乙型脑炎病毒SA_(14)-14-2减毒疫苗株的生物学和基因稳定性

Biological and Genetic Stability of Attenuated Japanese Encephalitis Vaccine Virus Strain SA_(14)-14-2

目的研究乙型脑炎病毒(JEV)SA14-14-2减毒疫苗株传代过程中的生物学和基因稳定性,为疫苗的安全性和免疫原性提供依据。方法将JEVSA14-14-2减毒疫苗株PHKC7代病毒在原代地鼠肾细胞(PHKC)上连续传代,检测各代次病毒的培养特性以及第8、12、17和22代病毒的安全性及免疫原性;对起止代次病毒(PHKC8和22代)进行全基因组序列测定,对中间代次病毒(PHKC12和17代)的结构蛋白基因进行测序,并与基因库乙脑病毒SA14-14-2株(D90195)比较。结果各代次病毒产生的细胞病变基本一致,病毒滴度在17代前较稳定;在BHK21细胞上形成的蚀斑形态一致;小鼠脑内的致病力未发生变化,乳鼠传代返祖试验高代次病毒毒力有升高趋势。8、12、17和22代病毒免疫小鼠后,对P3强毒攻击均具有保护作用。E蛋白核苷酸和氨基酸序列,PHKC8代与D90195完全一致;而PHKC12、17和22代出现单个碱基(第2142或2143位)突变,导致E389氨基酸改变(D→N或D→A),但并非回复突变。4个代次病毒的PrM-C区结构蛋白基因均未发生变化。PHKC8、22代病毒的其他核苷酸突变发生在基因组非结构蛋白区和3′端非编码区;全序列PHKC8和22代与D90195比较,发现7～8个核苷酸不同,导致3～4个氨基酸的差异,核苷酸和氨基酸的同源性分别大于99.93%和99.88%,且以上突变不存在于明确的相关减毒位点上。结论JEVSA14-14-2减毒疫苗株在PHKC上连续传15代,仍保持高度的生物学和基因稳定性,疫苗生产用病毒控制在10代以内较为安全。

Objective To explore the biological and genetic stability of attenuated Japanese encephalitis （JE） vaccine virus strain SA14-14-2 during subculture and provide basis for study on safety and immunogenicity of vaccine. Methods The strain SA14-14-2 of passage 7 was subcultured in primary hamster kidney cells （PHKCs） to passage 22. Each passage was tested for culture characteristics, and the passages 8, 12, 17 and 22 for safety and immunogenicity. The whole genome of passages 8 and 22 as well as the structural protein genes of passages 12 and 17 were sequenced, and the results were compared with those of JE virus strain SA14-14-2 （D90195） reported in GenBank. Results The CPE caused by strain SA14-14-2 of various passages were basically in agreement, and the virus titers of the strain within 17 passages were stable. The morphologies of plaques formed in BHK21 cells by strain SA14-14-2 of passages 8, 12, 17 and 22 were similar, and the neurotoxicity in mice of the four passages showed no significant difference. However, the toxicity reversion test in suckling mice proved that the toxicity of passage 22 showed an increasing tendency. The strain SA14-14-2 of passages 8, 12, 17 and 22 protected the mice against challenge with virulent P3 strain. Both nucleotide and amino acid sequences of E protein of strain SA14-14-2 of passage 8 were completely identical to those of D90195. However, the mutation of single base at site 2142 or 2143 were observed in passage 12, 17 and 22, resulting in the change of amino acid at site E389 （D→N or D→A）, though it was not a reverse mutation. No change was observed in the PrM-C region of structural protein gene of strain SA14-14-2 of passages 8, 12, 17 and 22, while the mutations of other nucleotides were observed in non-structural protein region and non-encoding region at 3＇-terminus of genomes of passages 8 and 22. Only 7-8 nucleotides in full-length gene of passages 8 and 22 were different from that of D90195, resulting in the difference of 3-4 amino acids. The homologies of nucleotides and amino acids of strain SA14-14-2 of various passages to those of D91095 were more than 99. 93% and more than 99. 88% respectively, and no mutations were observed at the relevant attenuation sites. Conclusion Attenuated JE virus strain SA14-14-2 showed high biological activity and genetic stability after subculture for 15 passages in PHKCs. It is safe to control the virus strain for vaccine production within passage 10.