两种猪腹泻相关病毒病原学特征研究

发布时间：2018-01-13 08:03

本文关键词：两种猪腹泻相关病毒病原学特征研究　出处：《浙江大学》2017年博士论文　论文类型：学位论文

【摘要】：仔猪腹泻持续在中国、韩国以及美国等地爆发,发病仔猪呈现腹泻、呕吐等症状,严重的能导致仔猪迅速脱水而引起死亡,死亡率较高,给养猪产业带来了巨大经济损失。目前,引起仔猪腹泻的病原还没有完全弄清,各国学者普遍认为猪流行性腹泻病毒(Porcine epidemic diarrhea virus,PEDV),猪传染性胃肠炎病毒(Transmissible Gastroenteritis Viru,TGEV),轮状病毒(Rotavirus,RV)以及其它肠道病毒性病原等为猪腹泻的主导。但是一些免疫过的猪场又会重新暴发腹泻,该现象引起各国学者反思。近年来一些新发腹泻病毒如猪丁型冠状病毒(Porcine deltacoronavirus,PDCoV),哺乳动物呼肠孤病毒(Mammalian orthoreovirus,MRV)等先后被报道,引起了人们的关注。本研究主要对两株新分离猪腹泻相关病毒的生物学特征开展了一系列研究。2013年,作者从腹泻仔猪粪便中分离到一株能在Vero细胞上增殖,并引起细胞肿胀、变圆等病变的未知病毒。通过RT-PCR检测,PEDV、TGEV、PDCoV以及RV均呈阴性,最终利用高通量测序技术确定该病毒为3型呼肠孤病毒,命名为MRV-ZJ2013。病毒大小约70-100 nm、无囊膜,感染细胞后整齐排列在细胞质中。MRV-ZJ2013毒株感染Vero细胞(MOI=0.01)后呈“S”型增长,在感染后第36h病毒量达到最高值(108.33TCID50/mL),随后进入平台期。MRV-ZJ2013对温度和紫外线比较敏感,在100℃处理1h或254nm UV照射30min后病毒完全失活。致病性试验显示,MRV-ZJ2013毒株感染组出生仔猪后出现轻微腹泻、软便、食欲不振等现象,有部分试验猪(3/6)在感染后的第1或2d有排毒现象。感染后,仔猪体内抗MRV3 IgG水平显著升高,直到感染后第14d,体内仍能检测到抗体。宰杀症状明显的仔猪,解剖发现其肠系膜轻微充血。组织病理切片显示MRV3感染组有小肠绒毛表层脱落,内层出现空洞等现象。免疫组化染色显示肠绒毛上存在病原。上述结果表明,MRV-ZJ2013毒株毒力较弱,尽管能够产生免疫应答,但不能引起明显症状。分子克隆MRV-ZJ2013基因组10个片段并上传至GenBank中(登录号为KY419120至KY419129)。MRV-ZJ2013毒株L1,M2,S1节段与3型代表株T3D相似度最高,其它的7个片段与1型代表毒株(T1L)相似度最高。氨基酸序列对比结果相似,λ1,μ1,μNS和σ1蛋白(分别由L1,M2,M3和S1编码)氨基酸序列与T3D毒株序列相似度最高,其它蛋白氨基酸序列与T1L毒株相似度最高。与所有已发表的猪MRV基因组序列比较分析发现,MRV-ZJ2013毒株与美国毒株相似度最低;MRV-ZJ2013毒株S1基因进化树结果显示其属于血清型3型,与亚洲分离的猪呼肠孤病毒亲缘关系最近;MRV-ZJ2013毒株基因组中有5个片段(M1,S3,M3,S2和S4)与中国蝙蝠MRV亲缘关系最近,L3,M2和S1与猪MRV亲缘关系最近,而L1和L2基因则与水貂MRV亲缘关系最近。将10个阶段串联之后利用RDP4重组分析,结果显示MRV-ZJ2013毒株S2,S3,L2,L3,M2,S1等片段存在重排,重排亲本可能来源于蝙蝠MRV、猪MRV、水貂MRV和未知来源MRV。因此,推测MRV-ZJ2013毒株可能是由蝙蝠MRV,猪MRV以及水貂MRV基因组重排而来。针对L1基因设计特异性引物建立猪呼肠孤病毒巢式RT-PCR检测方法,对从浙江、江西、山东、河南、黑龙江等省58个猪场共采集224份腹泻仔猪粪便样品进行检测。结果显示55个猪场(55/58,94.8%)共检出147个阳性样品(147/224,65.6%),其中第一轮从16个猪场检出阳性样品28个(28/224,12.5%)。建立基于衣壳蛋白δ1蛋白为包被抗原的间接ELISA(Enzyme linked immunosorbent assay,ELISA)检测方法,并检测从江苏、湖南、江西、河南、浙江、黑龙江和山东等省收集的1037份不同年龄段猪血清样品,MRV3抗体检出率为77%(OD450nm平均值为0.69±0.32)。母猪和1周龄以内仔猪血清样品阳性率和抗体平均水平都最高,后备母猪抗体水平最低。东部各省中,黑龙江省样品阳性率以及抗体平均水平均低于其它省份,而其它省份之间差异不显著。综合上述数据,本研究认为MRV广泛存在于中国东部猪场。猪丁型冠状病毒(Porcine deltacoronavirus)属冠状病毒科(Coronaaviridae),冠状病毒亚科(Coronaviriaae),是近年来新发现的一类冠状病毒,能感染各年龄段的猪群,引起腹泻、呕吐和脱水症状。本研究利用体外模型对PDCoV在细胞内复制过程进行研究。PDCoV感染5h后,细胞外开始检测到病毒,说明此时子一代病毒完成组装并释放到胞外。透射电镜观察PDCoV在细胞内复制过程发现,PDCoV通过膜融合进入细胞,随后在细胞质中复制和翻译。病毒在内质网和高尔基体中间体(ERGIC)完成病毒组装,随后被转运到高尔基体中完成“成熟”过程,最终被囊泡运输排出细胞。PDCoV感染LLC-PK1和IPEC-J2细胞引起细胞内膜重排(Membrane rearrangement),被感染细胞中能观察到双层膜结构(DMVs)、肿胀的内质网(swollenER)等。与此同时,还观察到自噬泡样结构,免疫印迹(Western-blot)方法也证实PDCoV侵染LLC-PK1细胞时诱导细胞自噬。
[Abstract]:Piglet diarrhea continues to Chinese, the outbreak of South Korea and the United States and other places, the incidence of piglet diarrhea, vomiting and other symptoms, serious can lead to rapid dehydration of piglets and cause death, the mortality rate is high, has brought huge economic losses to the pig industry. At present, cause diarrhea pathogen is not fully understood, many scholars generally believe that the swine epidemic diarrhea virus (Porcine epidemic diarrhea virus, PEDV), porcine transmissible gastroenteritis virus (Transmissible Gastroenteritis, Viru, TGEV), rotavirus (Rotavirus, RV) and other toxic pathogenic intestinal disease as the leading pig diarrhea. But some vaccinated pig diarrhea outbreak again, this phenomenon caused by many scholars in recent years reflect. Some new hair such as pig diarrhea virus hepatitis coronavirus (Porcine deltacoronavirus, PDCoV), mammalian reovirus (Mammalian orthoreovirus, MRV) etc. After being reported, attracted the attention of the people. The main biological characteristics of two strains of swine diarrhea virus carried out a series of.2013 years, the author from the faecal isolated strains in Vero cells, and causes cell swelling, unknown virus became round and through disease. PEDV, TGEV, RT-PCR detection, PDCoV and RV were negative, the final use of high-throughput sequencing technology to determine the virus type 3 reovirus named MRV-ZJ2013. virus, the size of about 70-100 nm, no envelope, cells infected with Vero.MRV-ZJ2013 strain infected cells aligned in the cytoplasm (MOI=0.01) after a "S" growth, reached the highest value in the 36h virus infection (108.33TCID50/mL), then into the platform of.MRV-ZJ2013 is more sensitive to temperature and ultraviolet, at 100 DEG 1H or 254nm UV 30min after irradiation the virus completely inactivated pathogenic. Test showed mild diarrhea, MRV-ZJ2013 virus infection group of piglets born after soft stool, loss of appetite, some pigs (3/6) have detoxification phenomenon after infection in first or 2D. After infection, piglet anti MRV3 IgG levels significantly increased until 14d after infection, the body can still be detected by the antibody the slaughter. Symptoms of piglets, found that the anatomy of the mesenteric slight hyperemia. Pathological sections showed that MRV3 infection group off the intestinal villi surface, the inner cavity phenomenon. Immunohistochemical staining revealed the presence of pathogenic intestinal villi. The results showed that the MRV-ZJ2013 strain was weak, despite the immune response, but not obvious symptoms. Molecular cloning of MRV-ZJ2013 genome 10 fragments and uploaded to GenBank (accession No. KY419120 to KY419129).MRV-ZJ2013 strain L1, M2, S1 segment and type 3 strains had the highest similarity to the T3D. 7 fragments of it and type 1 strains (T1L). The highest similarity of amino acid sequence comparison results similar to lambda 1, mu 1, mu 1 (sigma NS and protein respectively by L1, M2, M3 and S1 encoding) amino acid sequence and T3D strains had the highest similarity to other protein sequences, and the amino acid sequence of T1L strain similar is the highest. Compared with all published porcine MRV genome sequence analysis showed that MRV-ZJ2013 strain and the strain of the lowest similarity; MRV-ZJ2013 strain S1 gene phylogenetic tree showed that it belongs to serotype 3, and Asian isolate of porcine reovirus called the closest relationship; there are 5 fragments of MRV-ZJ2013 strain genome (M1, S3 M3, S2, and S4) and Chinese bat MRV had the closest relationship, L3, M2 and S1 MRV recently and pig genetic relationship, while L1 and L2 genes and mink MRV the closest genetic relationship. The 10 stage series after the use of recombinant RDP4 analysis results showed that MRV-ZJ2013 strains S2, S3, L2, L3, M2, S1 and other sections may be derived from the parent rearrangement rearrangement, bat MRV, porcine MRV, MRV and MRV. in mink of unknown origin, suggesting that MRV-ZJ2013 strains may be caused by the bat MRV, porcine MRV and mink MRV and genome rearrangement. According to the L1 gene specific primers were designed to establish the porcine reovirus nested RT-PCR detection method, from Zhejiang, Jiangxi, Shandong, Henan, Heilongjiang Province, 58 pig farms were collected 224 samples of feces samples of diarrhea piglets were detected. The results showed that 55 pig farms (55/58,94.8%) were detected in 147 positive samples (147/224,65.6%), in the first round from 16 pig farms were positive in 28 samples (28/224,12.5%). The establishment of capsid protein delta 1 protein based on indirect ELISA coated antigen (Enzyme linked immunosorbent assay, ELISA) detection method and detection from Jiangsu, Hunan, Jiangxi, Henan, Zhejiang and Shandong provinces, the black dragon collected 1037 different Age of swine serum samples, MRV3 antibody positive rate was 77% (the average value of OD450nm is 0.69 + 0.32). Within 1 weeks of age, the positive rate of sows and piglets serum samples and antibody level was the highest, gilts lowest antibody level. The eastern provinces, Heilongjiang province samples positive rate and antibody were lower than the average level of other provinces however, there are no significant differences between other provinces. Based on the above data, this study suggests that MRV exists widely in eastern China pig. Pigs (Porcine deltacoronavirus) is a type of coronavirus coronaviridae, coronavirus (Coronaaviridae) subfamily (Coronaviriaae), is a kind of new coronavirus infection can be found in recent years, the age of the pigs, causing diarrhea, vomiting and dehydration. The research on the process of PDCoV replication in cells of.PDCoV infection using 5h model in vitro, extracellular began to detect the virus at this time A generation of virus assembled and released into the extracellular. Transmission electron microscopy in cellular PDCoV replication process, PDCoV through membrane fusion into cells, then the replication and translation in the cytoplasm. The virus in the endoplasmic reticulum and Golgi intermediates (ERGIC) complete virus assembly, was subsequently transferred to the Golgi in "mature" the process, eventually vesicular transport from.PDCoV infected cells caused by cell membrane rearrangement and IPEC-J2 cell LLC-PK1 (Membrane rearrangement), the infected cells can double membrane structure was observed (DMVs), swelling of the endoplasmic reticulum (swollenER). At the same time, also observed autophagic vacuole like structure, Western blot (Western-blot) method confirmed PDCoV infection of LLC-PK1 cells induced by autophagy.

【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S852.651

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