登革4型病毒Ban18-30减毒株的生物学特性研究

Study on the biological characteristics of dengue viruses type 4,Ban18-30 attenuated strain

目的将登革4型病毒（DEN-4）Ban18原株及其减毒株Ban18-30株在vero细胞传代后进行生物学特性比较研究，以了解Ban18-30株的毒力和免疫原性及其是否为一株有希望的DEN-4减毒活疫苗候选株。方法Banl8原株及在原代地鼠肾细胞传代减毒的Ban18-30株在vero细胞中增殖，将两毒株进行中和实验，确证两株的型别，研究传代毒株的病毒滴度、毒力和免疫原性。结果两株病毒与抗DEN-4特异性抗体进行中和实验，中和指数≥316，证明两株病毒确为DEN-4；空斑滴定两株病毒，滴度约为2×10^6-2．5×106PFU／ml；Ban18-30株对乳鼠的脑内毒力较Banl8原株低，≥1．710gID50；Ban18-30株对幼鼠脑内接种不致病，而原株的log LD50值为4．3；腹腔免疫小鼠后用原株进行脑内攻击，能够抵抗大于1000 LD50致死剂量的病毒攻击。结论两株病毒在vero细胞内增殖良好，滴度较高，Ban 18-30对乳鼠和幼鼠的致病力明显减弱。在减毒的过程中仍保留了较高的免疫原性，有望成为一株DEN-4的减毒活疫苗候选株。

Objective Comparative study of the biological characteristics of two dengue 4 viruses（DEN-4）, parental Ban 18 swain and its attenuated swain Banl8-30. Clarify the neurovirulence in mice and immunogenicity of the two viruses. Methods The DEN-4 Ban18 strain and its attenuated strain were cultured in vero cells for 1 - 2 passages. The neutralization assays of the two viruses were performed using the anti-DEN-4 ascites tested in BHK21 ceils to identify the type of the two viruses. And then the virus titles, neurovirulence and immunogenicity of the two viruses were tested. Results The results of the neutralization assays showed that the both viruses were well neutralized by the antibody, neutralization index≥1000, indicating both the viruses were DEN-4. The plaque assay in BHK21 ceils of the two viruses showed that the titres of them were 2×10^6 - 2.5 × 10^6pFU/ml. Neurovirulenee tested in suckling mice showed that the LD50 of BanlS-30 was 1.7log lower than its parental virus Ban18. When tested in9-11g mice, the results indicated that the Ban18-30 virus didn＇ t kill any mouse by intracerebral inoculation whereas the parental Ban18 virus induced mice with high mortality, 4.3 logLD50. The immunogenicity of the Ban18-30 attenuated virus was tested in 7 - 9g mice by a single intraperitoneal vaccination and a challenge after 14 days with the Ban18 virus by intraperitoneal inoculation. The results indicated that the mice vaccinated with the Ban18-30 could protect against ≥ 1 000 LD500 of the Banl8 virus infection. This result revealed that Ban18-30 virus remained good immunogenicity. Conclusion Both the viruses are the DEN-4 and the outputs are high in vero cells. Experiments tested in mice demonstrated that Ban18-30 virus is highly attenuated and remains high immunogenicity. Ban18-30 virus is a promising live attenuated vaccine candidate of DEN-4.