LUNAM University, Oniris, (Nantes-Atlantic National College of Veterinary Medicine, Food Science and Engineering), Department of Research into the Health Risk and Biotechnology of Reproduction UPSP 5301 DGER, France.
Theriogenology. 2011 Jul 1;76(1):126-32. doi: 10.1016/j.theriogenology.2011.01.025. Epub 2011 Mar 12.
The three objectives of this study were to investigate whether cells of early goat embryos isolated from in vivo fertilized goats interact with bluetongue virus (BTV) in vitro, whether the embryonic zona pellucida (ZP) protects early embryo cells from BTV infection, and whether the 10 wash cycles recommended by the International Embryo Transfer Society (IETS) for bovine embryos effectively decontaminates caprine embryos exposed to Bluetongue Virus (BTV) in vitro. Donor goats and bucks were individually screened and tested negative for the virus by RT-PCR detection of BTV RNA in circulating erythrocytes. ZP-free and ZP-intact 8-16 cell embryos were co-cultured for 36 h in an insert over a Vero cell monolayer infected with BTV. Embryos were washed 10 times in accordance with IETS recommendations for ruminant and porcine embryos, before being transferred to an insert on BTV indicator Vero cells for 6 h, to detect any cytopathic effects (CPE). They were then washed and cultured in B2 Ménézo for 24 h. Non-inoculated ZP-free and ZP-intact embryos were submitted to similar treatments and used as controls. The Vero cell monolayer used as feeder cells for BTV inoculated ZP-free and ZP-intact embryos showed cytopathic effects (CPE). BTV was found by RT-qPCR in the ten washes of exposed ZP-free and ZP-intact embryos. In the acellular medium, the early embryonic cells produced at least 10(2.5) TCID(50)/ml. BTV RNA was detected in ZP-free and ZP-intact embryos using RT-qPCR. All of these results clearly demonstrate that caprine early embryonic cells are susceptible to infection with BTV and that infection with this virus is productive. The washing procedure failed to remove BTV, which indicates that BTV could bind to the zona pellucida.
调查从体内受精的山羊胚胎中分离的早期胚胎细胞是否在体外与蓝舌病病毒(BTV)相互作用;胚胎透明带(ZP)是否保护早期胚胎细胞免受 BTV 感染;国际胚胎移植学会(IETS)推荐的用于牛胚胎的 10 次洗涤循环是否能有效清除体外暴露于蓝舌病病毒(BTV)的山羊胚胎。供体山羊和公羊分别进行了筛选,并通过在循环红细胞中检测 BTV RNA 的 RT-PCR 检测呈阴性。ZP 无和 ZP 完整的 8-16 细胞胚胎在插入物中与感染 BTV 的 Vero 细胞单层共培养 36 小时。胚胎根据 IETS 对反刍动物和猪胚胎的建议洗涤 10 次,然后转移到 BTV 指示性 Vero 细胞的插入物中孵育 6 小时,以检测任何细胞病变效应(CPE)。然后将它们洗涤并在 B2 Ménézo 中培养 24 小时。未接种 ZP 无和 ZP 完整的胚胎进行了类似的处理,并用作对照。用作接种 BTV 的 ZP 无和 ZP 完整胚胎饲养细胞的 Vero 细胞单层出现细胞病变效应(CPE)。通过 RT-qPCR 在暴露的 ZP 无和 ZP 完整胚胎的 10 次洗涤中发现了 BTV。在无细胞培养基中,早期胚胎细胞至少产生了 10(2.5)TCID(50)/ml。通过 RT-qPCR 在 ZP 无和 ZP 完整胚胎中检测到 BTV RNA。所有这些结果都清楚地表明,山羊早期胚胎细胞易受 BTV 感染,并且该病毒感染具有生产力。洗涤程序未能去除 BTV,这表明 BTV 可以与透明带结合。