【摘要】：鴨病毒性肝炎(Duck viral hepatitis,DVH)是由鴨病毒性肝炎病毒(Duck hepatitis virus,DHV)引起的一種雛鴨的急性、高度致死性的傳染病,臨床上主要表現(xiàn)為食欲減退、神經(jīng)癥狀、突然死亡和肝臟腫大出血。鴨病毒性肝炎病毒I型屬于小RNA病毒科,命名為鴨甲肝病毒(Duck hepatitis A virus,DHAV),主要感染3周齡以內(nèi)的雛鴨,雛鴨發(fā)病后,死亡率急劇上升。病死鴨多呈角弓反張現(xiàn)象,剖檢肝臟特征性癥狀為出血性病變,是嚴(yán)重危害養(yǎng)鴨業(yè)的疫病之一。雖然我國(guó)目前已有DHV疫苗的生產(chǎn)和廣泛使用,但該病仍然在我國(guó)各地流行并造成肉鴨養(yǎng)殖業(yè)的重大損失。因此,尋找安全有效的理想疫苗毒株,研發(fā)高效的鴨病毒性肝炎疫苗,用于鴨肝炎病的預(yù)防或控制,依然是亟待解決的問(wèn)題。因?yàn)轼喖赘尾《镜淖儺悪C(jī)理尚不清楚,而3C蛋白作為功能性蛋白參與裂解功能,是否與毒力變化相關(guān)尚未了解。為了解鴨甲肝病毒1型(DHAV-1)基因的變異規(guī)律,本研究選擇在SPF雞胚上對(duì)病毒進(jìn)行傳代試驗(yàn),通過(guò)對(duì)3C基因的克隆測(cè)序及毒力的測(cè)定,為深入了解DHAV-1的遺傳變異規(guī)律提供依據(jù)。本研究將完成以下兩部分內(nèi)容:第一部分:5株DHAV-1病毒在SPF雞胚上的傳代致弱5株DHAV-1分別為ALY0901(1A),AZC14117(2A),A-病料毒(3A),A2-14(4A),ADNA-launch(5A)。將這5株毒經(jīng)過(guò)處理后作為初代毒,經(jīng)尿囊腔接種9-12日齡SPF雞胚,接種量為0.2 mL/胚。留存24小時(shí)后的死亡雞胚,在4℃冰箱冷卻4-12 h后,無(wú)菌收取雞胚尿囊液并觀察胚胎病變,將所收尿囊液對(duì)應(yīng)接入下一批雞胚,連續(xù)傳代60代。結(jié)果表明:雞胚傳代5代之前,24-96h之間雞胚并無(wú)死亡現(xiàn)象,胚體也無(wú)明顯病變;5代之后,到20代為止,雞胚開始出現(xiàn)死亡現(xiàn)象,并且胚體明顯有大量出血點(diǎn)。死亡時(shí)間集中在72h左右。20-50代72h內(nèi)雞胚無(wú)死亡現(xiàn)象,50-60代重新出現(xiàn)雞胚死亡。第二部分:DHAV-1各毒株各代次雞胚傳代毒3C基因的測(cè)序及序列分析完成對(duì)ALY0901(1A),AZC14117(2A),A-病料毒(3A),A2-14(4A),ADNA-launch(5A)五株毒每隔五代進(jìn)行3C基因的克隆測(cè)序,對(duì)得到的堿基及氨基酸序列進(jìn)行比對(duì),找尋基因變異的規(guī)律,研究發(fā)現(xiàn)堿基出現(xiàn)部分突變,位置基本一致,但并未發(fā)生氨基酸突變,故推測(cè)病毒毒力的變化與3C區(qū)間無(wú)關(guān)。
[Abstract]:Duck viral hepatitis (Duck viral hepatitis,DVH) is an acute and highly fatal infectious disease in ducklings caused by duck viral hepatitis virus (Duck hepatitis virus,DHV). Sudden death and liver swelling and bleeding. Duck hepatitis virus type I belongs to the small RNA virus family named duck hepatitis A virus (Duck hepatitis A virus,DHAV). It mainly infects ducklings within 3 weeks of age. The mortality rate of ducklings increases sharply after the onset of the disease. The dead duck showed angle arch retraction phenomenon, and the characteristic liver symptom was hemorrhagic lesion, which was one of the epidemic diseases that seriously endangers the duck industry. Although DHV vaccine has been widely used and produced in China, it is still prevalent in various parts of China and has caused great losses in duck breeding. Therefore, it is still an urgent problem to search for a safe and effective vaccine strain and to develop a highly effective duck viral hepatitis vaccine for the prevention and control of duck hepatitis. The mutation mechanism of duck hepatitis A virus (DHV) is unclear, and whether 3C protein, as a functional protein, is involved in the cleavage of duck hepatitis A virus (DHV) is not well understood. In order to understand the variation rule of duck hepatitis A virus type 1 (DHAV-1) gene, the virus was subcultured on SPF chicken embryo, and 3C gene was cloned and sequenced and its virulence was determined. To provide a basis for further understanding the genetic variation of DHAV-1. This study will complete the following two parts: the first part: five strains of DHAV-1 virus were subcultured on SPF chicken embryo. Five strains of DHAV-1 were ALY0901 (1A), AZC14117 (2A), A- virus (3A), A2-14 (4A), ADNA-launch (5A). The five strains were treated as primary viruses and inoculated into the allantoic cavity of 9-12 day-old SPF chicken embryos. The inoculation amount was 0.2 mL/ embryos. The dead chicken embryos were preserved after 24 hours. After 4 ~ 12 hours of cooling in 4 鈩，

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