CAJ | 학술논문

为了解近年来云南省师宗县蓝舌病病毒流行情况，2012年在师宗县五龙乡建立了10头蓝舌病血清学阴性黄牛的监控动物群。从2012年5~10月，每周采血1次，11~12月，每月采血1次，采用C-ELISA进行血清学监测。8月开始动物血清学检测结果转阳性，至11月，监控动物全部转为阳性。用转阳前1周、转阳本周、转阳后2~13周的经处理的红细胞静脉接种鸡胚，收获鸡胚肝脏，用PBS悬浮捣碎的鸡胚肝脏，上清接种于C6/36细胞一代、BHK-21三代后，出现细胞病变（cytopathic effect, CPE）。采用RT-PCR方法，针对蓝舌病较为保守的血清型群特异片段VP7设计了2对引物，扩增其相应片段。结果显示，共分离到86份疑似分离物，其中67份疑似分离物细胞培养液上清经RT-PCR扩增，均扩增出1156 bp片段，初步确认为蓝舌病病毒。采用国际24个蓝舌病标准毒及24个标准阳性血清对86份疑似分离物及其对应血清进行细胞微量中和试验，67份毒株为蓝舌病病毒，与RT-PCR结果一致。通过对2份经中和试验定型为BTV-1、BTV-16分离株的VP2基因测序分析发现，BTV-1株序列与同型Y863（登录号：KC879616）参考毒株的同源性为92%，BTV-16株序列与登录号为AB686221的毒株同源性为99%。结果表明共分离到67株蓝舌病毒株，分离株主要为BTV-1、BTV-9、BTV-16三个血清型。
위료해근년래운남성사종현람설병병독류행정황，2012년재사종현오룡향건립료10두람설병혈청학음성황우적감공동물군。종2012년5~10월，매주채혈1차，11~12월，매월채혈1차，채용C-ELISA진행혈청학감측。8월개시동물혈청학검측결과전양성，지11월，감공동물전부전위양성。용전양전1주、전양본주、전양후2~13주적경처리적홍세포정맥접충계배，수획계배간장，용PBS현부도쇄적계배간장，상청접충우C6/36세포일대、BHK-21삼대후，출현세포병변（cytopathic effect, CPE）。채용RT-PCR방법，침대람설병교위보수적혈청형군특이편단VP7설계료2대인물，확증기상응편단。결과현시，공분리도86빈의사분리물，기중67빈의사분리물세포배양액상청경RT-PCR확증，균확증출1156 bp편단，초보학인위람설병병독。채용국제24개람설병표준독급24개표준양성혈청대86빈의사분리물급기대응혈청진행세포미량중화시험，67빈독주위람설병병독，여RT-PCR결과일치。통과대2빈경중화시험정형위BTV-1、BTV-16분리주적VP2기인측서분석발현，BTV-1주서렬여동형Y863（등록호：KC879616）삼고독주적동원성위92%，BTV-16주서렬여등록호위AB686221적독주동원성위99%。결과표명공분리도67주람설병독주，분리주주요위BTV-1、BTV-9、BTV-16삼개혈청형。
The sentinel herd of 10 cattle was established in 2012 to monitor epidemiology of Bluetongue virus in Shizong county of Yunnan Province. Blood samples were taken weekly from May to October and then monthly from November to December 2014 and tested for serological response in C-ELISA and for virus isolation. The sero-conversion was revealed in August in some animals and then in November in whole sentinel herd. The erythrocyte preparations from blood samples were inoculated into chicken embryo veins. Chicken liver tissues were harvested, homogenized in PBS and centrifuged. The supernatants were inoculated into C6/36 cells and passaged in BHK-21 three times. The cytopathic effect (CPE) was observed in cell monolayers. The resulting 86 virus isolates were characterized in RT-PCR and virus neutralization (VN). The VP7 gene of Bluetongue virus was amplified in conventional RT-PCR using two pairs of primers designed according to the sequences in GenBank. A 1156 bp fragment of VP7 gene was amplified in RT-PCR from 67 out of 86 isolates. The virus neutralization was performed using 24 reference bluetongue viruses and 24 positive sera. Again, the same 67 isolates were confirmed as Bluetongue virus. The VP2 gene of two isolates was sequenced. One isolate was determined to be BTV-1 as it shared 92%identity with the reference strain Y863 (KC879616) and another isolate was BTV-16 as it shared 99%identity with the reference strain AB686221. The VN results also had an agreement with VP2 sequencing. In conclusion, 67 isolates isolated from the sentinel herd belonged to Bluetongue virus, indicating the virus transmission among bovine herds.