| Background and objective:Hand-foot-mouth disease (HFMD) is a worldwide infectious disease. In recentdecades, HFMD repeatedly epidemic outbreaks in the Asia-Pacific region. Among of themMalaysia, Singapore and Japan were countries with higher incidence of HFMD. In Japan,Coxsackie virus group A type16(Coxsachie virus A16, CVA16) infection was majorepidemic strain of HFMD outbreak from1969to1970. Enterovirus71(enteroviruses71,EV71) and CVA16alternately dominated and mixed infections appeared in HFMDepidemic from1997to2000. In China, since HFMD was firstly discovered in Shanghaicity in1981, HFMD was popular in several provinces of Anhui, Henan, Guangdong,Beijing, Taiwan, etc. In outbreak of HFMD in China Taiwan area in1998, CVA16infectionand EV71infection were alternately appeared. At present, the research has proved that bothEV71and CVA16are the main pathogen of HFMD outbreak. HFMD outbreak from EV71infection in Fuyang city, Anhui province, China in1998caused significant harm. At present,many EV71virus strains were isolated in our country, and basic and applied researcheshave been conducted in depth, and HFMD EV71vaccine will appear on the market.CVA16virus is another enterovirus caused severe HFMD, compared with EV71,however, CVA16virus is less discovered in HFMD cases and the understanding for it islate. The virus has not yet been attracted enough, but in the latest epidemiological survey,CVA16virus can also cause severe HFMD. At present, the domestic study of CVA16isincreasingly attached. The previous studies confirm CVA16virus genome variation andrestructuring in the process of popular rate is high, therefore, the research progress ofvaccines and drugs have been slow.Our laboratory found the virulence of CVA16-SHZH05is weak, but there is noin-depth research, while CVA16-CC has certain virulence.Because both CVA16-CC024 and CVA16-SHZH05are belong to CVA16, in order to further compare the vaccine safetyeffect difference. This research from the aspects of aspects of vaccine safety effect,analyzed the two subtypes of CVA16strain (CVA16-SHZH05and CVA16-CC) biologicalcharacteristics. On the basis of the successful experiences of EV71vaccine research anddrug design, an animal model used to evaluate the effectiveness of vaccines and drugs wasestablished. In the animal model, the characteristics of different strains of pathogenicviruses CVA16, the tropism of two virus strains, viral replication capacity and virusinfection cycle were studied in vivo. The virus tropism and viral replication capacity werecompared in vitro, Our study will provide a basis for fundamental research on CVA16virus,and will lay the foundation for research and development of vaccine.Methods:This study was divided into two parts. In the first part, the animal model wasestablished, the virus harvest fluid cultivated in cells was infected the brain cavity ofnewborn mice. The pathogenic, infection ability, histological location, and infection cyclein many strains circulating strains in Changchun (CVA16-CC) and early isolates inShenzhen (CVA16-SHZH05) were observed in infected neonatal mouse model. Thebiological characteristics of different viruses was compared. In the second part, the parallelcomparative study on infection ability of two virus strains (CVA16-CC andCVA16-SHZH05) was done in vitro, based on the first part study.Results:In the study we successfully established a neonatal mouse model of CVA16epidemicstrains fatal infection, and found the different pathogenic abilities of virus with similargenetic background; CVA16-CC and CVA16-SHZH05exhibited distinct characteristics invivo study. CVA16-SHZH05virus was no fatal and infectious, but CVA16-CC strains werelethal and infectious for newborn mice in vivo, with virus strain-and dose-relatived manner.The infected mice histopathological observation, immunohistochemical analysis and viralload determination proved that represented by CVA16-CC024virus, the replication ofCVA16-CC strains could proliferate in kinds of tissues of newborn mice, and resulted inserious injury in tissue, specially in the lung, hind leg muscle and spinal muscle. WhileCVA16-SHZH05could not replicate in newborn mice without tissue damage. Thecomparative study in vitro found both CVA16-SHZH05strain and CVA16-CC strains showed infectivity and cytopathic effect for BHK cells in vitro, but showed not infectivityfor C6, L929, ML-1cells. Genetic research results showed that5’-untranslated region(untranslated regions, UTR) in the amino acid sequence in the virus genome, and P1, P2andP3function protein coding regions had some diversity difference between CVA16-SHZH05and CVA16-CC024virus.Conclusions:CVA16-SHZH05virus was no fatal and infectious but CVA16-CC strains were lethaland infectious for newborn mice in vivo, with virus strain-and dose-relatived manner.CVA16-SHZH05could not infect newborn mice and did not replicate in newborn mice, sothe virus did not cause pathological tissue damage. Represented by CVA16-CC024virus,the replication of CVA16-CC strains in multiple tissues of mice were found besides causingillness and death, and the pathological damage had tissue specificity, and the lung, hind legmuscle and spinal muscle were specially damaged. Both CVA16-SHZH05strain andCVA16-CC strain showed infectivity and cytopathic effect for BHK cells in vitro, butshowed not infectivity for C6, L929, ML-1cells. Viral genome sequencing results showedthat5’-untranslated region (untranslated regions,5’UTR) in the amino acid sequence in thevirus genome, and P1, P2and P3function protein coding regions had some diversitydifference between CVA16SHZH05and CVA16-CC024virus. These diversity differencesleaded to difference of virus biological characteristics. The present study provided areference for the further study of CVA16molecular epidemiology, virulence variation andpathogenic site screening, and laid a foundation for vaccine and drug development. |
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