【摘要】：豬輪狀病毒(Porcine rotavirus,PRV)屬呼腸病毒科,輪狀病毒屬。豬輪狀病毒是引起仔豬病毒性腹瀉常見的病原之一,主要臨床癥狀表現(xiàn)為黃色水樣腹瀉并伴隨著嘔吐。因該病流行范圍廣,發(fā)病率高,常與其他疾病混合感染而使病情加重,導(dǎo)致高死亡率,給畜牧業(yè)造成嚴(yán)重經(jīng)濟(jì)損失。輪狀病毒血清群和血清型眾多,且不同毒株的生物學(xué)特性不同,故對(duì)輪狀病毒的分離鑒定及相關(guān)特性的研究,無(wú)論是對(duì)該病毒的基礎(chǔ)性研究亦或是對(duì)該病的應(yīng)用性研究均具有十分重要的意義。本研究對(duì)2012年從河北某發(fā)病豬場(chǎng)采集的仔豬腹瀉病料進(jìn)行了輪狀病毒的分離鑒定。首先對(duì)疑似病料,進(jìn)行了PRV VP6部分基因的PCR檢測(cè),對(duì)擴(kuò)增產(chǎn)物測(cè)序證明,獲得的275 bp的PRV VP6部分基因序列,同時(shí)對(duì)TGEV和PEDV進(jìn)行了RT-PCR擴(kuò)增,未見針對(duì)這兩種病毒的特異條帶。結(jié)果表明該病料中含有輪狀病毒。將PRV PCR檢測(cè)陽(yáng)性糞便10倍稀釋,經(jīng)病毒釋放和無(wú)菌處理后,分別接種于豬小腸上皮細(xì)胞IEC-2、恒河猴胎腎細(xì)胞MA-104和Marc-145,經(jīng)過(guò)一定代次的盲傳,在以上三種細(xì)胞基質(zhì)中均可觀察到細(xì)胞膜融合,細(xì)胞核固縮、空泡化,細(xì)胞脫落等細(xì)胞病變(CPE),且隨著傳代次數(shù)的增加,CPE也越發(fā)明顯;對(duì)該輪狀病毒地方流行株的細(xì)胞培養(yǎng)物進(jìn)行電鏡觀察,可觀察到典型的車輪狀病毒結(jié)構(gòu),病毒呈雙層衣殼,直徑大小約70~80 nm;免疫電鏡檢測(cè),可見有大量的典型的輪狀病毒粒子聚集在一起;病毒感染MA-104細(xì)胞后不同時(shí)間超薄切片電鏡觀察顯示,病毒接種30h在胞質(zhì)中可見典型的病毒粒子,60h時(shí)病毒明顯增多,90h后細(xì)胞失去固有形態(tài);間接免疫熒光檢測(cè)表明,接種該輪狀病毒分離株的IEC-2細(xì)胞、MA-104細(xì)胞和Marc-145細(xì)胞中均有特異性免疫熒光出現(xiàn);病毒中和實(shí)驗(yàn)表明,分離病毒所致病變,可被特異性豬輪狀病毒抗血清所抑制。為進(jìn)一步純化該病毒,將病毒感染的細(xì)胞培養(yǎng)物與氯仿連續(xù)作用5代后在MA-104細(xì)胞上擴(kuò)大培養(yǎng),利用雙層瓊脂法進(jìn)行蝕斑試驗(yàn),通過(guò)多次挑取蝕斑在細(xì)胞上培養(yǎng),達(dá)到了病毒的純化。本實(shí)驗(yàn)對(duì)所分離的毒株進(jìn)行了部分理化特性的測(cè)定。測(cè)定結(jié)果表明,該輪狀病毒地方流行分離株對(duì)脂溶性試劑表現(xiàn)出極度不敏感,pH3.0處理病毒1h后其感染能力略微下降,經(jīng)50℃水浴處理30min后,感染能力明顯下降。將分離毒細(xì)胞培養(yǎng)物灌服新生仔豬后,30h開始輕微腹瀉,70h后出現(xiàn)嘔吐和水樣腹瀉,感染后96h死亡。剖檢腸壁變薄,松弛胃內(nèi)充滿凝乳塊,對(duì)所收集的糞便樣品進(jìn)行PCR檢測(cè)和免疫熒光檢測(cè),均為陽(yáng)性,表明所分離的病毒對(duì)豬有一定致病性。該豬輪狀病毒的分離純化,為該病病原學(xué)、流行病學(xué)、及其免疫防治提供了重要的物質(zhì)基礎(chǔ)。
[Abstract]:Pig rotavirus (Porcine rotavirus,PRV) belongs to reenteroviridae and rotavirus. Pig rotavirus is one of the common pathogens causing viral diarrhea in piglets. The main clinical symptoms are yellow watery diarrhea accompanied by vomiting. Because of its wide epidemic range and high incidence, it is often mixed with other diseases to aggravate the disease, resulting in high mortality and serious economic losses to animal husbandry. There are many serotypes and serotypes of rotavirus, and the biological characteristics of different strains are different. Therefore, the isolation, identification and related characteristics of rotavirus are of great significance both for the basic research of rotavirus and for the application of the disease. In this study, rotavirus was isolated and identified from piglets with diarrhea collected from a pig farm in Hebei in 2012. Firstly, the PRV VP6 partial genes of suspected disease materials were detected by PCR, and the PRV VP6 partial gene sequences of 275 bp were confirmed by sequencing the amplified products. At the same time, TGEV and PEDV were amplified by RT-PCR, and no specific bands for these two viruses were found. The results showed that rotavirus was found in the diseased material. The positive feces detected by PRV PCR were diluted 10 times and inoculated into pig intestinal epithelial cells IEC-2, rhesus monkey fetal kidney cells MA-104 and Marc-145, after 10 times dilution and aseptic treatment, respectively. cell membrane fusion, nuclear pyknosis, vacuolation, cell shedding and other cytopathic (CPE), were observed in the above three cell matrices, and CPE became more and more obvious with the increase of passage times. The cell culture of the rotavirus local epidemic strain was observed by electron microscope. The typical rotavirus structure was observed. The virus was double capsid, the diameter was about 70 脳 80 nm; immunoelectron microscope, and a large number of typical rotavirus particles gathered together. Electron microscopic observation of ultra-thin sections of MA-104 cells at different time after virus infection showed that typical virus particles could be seen in cytoplasm at 30 h, the virus increased significantly at 60 h, and the cells lost their inherent morphology after 90 h. Indirect immunofluorescence assay showed that specific immunofluorescence appeared in IEC-2 cells, MA-104 cells and Marc-145 cells inoculated with the rotavirus isolate. Virus neutralization test showed that the disease caused by isolation of virus could be inhibited by specific swine rotavirus antiserum. In order to further purify the virus, the cell culture infected by the virus was cultured on MA-104 cells after 5 generations of continuous action with chloroform. The plaque test was carried out by double-layer Agar method. The plaque was cultured on the cell many times, and the purification of the virus was achieved. In this experiment, some physical and chemical properties of the isolated strains were determined. The results showed that the rotavirus isolates were extremely insensitive to fat-soluble reagents. The infection ability of the rotavirus was slightly decreased after 1 hour of pH3.0 treatment, and significantly decreased after 50 鈩，

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