福建省人感染高致病性禽流感病毒的分离及灭活病毒抗原的制备

目的从患者呼吸道标本中分离高致病性禽流感病毒(H5N1),摸索病毒灭活条件,用于制备安全和有效的高致病性禽流感病毒灭活抗原。方法鸡胚分离法分离高致病性禽流感病毒,应用甲醛和戊二醛两种灭活剂对分离物进行灭活。用鸡胚培养法进行病毒增殖试验,以确定灭活效果,同时测定灭活病毒的血凝效价。结果应用鸡胚分离法,并用电子显微镜及RT-PCR鉴定,确认从福建省人感染病例中分离出高致病性禽流感病毒。在应用甲醛和戊二醛对病毒进行灭活时,在1‰～5‰终浓度、4℃作用24h的条件下,两者均能完全灭活病毒;灭活病毒的血凝效价随着灭活剂的浓度增高而逐渐下降;灭活病毒随保存时间的延长,其血凝活性逐步下降;应用甲醛灭活后的病毒抗原,其血凝效价可维持更长时间。结论首次分离成功福建省人感染高致病禽流感病毒。应用1‰终浓度的甲醛可使高滴度的病毒完全灭活,并保持较好的血凝活性,适用于大量制备高致病性禽流感病毒灭活抗原。

Isolation and inactivation of high pathogenic avian influenza virus(H5N1) from a patient in Fujian province, China

To isolate the high pathogenic avian influenza virus (HPAIV,H5N1) from a human infected case and to determine of the virus inactivation conditions for the purpose of a safe and effective viral antigen. The specimen in respiratory tract from a patient was inoculated into the SPF embryonated chicken eggs for the isolation, the allantioic fluid was collected and confirmed to carry HPAIV by electron microscope and RT-PCR. The viral collection was inactivated by methanal or glutaraldehyde at variant final concentrations. The inactivated viral fluid was incubated into embryonated chicken eggs to evaluate the inactivation effect and detected its hemagglutinin activity by HA test. The results indicated that the virus could be completely inactivated by methanal or glutaraldehyde at the final concentrations ranging from 1% to 5% at 4℃ for 24 hours. The high concentration of the inactivation reagents used could affect the virus HA titer, and HA activity was gradually lost with raising concentrations of the inactivation reagents. The virus treated by methanal of final concentration of 1‰, at 4℃ for 24 hours, could maintain its HA activity for at least 4 weeks. The inactivation effect by methanal is better than that by glutaraldehyde in terms of maintenance of the viral HA activity. It shows that the method is suitable for preparation a large amount of safe and effective viral antigen for serodiagnosis of HPAIV.