Waldrop Julie G, Stringfellow David A, Galik Patricia K, Riddell Kay P, Riddell M Gatz, Givens M Daniel, Carson Robert L
College of Veterinary Medicine, Auburn University, 100 McAdory Hall, Auburn, AL 36849-5519, USA.
Theriogenology. 2004 Aug;62(3-4):387-97. doi: 10.1016/j.theriogenology.2003.07.028.
Early research indicated that bovine viral diarrhea virus (BVDV) would not adhere to zona pellucida-intact (ZP-I), in vivo-derived bovine embryos. However, in a recent study, viral association of BVDV and in vivo-derived embryos was demonstrated. These findings raised questions regarding the infectivity of the embryo-associated virus. The objectives of this study were to evaluate the infectivity of BVDV associated with in vivo-derived bovine embryos through utilization of primary cultures of uterine tubal cells (UTC) as an in vitro model of the uterine environment and to determine if washing procedures, including trypsin treatment, were adequate to remove virus from in vivo-derived embryos. One hundred and nine ZP-I morulae and blastocysts (MB) and 77 non-fertile and degenerated (NFD) ova were collected on day 7 from 34, BVDV-negative, superovulated cows. After collection, all MB and NFD ova were washed according to International Embryo Transfer Society (IETS) standards and exposed for 2h to approximately 10(6) cell culture infective doses (50% endpoint) per milliliter of viral strain SD-1. Following exposure, some groups of <10 MB or NFD ova were washed in accordance with IETS standards. In addition, an equivalent number of MB and NFD ova were subjected to IETS standards for trypsin treatment. Subsequently, NFD ova were immediately sonicated and sonicate fluids were assayed for presence of virus, while individual and groups of MB were placed in microdrops containing primary cultures of UTCs and incubated. After 3 days, embryos, media, and UTCs were harvested from each microdrop and assayed for BVDV. Virus was detected in the sonicate fluids of 56 and 43% of the groups of NFD ova that were washed and trypsin-treated, respectively. After 3 days of microdrop culture, virus was not detected in media or sonicate fluids from any individual or groups of MB, regardless of treatment. However, virus was detected in a proportion of UTC that were co-cultured with washed groups of MB (30%), washed individual MB (9%) and trypsin treated individual MB (9%), but no virus was detected in the UTC associated with groups of trypsin-treated embryos. In conclusion, virus associated with developing embryos was infective for permissive cells. Further, the quantity of virus associated with a proportion of individual embryos (both washed and trypsin treated) was sufficient to infect the UTC. In light of these results, an attempt should be made to determine if the quantity of a high-affinity isolate of BVDV associated with an individual embryo would infect recipients via the intrauterine route.
早期研究表明，牛病毒性腹泻病毒（BVDV）不会附着于体内来源的、透明带完整（ZP-I）的牛胚胎。然而，在最近的一项研究中，证实了BVDV与体内来源胚胎的病毒关联。这些发现引发了关于胚胎相关病毒感染性的问题。本研究的目的是通过利用输卵管子宫细胞（UTC）原代培养作为子宫环境的体外模型，评估与体内来源的牛胚胎相关的BVDV的感染性，并确定包括胰蛋白酶处理在内的洗涤程序是否足以从体内来源的胚胎中去除病毒。在第7天从34头BVDV阴性的超排母牛中收集了109个ZP-I桑葚胚和囊胚（MB）以及77个未受精和退化（NFD）的卵子。收集后，所有MB和NFD卵子均按照国际胚胎移植协会（IETS）标准进行洗涤，并暴露于每毫升病毒株SD-1约10⁶个细胞培养感染剂量（50%终点）下2小时。暴露后，部分小于10个MB或NFD卵子的组按照IETS标准进行洗涤。此外，对等量的MB和NFD卵子进行IETS标准的胰蛋白酶处理。随后，立即对NFD卵子进行超声处理，并检测超声处理液中是否存在病毒，而将单个和分组的MB置于含有UTC原代培养物的微滴中并进行培养。3天后，从每个微滴中收获胚胎、培养基和UTC，并检测BVDV。在分别经过洗涤和胰蛋白酶处理的NFD卵子组中，56%和43%的超声处理液中检测到病毒。微滴培养3天后，无论处理方式如何，在任何单个或分组的MB的培养基或超声处理液中均未检测到病毒。然而，在与洗涤后的MB组（30%）、洗涤后的单个MB（9%）和胰蛋白酶处理后的单个MB（9%）共培养的一部分UTC中检测到病毒，但在与胰蛋白酶处理后的胚胎组相关的UTC中未检测到病毒。总之，与发育中的胚胎相关的病毒对允许细胞具有感染性。此外，与一部分单个胚胎（包括洗涤和胰蛋白酶处理的胚胎）相关的病毒量足以感染UTC。鉴于这些结果，应尝试确定与单个胚胎相关的高亲和力BVDV分离株的量是否会通过子宫内途径感染受体。
