本文關(guān)鍵詞：兩種豬腹瀉相關(guān)病毒病原學(xué)特征研究　出處：《浙江大學(xué)》2017年博士論文　論文類型：學(xué)位論文

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【摘要】：仔豬腹瀉持續(xù)在中國、韓國以及美國等地爆發(fā),發(fā)病仔豬呈現(xiàn)腹瀉、嘔吐等癥狀,嚴(yán)重的能導(dǎo)致仔豬迅速脫水而引起死亡,死亡率較高,給養(yǎng)豬產(chǎn)業(yè)帶來了巨大經(jīng)濟(jì)損失。目前,引起仔豬腹瀉的病原還沒有完全弄清,各國學(xué)者普遍認(rèn)為豬流行性腹瀉病毒(Porcine epidemic diarrhea virus,PEDV),豬傳染性胃腸炎病毒(Transmissible Gastroenteritis Viru,TGEV),輪狀病毒(Rotavirus,RV)以及其它腸道病毒性病原等為豬腹瀉的主導(dǎo)。但是一些免疫過的豬場又會重新暴發(fā)腹瀉,該現(xiàn)象引起各國學(xué)者反思。近年來一些新發(fā)腹瀉病毒如豬丁型冠狀病毒(Porcine deltacoronavirus,PDCoV),哺乳動物呼腸孤病毒(Mammalian orthoreovirus,MRV)等先后被報道,引起了人們的關(guān)注。本研究主要對兩株新分離豬腹瀉相關(guān)病毒的生物學(xué)特征開展了一系列研究。2013年,作者從腹瀉仔豬糞便中分離到一株能在Vero細(xì)胞上增殖,并引起細(xì)胞腫脹、變圓等病變的未知病毒。通過RT-PCR檢測,PEDV、TGEV、PDCoV以及RV均呈陰性,最終利用高通量測序技術(shù)確定該病毒為3型呼腸孤病毒,命名為MRV-ZJ2013。病毒大小約70-100 nm、無囊膜,感染細(xì)胞后整齊排列在細(xì)胞質(zhì)中。MRV-ZJ2013毒株感染Vero細(xì)胞(MOI=0.01)后呈“S”型增長,在感染后第36h病毒量達(dá)到最高值(108.33TCID50/mL),隨后進(jìn)入平臺期。MRV-ZJ2013對溫度和紫外線比較敏感,在100℃處理1h或254nm UV照射30min后病毒完全失活。致病性試驗顯示,MRV-ZJ2013毒株感染組出生仔豬后出現(xiàn)輕微腹瀉、軟便、食欲不振等現(xiàn)象,有部分試驗豬(3/6)在感染后的第1或2d有排毒現(xiàn)象。感染后,仔豬體內(nèi)抗MRV3 IgG水平顯著升高,直到感染后第14d,體內(nèi)仍能檢測到抗體。宰殺癥狀明顯的仔豬,解剖發(fā)現(xiàn)其腸系膜輕微充血。組織病理切片顯示MRV3感染組有小腸絨毛表層脫落,內(nèi)層出現(xiàn)空洞等現(xiàn)象。免疫組化染色顯示腸絨毛上存在病原。上述結(jié)果表明,MRV-ZJ2013毒株毒力較弱,盡管能夠產(chǎn)生免疫應(yīng)答,但不能引起明顯癥狀。分子克隆MRV-ZJ2013基因組10個片段并上傳至GenBank中(登錄號為KY419120至KY419129)。MRV-ZJ2013毒株L1,M2,S1節(jié)段與3型代表株T3D相似度最高,其它的7個片段與1型代表毒株(T1L)相似度最高。氨基酸序列對比結(jié)果相似,λ1,μ1,μNS和σ1蛋白(分別由L1,M2,M3和S1編碼)氨基酸序列與T3D毒株序列相似度最高,其它蛋白氨基酸序列與T1L毒株相似度最高。與所有已發(fā)表的豬MRV基因組序列比較分析發(fā)現(xiàn),MRV-ZJ2013毒株與美國毒株相似度最低;MRV-ZJ2013毒株S1基因進(jìn)化樹結(jié)果顯示其屬于血清型3型,與亞洲分離的豬呼腸孤病毒親緣關(guān)系最近;MRV-ZJ2013毒株基因組中有5個片段(M1,S3,M3,S2和S4)與中國蝙蝠MRV親緣關(guān)系最近,L3,M2和S1與豬MRV親緣關(guān)系最近,而L1和L2基因則與水貂MRV親緣關(guān)系最近。將10個階段串聯(lián)之后利用RDP4重組分析,結(jié)果顯示MRV-ZJ2013毒株S2,S3,L2,L3,M2,S1等片段存在重排,重排親本可能來源于蝙蝠MRV、豬MRV、水貂MRV和未知來源MRV。因此,推測MRV-ZJ2013毒株可能是由蝙蝠MRV,豬MRV以及水貂MRV基因組重排而來。針對L1基因設(shè)計特異性引物建立豬呼腸孤病毒巢式RT-PCR檢測方法,對從浙江、江西、山東、河南、黑龍江等省58個豬場共采集224份腹瀉仔豬糞便樣品進(jìn)行檢測。結(jié)果顯示55個豬場(55/58,94.8%)共檢出147個陽性樣品(147/224,65.6%),其中第一輪從16個豬場檢出陽性樣品28個(28/224,12.5%)。建立基于衣殼蛋白δ1蛋白為包被抗原的間接ELISA(Enzyme linked immunosorbent assay,ELISA)檢測方法,并檢測從江蘇、湖南、江西、河南、浙江、黑龍江和山東等省收集的1037份不同年齡段豬血清樣品,MRV3抗體檢出率為77%(OD450nm平均值為0.69±0.32)。母豬和1周齡以內(nèi)仔豬血清樣品陽性率和抗體平均水平都最高,后備母豬抗體水平最低。東部各省中,黑龍江省樣品陽性率以及抗體平均水平均低于其它省份,而其它省份之間差異不顯著。綜合上述數(shù)據(jù),本研究認(rèn)為MRV廣泛存在于中國東部豬場。豬丁型冠狀病毒(Porcine deltacoronavirus)屬冠狀病毒科(Coronaaviridae),冠狀病毒亞科(Coronaviriaae),是近年來新發(fā)現(xiàn)的一類冠狀病毒,能感染各年齡段的豬群,引起腹瀉、嘔吐和脫水癥狀。本研究利用體外模型對PDCoV在細(xì)胞內(nèi)復(fù)制過程進(jìn)行研究。PDCoV感染5h后,細(xì)胞外開始檢測到病毒,說明此時子一代病毒完成組裝并釋放到胞外。透射電鏡觀察PDCoV在細(xì)胞內(nèi)復(fù)制過程發(fā)現(xiàn),PDCoV通過膜融合進(jìn)入細(xì)胞,隨后在細(xì)胞質(zhì)中復(fù)制和翻譯。病毒在內(nèi)質(zhì)網(wǎng)和高爾基體中間體(ERGIC)完成病毒組裝,隨后被轉(zhuǎn)運到高爾基體中完成“成熟”過程,最終被囊泡運輸排出細(xì)胞。PDCoV感染LLC-PK1和IPEC-J2細(xì)胞引起細(xì)胞內(nèi)膜重排(Membrane rearrangement),被感染細(xì)胞中能觀察到雙層膜結(jié)構(gòu)(DMVs)、腫脹的內(nèi)質(zhì)網(wǎng)(swollenER)等。與此同時,還觀察到自噬泡樣結(jié)構(gòu),免疫印跡(Western-blot)方法也證實PDCoV侵染LLC-PK1細(xì)胞時誘導(dǎo)細(xì)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.

【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：S852.651

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