【摘要】：豬傳染性胃腸炎病毒(Transmissible gastroenteritis virus of swine,TGEV)屬冠狀病毒科,冠狀病毒屬,是豬病毒性腹瀉的重要病原體之一。該病毒引起的豬傳染性胃腸炎(Transmissible gastroenteritis,TGE)以嚴(yán)重腹瀉、嘔吐和脫水為主要臨床特征。TGE發(fā)病急,流行范圍廣,且經(jīng)常與豬輪狀病毒(Porcine rotavirus,PRV)和豬流行性腹瀉病毒(Porcine epidemic diarrhea virus,PEDV)等其他病毒或細(xì)菌混合感染,從而加重了其發(fā)病和死亡,給畜牧業(yè)造成嚴(yán)重的損失。對TGEV流行毒株的分離與鑒定,無論是對該病的基礎(chǔ)性研究或病毒相關(guān)生物產(chǎn)品研制及疾病的科學(xué)防控都有重要意義。本研究以TGEV糞便病料為材料,比較了TGEV在豬原代睪丸細(xì)胞,傳代細(xì)胞系ST細(xì)胞以及PK15細(xì)胞上的增殖情況。病毒在三種細(xì)胞上連續(xù)傳代,傳至25代左右,提取核酸檢測,通過RT-PCR測定病毒在三種細(xì)胞的增殖情況,測定結(jié)果顯示,病毒在這三種細(xì)胞上均可擴(kuò)增出病毒特異條帶。通過對不同代次細(xì)胞毒的TCID50測定比較,在ST傳代細(xì)胞病毒滴度與其他兩種細(xì)胞滴度相當(dāng),且在ST細(xì)胞出現(xiàn)病變最早,故本實驗選擇ST細(xì)胞來進(jìn)行TGEV病毒的連續(xù)培養(yǎng)。當(dāng)病毒適應(yīng)ST細(xì)胞系后,用TGEV,PED,PRV特異性引物對病毒進(jìn)行檢測。經(jīng)過核酸檢測證明除TGEV外,細(xì)胞培養(yǎng)物中還可檢測到PEDV和PRV,表明在進(jìn)行TGEV傳代過程中,存在有三種病毒的細(xì)胞混合感染。為使TGEV得到純化,本研究采用蝕斑純化技術(shù)對病毒進(jìn)行了純化。通過對TGEV蝕斑形成試驗的摸索,確定了蝕斑形成條件為:瓊脂糖濃度為1.6%,培養(yǎng)液-瓊脂糖混合培養(yǎng)液的最適溫度約30℃,瓊脂糖厚度3mm,中性紅濃度為0.02%。通過核酸檢測為TGEV陽性的蝕斑毒繼續(xù)進(jìn)行蝕斑試驗,直致蝕斑大小形態(tài)一致,且核酸檢測TGEV陽性。對純化后的病毒在ST細(xì)胞上連續(xù)傳代,利用核酸檢測,證明病毒可在在細(xì)胞上的穩(wěn)定增殖。間接免疫熒光檢測接種TGEV病毒,可產(chǎn)生特異性熒光,電鏡觀察觀察表明。病毒直徑約150 nm左右,病毒粒子具有典型的冠狀結(jié)構(gòu);病毒感染ST細(xì)胞后進(jìn)行超薄切片電鏡觀察,可看到病毒彌散的存在于細(xì)胞質(zhì)中,直徑在60-100 nm。本實驗對純化后病毒的部分理化特性進(jìn)行了測定,結(jié)果顯示,病毒對熱敏感,60℃水浴30min即可完全失活;病毒不耐酸堿,在PH為3和PH為9的條件下病毒滴度明顯降低。該病毒對有機(jī)試劑如乙醚氯仿等較敏感。以純化后的細(xì)胞培養(yǎng)毒30 ml口服感染新生仔豬,仔豬在感染后30 h出現(xiàn)嘔吐,腹瀉,消瘦和脫水,表現(xiàn)為行動遲緩,腿部僵直等癥狀,仔豬在感染后8 d死亡,仔豬感染后不同時間收集糞便樣品,進(jìn)行RT-PCR檢測,感染仔豬72 h開始排毒。病死豬剖檢發(fā)現(xiàn)腸管擴(kuò)張,充滿液體,小腸絨毛變短,脫落。豬傳染性胃腸炎病毒的成功分離純化,為該病的流行病學(xué)、實驗室診斷及和病毒的生物學(xué)特征以及疫苗的制備等奠定了物質(zhì)基礎(chǔ)。
[Abstract]:Porcine infectious gastroenteritis virus (Transmissible gastroenteritis virus of swine,TGEV) belongs to coronavirus family and coronavirus genus, which is one of the important pathogens of porcine viral diarrhea. The infectious gastroenteritis (Transmissible gastroenteritis,TGE) caused by the virus is characterized by severe diarrhea, vomiting and dehydration. TGE is acute, widespread and often associated with porcine rotavirus (Porcine rotavirus,. PRV) and other viruses or bacteria, such as porcine epidemic diarrhea virus (Porcine epidemic diarrhea virus,PEDV), caused serious damage to animal husbandry. It is of great significance to isolate and identify the epidemic strains of TGEV, whether it is the basic research of the disease, the development of virus-related biological products, and the scientific prevention and control of the disease. In this study, TGEV fecal material was used to compare the proliferation of TGEV on primary testicular cells, ST cells and PK15 cells. The virus was subcultured on three kinds of cells and passed to about 25 generations. The nucleic acid was extracted to detect the nucleic acid, and the proliferation of virus in the three kinds of cells was determined by RT-PCR. The results showed that the virus could amplify the virus specific bands on the three kinds of cells. The titer of the virus in the ST passaged cells was similar to that of the other two cell lines, and the lesion was the earliest in the ST cells, so we chose the ST cells for the continuous culture of the TGEV virus. When the virus adapted to ST cell line, TGEV,PED,PRV specific primers were used to detect the virus. In addition to TGEV, PEDV and PRV, could also be detected in cell cultures, which indicated that there were three kinds of virus mixed infection in the course of TGEV passage. In order to purify TGEV, the virus was purified by plaque purification technique. The conditions of plaque formation of TGEV were as follows: agarose concentration 1.6, agarose mixed culture medium 30 鈩，

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