1型鸭甲肝病毒在SPF鸡胚上的传代及其3C基因变异规律的研究
发布时间：2018-11-11 07:41
【摘要】：鸭病毒性肝炎(Duck viral hepatitis,DVH)是由鸭病毒性肝炎病毒(Duck hepatitis virus,DHV)引起的一种雏鸭的急性、高度致死性的传染病,临床上主要表现为食欲减退、神经症状、突然死亡和肝脏肿大出血。鸭病毒性肝炎病毒I型属于小RNA病毒科,命名为鸭甲肝病毒(Duck hepatitis A virus,DHAV),主要感染3周龄以内的雏鸭,雏鸭发病后,死亡率急剧上升。病死鸭多呈角弓反张现象,剖检肝脏特征性症状为出血性病变,是严重危害养鸭业的疫病之一。虽然我国目前已有DHV疫苗的生产和广泛使用,但该病仍然在我国各地流行并造成肉鸭养殖业的重大损失。因此,寻找安全有效的理想疫苗毒株,研发高效的鸭病毒性肝炎疫苗,用于鸭肝炎病的预防或控制,依然是亟待解决的问题。因为鸭甲肝病毒的变异机理尚不清楚,而3C蛋白作为功能性蛋白参与裂解功能,是否与毒力变化相关尚未了解。为了解鸭甲肝病毒1型(DHAV-1)基因的变异规律,本研究选择在SPF鸡胚上对病毒进行传代试验,通过对3C基因的克隆测序及毒力的测定,为深入了解DHAV-1的遗传变异规律提供依据。本研究将完成以下两部分内容:第一部分:5株DHAV-1病毒在SPF鸡胚上的传代致弱5株DHAV-1分别为ALY0901(1A),AZC14117(2A),A-病料毒(3A),A2-14(4A),ADNA-launch(5A)。将这5株毒经过处理后作为初代毒,经尿囊腔接种9-12日龄SPF鸡胚,接种量为0.2 mL/胚。留存24小时后的死亡鸡胚,在4℃冰箱冷却4-12 h后,无菌收取鸡胚尿囊液并观察胚胎病变,将所收尿囊液对应接入下一批鸡胚,连续传代60代。结果表明:鸡胚传代5代之前,24-96h之间鸡胚并无死亡现象,胚体也无明显病变;5代之后,到20代为止,鸡胚开始出现死亡现象,并且胚体明显有大量出血点。死亡时间集中在72h左右。20-50代72h内鸡胚无死亡现象,50-60代重新出现鸡胚死亡。第二部分:DHAV-1各毒株各代次鸡胚传代毒3C基因的测序及序列分析完成对ALY0901(1A),AZC14117(2A),A-病料毒(3A),A2-14(4A),ADNA-launch(5A)五株毒每隔五代进行3C基因的克隆测序,对得到的碱基及氨基酸序列进行比对,找寻基因变异的规律,研究发现碱基出现部分突变,位置基本一致,但并未发生氨基酸突变,故推测病毒毒力的变化与3C区间无关。
[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，
本文编号：2324203
[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，
本文编号：2324203
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