Molecular Epidemiological Characteristices Of Enteroviruses In Inner Mongolia And Development Of Enterovirus 71 And Rotavirus Combined Vaccine

The enterovirus 71(EV71)and rotavirus(RV)are the two main pathogens that have chronically threatened children's health.Both pathogens are viruses of the intestinal genus(or called enteric viruses),their major route of transmission is fecal-oral transmission and the susceptible population are children under 5 years old.EV71 is a type of neurotropic viruses,which normally causes children with fever,hand-foot-mouth disease(HFMD),herpangina and other clinical symptoms,it can also lead to neurological and heart diseases or even death in some severe cases.RV mainly causes viral diarrhea in children,and approximately 480,000 to 640,000 infants die from RV infections worldwide each year.In order to control the infectious diseases caused by EV71 and RV,preventive vaccines against the two viruses have been developed from the 1970 s to the 1980 s.So far,there have been 3 types of inactivated EV71 vaccines and 7 types of oral RV live vaccines are on the market.The three types inactivated EV71 vaccines were all produced in China and only launched in 2016,resulted in relatively low vaccination coverage rate and the limitation of vaccination quantity.It requires more time to evaluate the safety and immunity durability of the vaccines.The first RV vaccine in the world was launched in the United States in 1998,but it was forced to disuse due to the great relevance to severe adverse reactions such as pediatric intussusception.There have been a total of 6 oral RV attenuated vaccines have been marketed globally since the launch of Live Rotavirus Vaccine? in China in 2000.However,the RV vaccine is a second-class vaccine that is voluntarily vaccinated at own expense and has not been included in the national immunization program in China,the size of vaccinated population is constrained.At present,whether the existing live RV vaccine is the implication of serious adverse reactions such as intussusception in children is still remain indeterminated.The urgency of developing brand new EV71 vaccines and RV vaccines has been emerged under this circumstances for the purpose of avoiding the temporary absence of which the existing vaccines cannot be used due to the critical side-effects.This study focuses on the similarities between EV71 and RV to explore a new type of EV71/RV combined vaccine through the expression of recombinant chimeric protein,after a series of immunological experiments,the study has reached some expected achievements.Materials and Methods:1.The analysis of molecular epidemiological characteristics of main enteroviruses in Inner Mongolia.Through the methods of epidemiological investigation and laboratory pathogenic microorganism detection,monitoring and analyzing the incidence of HFMD and the epidemiological characteristics of EV71 virus in Inner Mongolia from 2014 to 2018;based on the investigation of viral diarrhea and pathogen detection in children under 5 years old from the largest children's hospital in Inner Mongolia-Inner Mongolia Maternal and Child Health Care Hospital,comprehensive hospitals-Inner Mongolia People's Hospital and the Affiliated Hospital of Inner Mongolia Medical University,military hospital-Inner Mongolia General Armed Police Hospital,and county-level comprehensive hospital-Tuo Ke Tuo County Hospital,to perceive the epidemiological characteristics of rotavirus.By means of on-site sampling,field investigations and laboratory tests of intestinal flora of healthy children under five in all prefectures cities in Inner Mongolia,the intestinal etiological characteristics of children in fitness have been carried out.2.The establishment of animal model of mouse.To culture the mouse-adapted strains of EV71 and RV through continuous passage and use the mouse-adapted strains to construct animal models of the two viruses.3.Research on mechanism of cellular immunology.To explore the immunological mechanism at the cellular level through the transfection of the Vero cells with the mouse-adapted strain EV71 C33?.4.Research on the construction and immunological properties of recombinant combined vaccine.Based on the highly conserved histone VP6 protein of rotavirus,an expression vector carrying the vp6 gene was successfully constructed.The epitope SP70 and SP28 with strong antigenicity were selected respectively from the main antigen proteins VP1 and VP2 of EV71,and inserted into VP6,constructing and expressing the two chimeric recombinant proteins VP6/SP70 and VP6/SP28 based on VP6.After the two recombinant proteins are used as a combined vaccine to immunize animals,it was verified by methods such as ELISA,neutralization test,and animal protection experiment.Results and Conclusion:The study has a great understanding of epidemiological features of the incidence of HFMD in Inner Mongolia from 2014 to 2018,A total of 70,601 cases of HFMD were reported within 5 years,with an average annual incidence of 56.48/100,000.There were 6561 laboratory diagnosed cases,of which the detectable rate of EV71 was 32.57%,and it was the primary pathogens that caused HFMD which was mainly prevalent in June to July.Children aged 5 and under showed the highest incidence with 59,890 cases reported in 5 years,accounting for 84.83% of the total number of cases.By molecular evolution analysis,all the prevalent subtype of EV71 in Inner Mongolia is C4,of which is homologous to domestic strains.After analyzing the viral molecular epidemiological characteristics of patients from 4 grand hospitals and 1 county-level hospital,the rotavirus which is accounting for 30.76% has been proved to be the dominant pathogen that infects children under 5.Rotavirus infections are mainly concentrated in cold seasons,with 59.22% of the cases from November to next February.The serotype(G type)and genotype(P type)of RV strains in Inner Mongolia are tested,consequenced in the detection of five serotypes of G and four genotypes of P,of which the serotype G9 and the genotype P[8] that accounting for and 82.55% and 88.04% respectively are the most prevalent strains.The carrier rate of enterovirus in healthy children under 5 in Inner Mongolia is 7.50%,a total of 5 types of enteroviruses have been detected,including EV71 virus,Coxsackie virus and Echo virus.The obtainment of mouse-adapted strains of EV71 and RV can infect mice and show typical clinical symptoms after continuous passages.The animal model of EV71 and RV were successfully constructed using Kunming mice.The results of cellular immunological mechanism research found that the rising level of gene expression of immune-related factors can be detected in Vero cells infected with EV71.The chimeric protein expression vector based on RV VP6 protein was successfully constructed,and two recombinant proteins of VP6/SP70 and VP6/SP28 were reaped.The recombinant protein has been demonstrated with strong immunogenicity after testing,the specific Ig G antibody of EV71 and RV VP6 can be detected in the antiserum gathered from the immunized mice;the antiserum can effectively neutralize the EV71 virus and the titer of neutraliztion is 1:64?1:128.The two recombinant proteins also have a strong ability to stimulate cellular immunity,the up-regulation of four immune factors,IFN-?,CD4,CD8 and IL-1? in the immunized mice can be detected.The effects of oral administration and intramuscular injection in mice are equivalent.Aluminum hydroxide adjuvant can effectively enhance the effect of the recombinant combined vaccine in this study.The recombinant combined vaccine can effectively resist the EV71 and RV infection with a high level of protection.