【摘要】 目的建立甲型H1N1流感病毒鸡胚分离方法,了解病毒分离株特征,为疫苗制备和开展实验室常规监测等奠定基础。方法呼吸道标本接种鸡胚尿囊腔;血凝(HA)法测定收获的尿囊液中病毒滴度;逆转录PCR法检测病毒亚型特异性。使用甲醛灭活鸡胚分离物,灭活效果采用鸡胚接种法评价。血凝抑制(HI)实验检测急性期和恢复期血清,评价检测病毒感染后的免疫应答情况。结果从13份患者的呼吸道标本中分离出12株甲型H1N1流感病毒。多数病毒可在第二次传代鸡胚中分离到。病毒分离株的血凝滴度较低,为1∶1～16。阳性分离物再次接种鸡胚无法显著提高其HA效价。同鸡和人"O"型红血球比较,使用豚鼠红细胞可得到更高的血凝滴度。1‰甲醛可在24h内完全灭活病毒,但同时病毒的HA效价显著降低。病毒感染后,恢复期的HI滴度较急性期有2～16倍升高,HI抗体4倍增长需要约3w时间。结论鸡胚分离可应用于甲型H1N1流感病毒的分离,病毒的HA滴度较低。尽管甲醛可迅速灭活病毒,但灭活后病毒抗原血凝效价难以维持。病毒感染后,特异性血凝抗体产生较慢,同时滴度较低。基层实验室采用豚鼠血球更适合于病毒的HA滴度测定。更多还原

【Abstract】 To isolate influenza type A H1N1 virus by using chicken embryo inoculation and to characterize its biological properties in order to provide a scientific basis to establish the preventive measures,the respiratory specimens were inoculated into the allantoic cavity of developing chicken embryo for virus propagation and the viral titer was measured by hemagglutination (HA) assay. The sub-type specificity of virus was determined by reverse transcription PCR (RT-PCR). Formaldehyde was used to inactivate the virus isolated from chicken embryo and the effect of inactivation of virus was evaluated by chicken embryo inoculation method. The immune response profile following viral infection was analyzed by hemagglutination inhibition (HI) test for both acute and convalescent phase sera of the confirmed cases. It was found that 12 strains of influenza type A H1N1 virus were isolated from 13 respiratory specimens of the clinically confirmed cases,and most of which was isolated at the second passage with lower HA-titers ranged from 1∶1 to 1∶16. Moreover,the HA titer of the viral isolate was not increased dramatically at second passage in chicken embryo. Compared to chicken or human "O" erythrocytes,the guinea pig erythrocytes were proved to be more sensitive when the HA titer of isolate was measured. The 1‰ formaldehyde could completely inactivate the virus within 24 hours,and simultaneously,the viral HA titer lost dramatically. The HI titer in convalescent phase sera showed a 2 to 16-fold increase in comparison with that in the paired acute phase sera,but the 4-fold increase in HI titer required approximately a period of 3 weeks. It is evident that the chicken embryo inoculation method can be applied for the isolation of influenza type A H1N1 virus,but the HA titer of the isolates is lower. Although formaldehyde can rapidly inactivate the virus isolated,the HA titer of viral antigen after inactivation is difficult to maintain at a high level. The production of the specific HA antibody is rather slow with lower titer. For the local laboratory,it is better to use guinea pig erythrocytes to detect the viral HA titer.更多还原