| Hand, foot and mouth disease(HFMD) has become a global widespread acute infectious disease. Enterovirus 71 (EV71) is one of the main pathogens, which mainly infect infants and young children under the age of five, and it can cause severe in a minority of patients with neurological syndrome,or even lead to death. In recent years, There were a lot of research about EV71 attenuated vaccine and attenuated mechanism, but it was not fully difined. How to reduce the virulence of EV71 and evaluate its virulence decrease is pivotal in attenuated EV71 vaccine research.In this paper, we separated one EV71 strain from many clinical samples, which has a higher infectious titers and could make 100% suckling mouse dead, after five generation serial passaging in vivo,we found that there was no significant decrease of pathogenicity to the suckling mice, But compared with the second generation, the time for virus of the fifth generation making the KMB17 lesion completely was significantly extended. It means that the adaptability of virus in KMB17 decreased after successive batches in the suckling mice.While,alternating passaging to the fourth generation,the pathogenicity of the the strain has a weak decreasing trend.According to the study of FY23-K strain passaging in different temperature and ways in vitro,we found:(1)after serial passaging in KMB17 cells at 33 ℃, the strain’s infectious titers decreased gradually, the time to reach 75% cytopathic effect delayed with passaging; while at 35℃,viruses were able to grow stably, maintain good growth curve.So as a low temperature,35 ℃ might be more suitable for FY23-K strain to extend on KMB17 Cells.(2) Using limited dilution method, we choose a clone with higher infectious titer and better immunogenicity.After intracranial injecting 1-2-day ICR mice,there’s no morbidity and mortality,even no difference on their growth and development compared with the control group.Through the analysis of pathology in the brain, spinal cord and muscle tissue,we found there’s no obvious pathological damage in all the organization cell,only appearing a few inflammatory cells infiltration in the muscle tissue clearance and some glial nodule at the frontal lobe of the brain.(3)When FY23-K strain was serial passaged at 35℃ and 37℃ respectively, there’s no significant difference and change on its infectious droplets, immunogenicity and antigen content. passaging at 35℃, form the 25th to the 35th generation,there’s only three bases of mutation on the whole genome and one base mutation in VP1 area.At 37℃,from the 33th to the 50th generation,there’s also three bases of mutation on the whole genome and one base mutation in VP1 area, Therefore, the strain keep a stable genetic property in both 35℃ and 37℃.(4) Through intraperitoneal, muscle and subcutaneous injection respectively to the 4-6-week ICR mice with the 50th generation live virus passaging at 37℃,we obtained: after 7 days with subcutaneous booster immunization, the seroconversion of the mice was only 37.5%, GMT was 1:8. while the seroconversion were both 100% in another immune way, the GMT of intraperitoneal injection was 1:109.42, which was higher than muscle injection (GMT 1:42.22). all the results suggest that the effects of mice immune response caused by intraperitoneal injection was better than another two injection ways,which could be use to evaluate the immunogenicity of EV71 attenuated vaccine preliminarily.(5) Injecting respectively to 1-2-day ICR mice and 6month rhesus monkeys with the 28th generation samples passaging at 35 ℃,we found that there’s no morbidity and mortality for all the mice,even no difference on their growth and development, Which means the pathogenicity had reduced comparing with the original generation. While on the rhesus monkeys, sores or suspected herpes appeared in the hand, foot and mouth, the pulmonary edema and congestion appeared in the lung.and even with obvious viremia in the blood.Results suggest:6mouth rhesus monkey can be used as a sensitive animals model for evaluating live attenuated EV71, and the mice are not the best animal models. To the 28th generation passaging at 35 ℃, FY23-K strain was still not meet the requirements of ideal attenuared strain,attenuated work needs to be continued.(6)Compare with the originnal generation, FY23-K strain passaging to the 25th genaration at 35℃ has 48 bases mutation that was stable exist at the 33th genaration at 35℃ and the 33th and the 50th generation at 37℃,which led to 10 amino acids changes, and might be the cause of the decrease of the strain pathogenicity. In addition,we speculated the 145th amino acid in VP1 of EV71 may be a virulence related site, turning glutamate into glutamine,could weaken the virulence of EV71. |
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