Identification Of Coxsackievirus A2 And Construction Of An HFMD Mouse Model For A Coxsackievirus A2 Strain

BackgroundHand-foot-mouth disease(HFMD)is a pediatric infectious disease mainly caused by enterovirus infection.As there is no effective treatment,the annual incidence of HFMD is still the top one C type infectious disease in China,which has become an important public health issue and pose a threat to infants and young children.In recent years,significant mutation have taken place in the pathogenic spectrum of HFMD.In addition to the common enterovirus 71(EV71)and Coxsackievirus A 16(CVA16),Coxsackievirus A 2(CVA2),coxsackievirus A 10(CVA10)and Coxsackievirus A 6(CVA6)are emerging new enterovirus that can cause HFMD epidemic.Therefore,to elucidate the pathogenesis and immune mechanism of HFMD caused by new enterovirus and establish a stable animal infection model is of great significance for preventing HFMD.Objective1.To study the pathogen spectrum characteristics of HFMD in the First Affiliated Hospital of Xinxiang Medical University.2.To identify clinically derived CVA2 strains and analyze its virulence and effects on innate immune response in vitro.the prevalence of CVA2 virus strains in Xinxiang area,so as to provide basis for understanding the changes of pathogenic spectrum in this area.3.To establish a neonatal mouse model for CVA2 infection,which will be useful for studying the pathogenesis of CVA2 infection.Method1.From May 2017 to August 2017,19 stool specimens of HFMD cases were collected from the First Affiliated Hospital of Xinxiang Medical College.Viruses that might exist in the samples were isolated and amplified by RD cells.Then,viral RNA was extracted infected cells.The types of enterovirus were identified by RT-PCR and sequencing.Evolutionary trees were constructed for 3 strains of CVA2 strains.2.RD cells were infected by CVA2(11-1)with MOI of 0.1,the expression of CVA2 mRNA was determined at 0,4,8,16,24,48 hours post infection.,RD cells were infected by CVA2(11-1)with MOI of 0,0.1,0.25,0.5,1 and 2 and at 24 hours post infection,the expression levels of cytokines including IFN-β1,IL-1β,IL-6 and apoptotic genes including Caspase-8,Caspase-9 and cyto C were detected by qPCR.Western blot was used to determine the expression levels of RIG-I,MAVS,IRAK1,TRAF6,pTBK1,TBK1,Caspase-3 and Cleaved Caspase-3.3.5-day-old BALB/c suckling mice was infected by a CVA2(11-1)strain with intracranial inoculation and the survival of infected mice was recorded.At 7 days post infection,brain,lung,heart,spleen,skeletal muscle,small intestine,spleen,liver and kidney tissues of infected mice were taken out,and hematoxylin-Eosin(H&E)staining was performed to observe the histopathological changes.Result1.The sequencing results of pathogens from 19 HFMD cases showed that the percentage of other enterovirus,EV71,CVA16 and CVA2 was 36.84%,10.53%,36.84%,15.79%,respectively.RT-PCR and sequencing of 19 samples showed that the results of PCR electrophoresis of 7,11 and 12 samples were inconsistent with those of sequencing.Further sequencing revealed that 7,11 and 12 samples were CVA2 type.2 After CVA2 infection,the viral titter of RD cells was increased.Additionally,the expression levels of inflammatory cytokines including IL-1β and IL-6 mRNA in RD cells were significantly increased(P < 0.05),while the expression level of antiviral gene IFN-β1 mRNA was significantly decreased(P < 0.05).The results of apoptosis-related genes showed that the expression levels of Caspase-8 and Caspase-9 in RD cells were significantly increased after CVA2 infection(P < 0.05),while the expression level of Cyto C mRNA was first increased and then was decreased.The results of innate immune signal detection showed that the expression levels of RIG-I,MAVS and pTBK1 in RD cells were increased after CVA2 infection,while the expression levels of IRAK1 and TRAF6 were decreased.The expression level of the apoptotic protein Caspase-3 was decreased,and the expression level of Cleaved Caspase-3 was clearly increased.3.Significant central nervous system symptoms appeared in mice with CVA2 infection.HE staining showed neuronal degeneration and inflammatory cell infiltration in the brain tissue.The lung tissue was congested and swollen.The alveolar space was enlarged,the interstitial lung was thickened,and a large number of red blood cells and inflammatory cells were infiltrated.Myocardial tissue manifests as a change in myocarditis,manifested by right atrial dilatation,thickening of the atrioventricular wall,and inflammatory cell infiltration.Skeletal muscles were clearly dissolved and necrotic with inflammatory cell infiltration.The small intestine villi were broken and appear irregularly arranged.Inflammatory cell infiltration occured in the kidney and liver.Conclusion1.CVA2 was identified in the pathogenic spectrum of 19 patients with HFMD.2.The CVA2 strain isolated in this study showed a strong virulence in vitro and can affect the innate immune signal transduction..3.CVA2 can successfully infect 5 day old BALB/c suckling mice with neurological and pulmonary lesions.