| Bovine viral diarrhea is casued by Bovine viral diarrhea virus(BVDV) which can result in significant economic losses in the cattle industry worldwide. BVDV is widely distributed and has been detected in more than 20 provinces in China. To assess the overall prevalence of BVDV in Chinese free-roaming beeves, the free-roaming beeves in the Western of China were used in the present study by the methods of serological and bioinformatic analysis. Then, the genetic diversity of BVDV was analyzed. This study can provide theoretical basis for the development of reasonable prevention and control, and for the vaccine development.1332 sera were collected in Yunnan, Xinjiang, Gansu and Inner Mongolia from 2014 to 2015.These samples were analyzed by the commercial antibody(Ab) detection kits, and the Ab positive rate of BVDV in various regions were summarized up. Meanwhile, the positive or suspicious samples,whose S/P were less than 60% were further detected by the Ag detection kit. Then, to isolate BVDV, the positive samples were inoculated into the MDBK cells and passage by five generations. Identify genotype of clinical strains by RT-PCR based on the 5’-UTR and Npro, respectively sequencing and phylogenetic analysis. Then, a virus(designated as BVDV-NM1369) would be cultured adaptively in MDBK and TCID50 trial also would be conduted. Refering to NADL standard strain sequence, a set of primers were designed. The genome sequence was abtained by PCR and sequcing. Last, the genome sequence of BVDV-NM1369 was analysised compareing with the domestic and overseas strains by BLAST.Results showed that the average Ab positive rate of BVDV was 33.93% in the four detected provincial regions. Among them, the Inner Mongolia positive rate was the highest(71.43%), with Xinjiang taking the second place(57.69%), Gansu the third(16.54%) and Yunnan the lowest(10.0%).The average Ag positive rate of BVDV was only 1.58%, with Xinjiang the highest(5.49%). In this study,a total of 13 BVDV strains were isolated, they were named as Gansu 120, Mongolia 1369, Yilihuoqing12953, Yillihuoqing 12960, Yilihuoqing 12981, Bozhoujinghe 13001, Bozhoujinghe 13023,Bozhoujinghe 13033, Xinjiangqitai 13041, Xinjiangqitai 13042, Xinjiangqitai 13191, Xinjiangqitai13220, Xinjiangqitai 13251, respectively. The polygentic analysis indicated that these isolated could be mainly classified into four subtypes: BVDV-1q and BVDV-1f(isolated in Xinjiang), BVDV-1m(isolated in Inner Mongolia) and BVDV-1u which was a new subtype isolated in Gansu. The result of sequencing of NM1369 showed that it was composed of 12268 nucleotides in length, deduced 3902 amino acid. The 5’-UTR and Npro sequences were analyzed by Clus W and construction of phylogenetic trees, which revealed that BVDV-NM1369 belonged to BVDV-1m. Compared with other reference strains, nucleotide homology range was between 74.0% and 94.8%. Among them, the strains of highest homology which was 94.8% were ZM-95 and SD-15 isloated in China. NM1369 was Inoculated into monolayer MDBK cells and passaged serially. The titer of NM1369 was 105.24 TCID50/0.1mldeterminated by indirect immunoinfluscent assay(IFA)。The whole level of BVDV infection among free-range beef cattles was lower than that in the scale dairy farms, however there was a big variation of prevalence level with different provinces. We should make overall layout, but prevention discriminatingly. In addition, high genetic variability of bovine viral diarrhea virus might be concerned, therefore, identification and removal of persistent infection animals and cutting off transmission routes by strengthen management are necessary. |
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