兔出血症病毒2型浙江株鉴定及致病性研究  

Identification and Pathogenicity Study of a Strain of Zhejiang Rabbit hemorrhagic disease virus type 2

作　　者：郝方姣 季权安[2] 黄叶娥 崔雪梅 胡子喆 宋厚辉 鲍国连 刘燕[1,2] HAO Fang-Jiao;JI Quan-An;HUANG Ye-E;CUI Xue-Mei;HU Zi-Zhe;SONG Hou-Hui;BAO Guo-Lian;LIU Yan(College of Animal Science and Technology•College of Veterinary Medicine,Zhejiang A&F University,Hangzhou 311300,China;Institute of Animal Husbandry and Veterinary Science,Zhejiang Academy of Agricultural Sciences,Hangzhou 310021,China)

机构地区：[1]浙江农林大学动物科技学院•动物医学院,杭州311300 [2]浙江省农业科学院畜牧兽医研究所,杭州310021

出　　处：《农业生物技术学报》2024年第11期2649-2661,共13页Journal of Agricultural Biotechnology

基　　金：浙江省自然科学基金(LTGD24C180001);浙江省重点研发项目(2021C02068-7);国家兔产业技术体系项目(CARS-43-C-2)。

摘　　要：兔出血症病毒2型(Rabbit hemorrhagic disease virus type 2,RHDV2)严重威胁家兔(Oryctolagus cuniculus)养殖生产安全。本研究旨在鉴定浙江省某肉兔养殖场疑似RHDV2感染疫情的病原,明确分离株的致病特性及其对兔肠道菌群结构的影响。首先采集病死兔肝脏样品,采用RT-PCR技术扩增RHDV2的衣壳蛋白基因VP60(capsid protein),通过测序及系统进化树分析进行病原鉴定;进一步通过动物回归试验明确分离毒株的感染和致病特性,采用16S r DNA测序技术分析空白对照组和RHDV2攻毒组的组间盲肠菌群结构变化。结果显示,测序及同源性分析证实RT-PCR扩增获得的目的片段为RHDV2的VP60部分序列(GenBank No.PQ215520),与国内的RHDV2毒株(GenBank No.OQ570964.1)同源,一致性为98.66%。动物回归试验显示,50日龄幼兔在感染48 h内全部死亡,呈现口鼻出血等典型临床症状;组织切片染色可见肝脏组织结构紊乱,肝实质内有明显的肝细胞片状坏死,肺泡壁有较多炎性细胞侵润,出现大范围肺水肿;提取感染兔肝脏RNA,经RT-PCR扩增及测序,发现目的片段与发病兔场的分离毒株一致。基于16S rDNA的高通量测序结果表明,RHDV2感染组家兔的肠道菌群结构和相对丰度与健康对照组比较存在显著差异,在属水平上筛选出13个差异显著的物种,表明RHDV2感染改变了兔肠道原有的微生态。本研究首次对浙江省RHDV2分离株进行了致病性研究,为RHDV2防控和诊治提供参考依据。Rabbit hemorrhagic disease virus type 2(RHDV2)seriously threatens the safety of Oryctolagus cuniculus breeding and production.This study aimed to identify the pathogen of suspected RHDV2 infection in a meat rabbit farm in Zhejiang province,and clarify the pathogenic characteristics of isolated strain and its influence on the structure of rabbit intestinal flora.The liver samples of dead rabbits were collected firstly,the capsid protein VP60 gene of RHDV2 was amplified by RT-PCR,and pathogens were identified by sequencing and phylogenetic tree analysis.Animal regression test was used to clarify the infection and pathogenic characteristics of isolated strain,and 16S rDNA sequencing technology was used to analyze the structural changes of cecal microbiota between the blank control group and RHDV2 group.The results of the sequencing and homology analysis showed that the target fragment amplified by RT-PCR was RHDV2 VP60 partial sequence(GenBank No.PQ215520),which was homologous to China RHDV2 strain(GenBank No.OQ570964.1)with a consistency of 98.66%.The results of animal regression test showed that all 50-day-old rabbits died within 48 h of infection,and presented typical clinical symptoms such as mouth and nose bleeding.Staining of tissue sections showed disorders of liver tissue structure,and there were obvious flake necrosis hepatocytes in the liver parenchyma,more inflammatory cell infiltration in the alveolar wall,and large-scale pulmonary edema.The liver RNA of infected rabbits was extracted for PCR amplification and sequencing,and the results showed that it was consistent with the isolated strain from the diseased rabbit farm.The results of 16S rDNA high-throughput sequencing showed significant differences in the structure and relative abundance of intestinal flora between the RHDV2 group and healthy control group,and 13 species with significant differences were screened at the genus level,indicating that RHDV2 infection altered the original microbiome of the rabbit's intestine.This study explored the pathogen

关 键 词：兔出血症病毒2型(RHDV2) RT-PCR 病理学观察 肠道菌群 

分 类 号：S858.291[农业科学—临床兽医学]