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研究从患病草鱼中新分离到一株草鱼呼肠孤病毒(Grass carp reovirus,GCRV),对其进行了病毒纯化与电镜观察、基因组RT-PCR分型以及病毒量定量分析等,并在此基础上探索了一种新的病毒人工感染方法。取病鱼肌肉组织进行病毒纯化与电镜观察,观察到大量病毒粒子,直径在70—80 nm。病毒基因组RT-PCR扩增结果表明,该草鱼呼肠孤病毒新分离株属基因型Ⅱ型GCRV;通过绝对定量的方法,对病毒悬液的浓度进行了测定,为2.97×103 copy/μL。通过灌胃法,对3个组别的实验鱼分别感染不同浓度的病毒液,同时设置灌胃PBS的组别作为对照组。结果显示,3个实验组死亡率均在80%左右,而对照组仅出现一例死亡个体。实验组死亡个体体表发黑,腹部、鳍条基部以及鳃盖处均有明显的出血症状,为草鱼出血病的典型症状。随机选取死亡的个体进行RT-PCR检测,均能检测出Ⅱ型GCRV的条带。以上结果说明,灌胃法可以作为一种新的方法,用于草鱼的GCRV人工感染实验。
A newly isolated grass carp reovirus (GCRV) was isolated from sick grass carp. The virus was purified by electron microscopy, genotyping by RT-PCR and quantitative analysis of the virus Based on the exploration of a new method of artificial infection of the virus. Take diseased fish muscle tissue for virus purification and electron microscopy, observed a large number of virus particles, the diameter of 70-80 nm. The results of RT-PCR showed that the new isolate of reovirus was a genotype Ⅱ GCRV. The concentration of the virus suspension was determined by absolute quantification, which was 2.97 × 103 copies / μL. The gavage method was used to infect 3 groups of experimental fish with different concentrations of virus solution respectively, and PBS group was set as the control group. The results showed that the mortality rate of three experimental groups were about 80%, while the control group, only one death occurred. The dead individuals in the experimental group were black, with obvious bleeding symptoms at the abdomen, the base of the fin and the gill cap, which were the typical symptoms of grass carp hemorrhage. Randomly selected dead individuals were detected by RT-PCR, can detect type Ⅱ GCRV bands. The above results indicate that gavage can be used as a new method for artificial infection of GCRV in grass carp.