The Pathogenesis And Evaluation Of Vaccine Candidate In A Mouse Model With Enterovirus71

Hand, foot and mouth disease (HFMD) is a multiple enterovirus caused diseasewhich have been circulating worldwide since it was reported. Children less-than5years old is the susceptible population. Most of the HFMD patients only exhibit mildsymptoms，including fever, skin rashes, herpes, ulcer in hand, foot, and oral mucosa,and generally will self-cure and recover within2weeks. Few severe cases maysuffer a series of complications such as meningitis, pneumonedema, myocarditis,acute flaccid paralysis and even death, and the survivor may sustain sequela. In therecent decades, circulating enterovirus71(EV71)-associated HFMD is on the rise inthe Asian-Pacific region. Shanghai city reported HFMD in1981for the first time inmainland of China. Subsequently, twice outbreaks of HFMD occurred in Tianjin cityof China in years1983and1986. In1998and2008, a large outbreak of enterovirusinfection have occurred in Taiwan and Anhui province, separately. The numbers ofHFMD morbidity shown an up trend according to Ministry of Health in China. Atpresent, there are no effective antiviral drugs available for severe EV71infection andsymptomatic treatment is the major method.There are more than20types of human enterovirus can induce HFMD,including enterovirus71, coxsackievirus A2、4、5、7、9、10、16and coxsackievirusB1、2、3、4、5, as well as echovirus and so on. Among of them EV71and CA16aretwo major pathogens in most previous outbreaks of HFMD.Human enterovirus71(EV71) is a non-enveloped, single-strandedpositive-sense RNA virus that belongs to the Enterovirus species A genogroup in thePicornaviridae family. The lenghth of complete genome is about7411nt, and iscomposed of5′and3′terminal untranslated region(UTR) and an open readingframe(ORF). Virus genome encode a polyprotein containing2194amino acidresidue. The inactive polyprotein could be further hydrolyzed into three precursorprotein P1, P2and P3. P2and P3protein are split into7non-structural proteins(2A、2B、2C and3A、3B、3C、3D)，which take part in regulating the processof viral polyprotein, RNA replication and synthesis of host cells. P1protein is splitinto VP0, VP1and VP3proteins by3C and3D, then, VP0is split into VP2and VP4.VP1（α）、VP2（β）、VP3（γ） and VP4（δ）assemble into virus capsid in vivo. VP1region determine the viral virulence and neutralization features. The VP1gene haveequivalent genetic diversity with virual serotype, so it not only can be used for thevarious serotype classification of enterovirus but also for classification of enterovirusspecies in the picornaviridae family. Therefore, the VP1gene sequence is the majorregion for enterovirus71genic classification and phylogenetic analysis. At present,on the basis of VP1gene nucleotide sequence diversity, EV71can be classified intothree genotypes of A, B, C and many subtypes (A, B0, B1-B5, and C1-C5). Viralinfection process is the specific binding of virus with receptor located on the cellsurface, and then the virus release nucleic acid after endocytosis of the cell.In this study, we established a lethal mouse model using primary EV71isolatedfrom HFMD patients that are currently circulating in humans in Changchun, China.We revealed that the circulating EV71are recombinant viruses of C4subtype aftercharacterizing the virus molecular genetic features. In the isolated primary EV71infectious neonatal mouse model, we systematiclly studied the viral pathogenicmechanism and protection effect of vaccine candidate.In viral pathogenesis study, the primary EV71could infect and induce theone-day-old ICR mice exhibited pathogenic changes in specific tissues. Throughmonitoring the time point of morbidity, mean clinical symptoms, and survival ratesof the infected mice, we found the viral strain-related and dose-related morbidity andlethality in infected mice. The Changchun isolated circulating EV71viruses hadstronger virulence than the prototype BrCr, Fuyang isolated strain of FY0805andShenzhen strain SHZH98which are non-circulating viruses. For the tissueshistopathological and immunohistochemical analysis, viral loads detection of theinfected mice, we understand the pathogenic features and distribution of virus anddescribed the virus pathogenic mechanisms in the neonatal mouse model. In this article, We screened a vaccine candidated from Changchun isolatedEV71viruses, and developed an inactved EV71vaccine. Analysis of humoralimmunity showed that the vaccine generated better immunogenicity and broadcross-neutralization by cytopathic effect (CPE) in vitro. The antiserum of immunizedfemal mice and the neonatal mice can neutralize various viruses. The vaccine canwell protect the neonatal mice born to immunized female mice from lethal-dosechallenge with a series of EV71viruses in vivo. This lethal mouse model for EV71and the broad cross-protection vaccine candidates could be useful tools for the futuredevelopment and evaluation of EV71vaccine.