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目的对柯萨奇病毒A组16型(coxsackievirus A16,CA16)减毒活疫苗候选株进行初步分析,为CA16减毒活疫苗的研制奠定基础。方法将两株CA16疫苗候选株(KM168和KM154)进行低温连续传代减毒,对不同代次病毒进行减毒特性的分析。将候选株病毒收获液免疫ICR乳鼠(颅内注射)及ICR成鼠(腹腔注射),分别评价减毒疫苗候选株的毒力、免疫原性以及乳鼠作为减毒活疫苗残余毒力评价动物模型的可行性;对病毒进行VP1片段的测序,分析遗传稳定性。结果两个候选株经低温连续传代后,均具有稳定的生长特性及良好的免疫原性,感染性滴度均保持在约7.0 Lg CCID50/ml,免疫小鼠后抗体阳转率达100%,最高GMT约1∶30。乳鼠及成鼠感染病毒后,在大部分组织中能检测到CA16病毒;KM154株在第12代后未对乳鼠产生临床致病性及致死率,病理检测结果主要表现为少量炎性细胞集结,无明显组织损伤,随着传代增加,组织损伤明显减少。两个候选株经低温连续传代后,能保证VP1片段的遗传稳定性。结论低温连续传代可使CA16的毒力明显降低,有望筛选出减毒活疫苗的候选株;1~2日龄ICR乳鼠可以作为CA16减毒活疫苗的候选株毒力评价的动物模型。
Objective To preliminary analyze the live attenuated vaccine strain of coxsackievirus A16 (CA16) in group C, and lay a foundation for the development of live attenuated CA16 vaccine. Methods Two strains of CA16 vaccine (KM168 and KM154) were continuously passaged and attenuated at low temperature, and the attenuated characteristics of different generations of viruses were analyzed. Candidate virus harvested liquid was used to immunize ICR suckling mice (intracranial injection) and ICR into mice (intraperitoneal injection) to evaluate the virulence and immunogenicity of attenuated vaccine candidate strains and evaluate the residual virulence of suckling mice as live attenuated attenuated vaccine Feasibility of animal model; sequencing VP1 fragment of virus to analyze genetic stability. Results Both of the two candidate strains had stable growth characteristics and good immunogenicity after being continuously passaged at low temperature. The infectivity titer was kept at about 7.0 Lg CCID50 / ml. After immunization, the positive rate of the antibody was 100% The highest GMT is about 1:30. CA16 virus could be detected in most tissues of suckling mice and adult mice. KM154 strain did not produce clinical pathogenicity and lethality in neonatal rats after passage 12, and the results of pathological examination mainly included small amount of inflammatory cells Assembled, no obvious tissue damage, with the increase of passage, tissue damage was significantly reduced. After two candidate strains were passaged at low temperature continuously, the genetic stability of the VP1 fragment was guaranteed. CONCLUSION: The low toxicity of CA16 could significantly reduce the virulence of CA16, and it is expected to screen candidate strains of attenuated live attenuated vaccine. ICR suckling mice aged 1-2 days can be used as animal models for evaluating virulence of CA16 attenuated live vaccine.