锦鲤疱疹病毒保存方法的筛选及比较

Screening and comparison of methods for preservation of Koi herpesvirus

目的筛选并比较锦鲤疱疹病毒(Koi herpesvirus,KHV)的保存方法。方法将患锦鲤疱疹病毒病(Koi herpesvirus disease,KHVD)的患病鱼组织分别采用-80℃超低温保存法、液氮超低温保存法、50%磷酸甘油缓冲液保存法、乙醇保存法及异丙醇保存法保存90 d,进行PCR鉴定,同时检测不同方法保存的患病组织对感染健康鱼及CCB细胞的影响。用KHV感染CCB细胞后,收集病毒液,分别于4、-20、-80℃及液氮中存放90 d,检测病毒感染力(TCID50)。结果 5种方法保存的患病鱼组织经PCR检测均为KHV阳性。于50%磷酸甘油缓冲液、乙醇及异丙醇中保存的患病鱼组织失去了感染健康鱼的能力,而于-80℃及液氮中保存的患病鱼组织均可使健康锦鲤感染KHV;仅保存于50%磷酸甘油缓冲液中的患病鱼组织可感染CCB细胞。于4及-20℃保存的CCB细胞病毒液的病毒感染力分别下降61%和50%,而于-80℃及液氮中保存的CCB细胞病毒液的病毒感染力仅下降7%。结论利用-80℃及液氮超低温保存法可长期保存患病组织病料和细胞病毒液,于50%磷酸甘油缓冲液保存的组织病料有利于进行病毒的细胞分离。

Objective To screen and compare the methods for preservation of Koi herpesvirus（KHV）. Methods The tissues of Koi fish with KHV disease（KHVD） were preserved at-80 ℃, in liquid nitrogen, in 50% glycerol phosphate buffer, in ethanol solution and in isopropyl alcohol for 90 d respectively, and identified by PCR, based on which the effect of tissues preserved by various methods on infected fish and CCB cells were evaluated. CCB cells were infected with KHV,and virus liquid was harvested, stored at 4,-20 and-80 ℃ and in liquid nitrogen for 90 d respectively, and determined for titer（TCID50）. Results The tissues of Koi fish with KHVD preserved by five methods were proved as positive for KHV by PCR. The tissues preserved at-80 ℃ and in liquid nitrogen showed the ability to infect healthy Koi fish. However, the tissues preserved in 50% glycerol phosphate buffer, ethanol and isopropyl alcohol failed to infect Koi fish. The tissues preserved in 50% glycerol phosphate buffer only infected CCB cells. The titers of virus harvested from infected CCB cells decreased by 61% and 50% after storage at 4 and-20 ℃ respectively, while decreased by only 7% after storage at-80 ℃and in liquid nitrogen. Conclusion The cryopreservation at-80 ℃ and in liquid nitrogen were suitable for long-term preservation of infected tissues and virus liquid, while preservation in 50% glycerol phosphate buffer were suitable for the separation of KHV from cells.