Isolation, Detection Of Enterovirus And Establishment Of Quick, High-throughput EV71 Neutralization Assay

Hand-foot-mouth disease(HFMD) is popular in children,which is caused by many enteroviruses.EV71 is the main agents of HFMD in china these years.The HFMD caused by EV71 sometimes is associated with the hurt in central neural system,but there are no diagnostic reagents and effective vaccine against EV71,so the research of EV71 is very important to the development of EV71 diagnostic reagent and vaccine.In this research,we have build a platform of enterovirus isolation and identification,and a quick,high-throughput EV71 neutralizing method,we also do some basic research on the EV71 animal model.All these work will make a contribution to the research of EV71 diagnostic reagents and vaccine.In this research,we build a panel of enterovirus through virus isolation and culture,including 37 strains of EV71,9 strains of CA16 and 11 strains of non EV71＆CA16.We can do a large scale of EV71 production,concentration and purification.Now we have achieved EV71 virus with a high titer and purity.EV71 neutralization assay is important in the detection of ev71 infection.Virus isolation,RT-PCR and neutralizing test are gold criterions of EV71 indentify.The virus isolation and RT-PCR sometimes are difficult because of the virus titers are too low,so neutralization assay is a credible method to detect someone who have ever infected by EV71.In addition,the level of neutralizing antibody is important in EV71 vaccine development.The TCID₅0 test is a general method now used to detect the EV71 neutralizing antibody.This method is tome-consuming,need 7 days to wait for the results.The workload is too heavy to do enough samples.The manual estimation may cause subjective error.So the TCID₅0 test can not afford the quick and high-throudhput EV71 neutralization assay.In this research,we eatablished a new method for EV71 neutralization assay.This method is based on Elispot and CTL automated spot-scanning.We used a EV71 antibody marked with HRP to capture the EV71 which infected RD cells,then add TMB to make the infected cells be blue spots.We can achieve the results through the CTL automated spot-scanning apparatus.This method is quick and high-throughput, we need only 30 hours to get the results.The automated scanning is more creditable and stable then TCID₅0 method.We have do some work to optimize the new EV71 neutralization assay to make it more stable.This method has a good relativity with TCID₅0 method,the sensitivity,speciality and repeatability of new method is good, this research make a proof for the use and popularize of the new EV71 neutralization assay.Now,there are no good vaccine for enterovirus except the IPV and OPV for poliovirus.The EV71 vaccine development is important,the EV71 animal model is the basement of vaccine development.This research also do some experiment about the EV71 animal model.We make ICR neonatal mice as a EV71-infected model,the mice infected by EV71 show some syndromes of weigh-lost,tremble,paralysis and even death.The immunoassay of tissues show that there are EV71 in the brain of the dead mice,this basic work is useful for the later EV71 animal research and vaccine development.