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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 preservation methods of Koi herpesvirus (KHV). Methods The diseased fish tissues suffering from Koi herpesvirus disease (KHVD) were treated with cryopreservation method at -80 ℃, cryopreservation method under liquid nitrogen, 50% phosphoglycerate buffer preservation method, ethanol preservation method and isopropyl The alcohol preservation method was stored for 90 days for PCR identification. At the same time, the effects of different methods on the infected healthy fish and CCB cells were also tested. After infection of CCB cells with KHV, the virus solution was collected and stored at 4, -20, -80 ℃ and liquid nitrogen for 90 days respectively to detect the virus infectivity (TCID50). Results The diseased fish tissues preserved by five methods were both positive for KHV by PCR. The diseased fish tissues preserved in 50% phosphoglycerin buffer, ethanol and isopropanol lost their ability to infect healthy fish, whereas diseased fish tissues stored at -80 ° C and liquid nitrogen all caused infection of healthy Koi KHV; diseased fish tissue stored in 50% phosphoglycine buffer can infect CCB cells. The virus infectivity of CCB cell virus stocks preserved at 4 and -20 ° C decreased by 61% and 50%, respectively, whereas the viral infectivity of CCB cell virus stocks stored at -80 ° C and liquid nitrogen decreased only by 7%. CONCLUSION: Preservation of diseased tissues and cytomegalovirus using -80 ℃ and cryopreservation of liquid nitrogen can save virus tissue in the 50% phosphoglycerate buffer.