the genetic evolution relationship between L2 gene and foreign strains,in this study,300 bovine heparin sodium anticoagulated bloods were taken from Jiangcheng county for extraction of red blood cells,and 10 days old chicken embryos were inoculated intravenously.The collected chicken embryo livers were crushed and centrifuged,and the supernatant was inoculated into C6/36 and BHK21 cells for passage.For the cytopathic samples,the group-specific VP7 fragment primers were used for RT-PCR detection.The BTV-16 L2 gene-specific primers were used to perform RT-PCR amplification and sequencing of the BTV nucleic acid positive samples.The nucleotide and amino acid homology alignments and genetic evolution analysis of the obtained L2 gene coding region sequences were performed using DNAStar and Mega 6.0 softwares.At the same time,the isolated virus was neutralized by BTV-16 standard positive serum.The results showed that 30 cytopathic samples were found in Jiangcheng county,17 of which were initially identified as BTV by RT-PCR;6 of them were identified as BTV-16 strains by L2 gene sequence analysis and neutralization test;The nucleotide and amino acid homology alignment analysis showed that the nucleotide and amino acid homology of the 6 strains were 93.4% to 98.0% and 94.2% to 99.1%,respectively;Genetic evolution analysis found that 5 of them were closely related to the BTV-16 strains isolated in Japan from 2001 to 2008 and in India from 1982 to 2011,one strain was closely related to the BTV-16 strain isolated from Japan in 1985 to 1990.This study found that the BTV-16 strain in Jiangcheng county of Yunnan province showed a cross-continuous epidemic of new and old strains,but the genetic variation in natural evolution was small and had certain stability.This study at the molecular level clarified the local popular BTV from Jiangcheng county in Yunnan province.The genetics and differences between the BTV-16 L2 gene provided a scientific basis for further research of BTV molecular epidemiology and detection.

Key words: Bluetongue virus (BTV); virus isolation; L2 gene; sequence analysis

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