猪细小病毒PPV2010株对初产母猪繁殖障碍的影响及其机制研究

Porcine parvovirus infection (PPI) is one of the major diseases in reproduction failure caused by porcine parvovirus (PPV) in swine, characterized by embryonic death, stillbirths, mummified and weak fetuses, infertility and fatal malformation. Studies indicate that PPV infection can result in a high virus titer in the uterus, and virus can invade the placenta and easily cross the placental barrier, causing fatal infection. Fatal infection is the clinical consequences of PPV infection, but how the virus invades the placenta and to break through the placenta barrier followed by fatal infection is unclear. Virus infection can cause proteome changes and to survival in the host with up or down regulation of some specific proteins in host tissues and cells. To explore the effects of the newly emerged porcine parvovirus stain PPV2010 on the early pregnancy of primiparous gilts and its possible mechanism, an animal model induced by PPV2010 strain will be developed to investigate its effects on primiparous sows as well the fetuses. On the basis of this animal model, comparative proteomics technologies isobaric Tags for Relative and Absolute Quantitation（iTRAQ）labeling and Liquid chromatography tandem mass spectrometry （LC-MS/MS）-based proteome profiling will be used to study the proteome changes in the endometrium and placenta, aiming to discover and identify differentially expressed proteins after PPV infection. The differentially expressed proteins will be further validated on the levels of their corresponding RNA and protein to determine the specific proteins. Moreover, to explore the roles of the selected proteins in regulation of cell functions during the process of virus infection, primary porcine umbilical vein endothelial cells (PUVEC) will be isolated and cultured. Cell funtions including proliferation, migration and vasular formation will be assayed using MTS assay, transwell matrigel migration assay and vasculogenic mimicry. The relevant findings will be helpful to understand the pathogenic mechanism of PPV in sow reproductive disorders, and also provide new ideas to study the livestock reproductive failure caused by viral infectious diseases.

猪细小病毒病是由猪细小病毒（PPV）引起的猪主要繁殖障碍性疾病之一，以初产母猪发生胚胎死亡、不孕、产死胎、畸形胎、木乃伊胎及弱仔等为特征。PPV感染怀孕母猪后在子宫内含毒量很高，病毒通过侵袭胎盘并突破胎盘屏障使胎儿感染而导致临床后果，目前对于其作用机制不是很清楚。病毒感染后引起组织细胞内关键功能蛋白质发生变化可能是构成胎盘屏障遭受破坏的重要原因。PPV2010是最近分离鉴定的PPV毒株，具有致病性PPV和疫苗株的特性，为探讨其对初产妊娠母猪的影响及其可能作用机制，本项目在建立动物模型的基础上，运用比较蛋白质组学技术iTRAQ、LC-MS/MS等分析PPV在感染子宫内膜、突破胎盘屏障等过程中差异蛋白质表达规律；并通过离体研究探讨相关差异表达蛋白在病毒感染过程中对细胞功能的调节。相关研究结果将为研究PPV引起母猪繁殖障碍的致病机制奠定基础，也为病毒性疾病引起的家畜繁殖障碍研究提供新的思路。

猪细小病毒病是由猪细小病毒（PPV）引起的猪主要繁殖障碍性疾病之一，对于其致病机制的研究可为该病的防治奠定基础。在早期妊娠过程中，胎盘的正常发育是维持正常妊娠的关键，PPV感染怀孕母猪后在子宫内含毒量很高，病毒通过侵袭胎盘并突破胎盘屏障使胎儿感染而导致临床后果，这与病毒感染后组织细胞内关键功能蛋白发生变化密切相关。本研究通过体内动物实验将PPV2010病毒株感染初产怀孕母猪，发现在感染一周后病毒对母猪的妊娠影响并不明显，而在感染两周后，引起死胎增多，并在子宫内膜和胎盘组织检测到较高含量的病毒核酸，但运用iTRAQ蛋白组学技术检测并未在相关组织中获得可验证的差异表达蛋白，这可能与样品数量及取材部位相关。体外研究发现，用病毒感染细胞后能获得大量的差异表达蛋白，其中130个为感染后下调，88个为感染后上调，对部分差异蛋白从基因和蛋白水平进行验证，其变化趋势与所检测的基本一致。进一步以原代猪脐静脉血管内皮细胞（PUVEC）为模型，研究了差异蛋白HSP90-α 在PPV感染过程中的作用，结果显示：下调HSP90-α可在一定程度上抑制PPV病毒的繁殖，同时对PPV所引起的血管内皮细胞的增殖与迁移能力下降也能在一定程度上得到抑制。以上结果提示，在PPV感染过程中，一些蛋白如HSP90-α可通过影响血管内皮细胞功能而影响胎盘发育导致流产症状，该研究成果将为进一步揭示PPV的发病机制提供科学依据。