S_(79)株流行性腮腺炎病毒不同代次的序列研究

Sequence Research on S79 Strain of Mumps Virus During the Successive Passaging

目的观察流行性腮腺炎(腮腺炎)病毒(MuV)S79株各代病毒的蛋白基因在传代过程中是否发生变化,对比S79株主代病毒(S79-1-3)、蚀斑纯化病毒(S79-1-P7-3③)及ME(Enders株)病毒的基因差异。方法S79株主代病毒及蚀斑纯化病毒在原代鸡胚细胞(CEC)上连续传递12代,观察每代病毒培养特性及高变区SH基因序列,又选择其中的CEC5、6、10、15代,以及在鸡胚尿囊腔中传代的ME株病毒,测定各个主要基因序列(N、NS1/P、M、F及HN基因),对核苷酸序列和它相应的氨基酸序列以及基因差异进行计算机分析。结果S79株主代病毒及其蚀斑纯化病毒在CEC上连续传递12代,其培养特性、致细胞病变(CPE)及病毒滴度基本稳定。S79株主代病毒及其蚀斑纯化病毒与ME株病毒同属A基因型。S79株疫苗各个代次之间高变区SH基因未见突变。S79株的5、6、10、15代病毒在各个主要基因区域有散在基因的有意义突变比例极低。S79-1-3中不同亚株之间的比例可能会随传代而发生变化,比较认为经过蚀斑纯化的S79-1-P7-3③株在传代中较S79-1-3株更为稳定。结论S79株病毒与文献报道的JL2株十分相似,经连续传代总体的核苷酸差异<0.5%。S79-1-3主代病毒在药典规定代次范围内甚为稳定。经纯化的各代S79病毒则更为稳定,从分子水平上提示其在CEC15代内都可作为工作代毒种使用。

Objective To explore the gene changes of the mumps vaccine S79 strain during the successive 12 passages,and compare the sequence among the three viruses. S79 master seed virus （S79-1-s） ,S79 plaque-purified strain（S79-1-P7-3③ ）and ME （Enders） strain, a mumps virus strain cultivated in chicken embryonic allantoic fluid,in the laboratory. Method The mumps vaccine S79 strain was cultivated through CEC from CEC4 to CEC15 passage. Observe the changes of the cultivation characteristic and the sequence of SH gene,the most variable part of the mumps virus genome, some of the passages vaccines（5th, 6th, 10th, 15th）and ME strain, were selected to amplify in N,NS1/P,M,F and HN gene,then assessed the difference of nucleotides and some protein sequences by analysis software. Results The statistic of the cultivation characteristic,cytopathic effect （CPE） and the virus titers of successive passages of S79-1-3 and S79-1-P7-3③ strain were basically similar. All of the three mumps virus strain belong to genotype A. There were no gene changes on SH gene through CEC for 12 successive passages of S79-1-P7-3③ strain and only two changes on 12th of S79-1-3 strain. The transformations of S79 strain did not change,the significant mutation proportion in the main gene is low. Passaging S79-1-3 strain in CEC cell cultures may result in selection of its substrain compositions, S79-1-3 strain was less stable than S79-1-P7-3③ strain. Conclusion From the sequence of all genes the S79 strains were very similar to JL2 strain,a vaccine strain widely used in the world. The nucleotide difference between the two strains was less than 0. 5%. The S79-1-3＇S passages were stable before 10th. All passages of the S79-1-P7-3③ could be used in vaccine-manufacture theoretically as the genes of 12 successive passages were relatively stable.