Identification Of EV71 In Xi'an And Preparation Of Monoclonal Antibody Against EV71

Enterovirus 71 (EV71) belongs to human Enterovirus A species of the Enterovirus genus, Picornaviridae family. It was first isolated from cerebrospinal fluid by Schmidt and now its spread is worldwide. Recently in China is increasing year by year. After infection EV71, besides can cause HFMD, it can lead to some symptom of acute central nervous system , such as aseptic meningitis,encephalitis, poliomyelitis-like paralysis, and neurogenic pulmonary edema, myocarditis. It is a serious threat to the life and health of children.The EV71 genome was constituted by 60 of the same subunits, and each of them contains the VP1~VP4 genes. On the basis of VP1 nucleotide sequence comparisons, three genogroups have been distinguished:A, B, and C, and 9 subgenotypes, which were B1~B5 and C1~C4 were identified. Currently ,the molecules epidemiological studies of EV71 is aimed at the VP1,VP4 genes and the variability of 5'u?ntranslated region(5'-UTR). Compared with other members, capsid protein VP1 of EV71 is more valuable than other regions in the genome. Besides, the specific epitopes responsible for serotypic specificity are clustered mainly on VP1, and the 5'-UTR is involved in several aspects of the function, such as host range and virus toxicity.EV71 type genetics have variability in the same area and different time , which lead to clinical symptoms were differences in different areas.Most children can recover within a week, but a few young children may only show the central nervous system symptoms of infection, so it increased the difficulty of the early diagnosis. And now, we just have little knowledge about the molecular mechanisms of disease of EV71.The receptor through the host cell is unknown. We haven't developed a safe and effective drug and vaccines. So, the rapid detection of pathogens in time has an important role of the diagnosis and treatment of HFMD disease.ObjectiveTo isolate the prevalent strain of enterovirus 71(EV71)in Xi'an area. After the preliminary purification, establish a new fast and effective detection methods of ELISA for EV71 antibody. Then use the purified virus produces anti-EV71 monoclonal antibody, Construct stability of hybridoma cell strains and determine the potency of ascites.Methods1. To detect and identify the EV71 virus from throat swabs of children suspected with hand-foot-and-mouth disease in Xi'an. Then use reverse transcription polymerase chain reaction(RT-PCR)and immunofluorescent technique in detecting EV7 l. Analyse the nucleotide sequence of viruse.2. Use the precipitation method of polyethylene glycol to purify the virus. Test the positive serum of patients saved in our laboratory by ELISA to identify the immunological activity of the virus. 3. Female Balb/c mice were immunized with the purified EV71 virus. The splenocytes of immunized mice were fused with SP2/0 myeloma cells by the routine method. Then the hybridomas were selected in HAT medium. After the hybridoma cells secreting specific antibody were detected by ELISA, inoculate the hybridoma cells to the intra-abdominal of Balb/c mice for preparing lots of monoclonal antibody and determine the potency of ascites.Results1. The positive rates of RT-PCR on the 22 samples of throat swabs were 31.8%(7/22). Among the 22 samples of throat swabs inoculated on RD cells, the positive rates were 45.4%(10/22). The positive rates was 68.1%(15/22) after the viral isolation.2. The nucleotide sequence of the isolated virus was the same to the EU81246 in the NCBI Genbank. The results of immunofluorescence showed that the purified virus were enterovirus 71.3. Use the established method of ELISA to test the positive serum of patients, the positive rates were 85% , and the results of serum of patients infected CoxA were all negative.4. Obtained two hybridoma cell strains designated as EM1,EM2, and the potency of ascites is 10-4 .Conclutions1. The unification of viral isolation and RT-PCR can enhance the positive detection rates of EV71, which can offer a powerful way to the clinical diagnosis. There is statistical significance in the distinction of consequence(χ2 =4.9,P<0.05).2. Established a new fast detection methods of ELISA for EV71 antibody. It show that the purified virus have immunological activity. 3. Obtained hybridoma cell strains and prepared lots of monoclonal antibody of ascites, which may lay the foundation for further research function of EV71 and set up the dectect Kit of EV71.