In vitro interaction of bovine herpesvirus 1 with uterine tube epithelial cells and oocytes

The aims of this study were to assess in vitro if bovine oocytes and oviductal epithelial cells from slaughterhouses for in vitro fertilization use may be infected with bovine herpesvirus 1; to analyze whether the treatment with trypsin according to the International Embryo Transfer Society guideline is efficient to inactivate the bovine herpesvirus 1; to morphologically study the virus-oocyte interaction through optical microscopy. In this study, Madin Darby Bovine Kidney (MDBK) cells that were co-cultured with oocytes matured in vitro and exposed to bovine herpesvirus 1 showed a cytopathic effect. The nested polymerase chain reaction for the supernatant was positive for the bovine herpesvirus 1, thus suggesting that the cytopathic effect observed in the MDBK monolayer was seen due to virus replication and not because of any culture toxicity. It was also observed cytopathic effect and positive nested polymerase chain reaction in MDBK cells co-cultured with in vitro maturated oocytes free of virus, but that were co-cultured in uterine epithelial cells pre-infected with bovine herpesvirus 1 and washed or not with trypsin, demonstrating an oocyte contamination by the virus. When trypsin-washing efficacy was evaluated, we could observe that the trypsin treatment was not able to eliminate the bovine herpesvirus 1 of the oocytes, and it was not observed any morphological difference in the infected oocytes.

本研究旨在达成三项目标：其一，体外评估屠宰场采集的、用于体外受精的牛卵母细胞与输卵管上皮细胞是否可感染牛疱疹病毒1型（bovine herpesvirus 1）；其二，分析按照国际胚胎移植学会（International Embryo Transfer Society）指南实施的胰蛋白酶处理能否有效灭活牛疱疹病毒1型；其三，借助光学显微镜从形态学层面探究病毒与卵母细胞的相互作用。本研究中，与体外成熟卵母细胞共培养且暴露于牛疱疹病毒1型的Madin-Darby牛肾细胞（Madin Darby Bovine Kidney, MDBK）出现了细胞病变效应。对上清液开展的巢式聚合酶链反应（nested polymerase chain reaction）检测结果呈牛疱疹病毒1型阳性，由此提示，MDBK细胞单层中观察到的细胞病变效应系病毒复制所导致，而非培养体系的毒性作用所致。此外，在与无病毒的体外成熟卵母细胞共培养的MDBK细胞中同样观察到细胞病变效应与巢式聚合酶链反应阳性结果；这些卵母细胞与预先感染牛疱疹病毒1型的子宫上皮细胞共培养，且经或未经胰蛋白酶洗涤，这证实卵母细胞可被该病毒污染。在评估胰蛋白酶洗涤的有效性时，我们发现胰蛋白酶处理无法清除卵母细胞携带的牛疱疹病毒1型，且感染该病毒的卵母细胞未显现任何形态学差异。