Establishment Of An EV71 Type Hand-foot-mouth Disease Animal Model Using C57 Suckling Mice

BackgroundHand,foot and mouth disease(HFMD)is a common acute intestinal infectious disease in children.Infants under five are generally susceptible.Most of the children had mild symptoms,with hand,foot,mouth,and hip rash as the main clinical manifestations,and the prognosis was good.However,some children with severe hand-foot-mouth disease may be accompanied by CNS damage and serious complications such as neurogenic pulmonary edema.Enterovirus Type 71(EV71)and Coxsackievirus 16(CA16)are the major pathogens of HFMD,and EV71 infection is the culprit of severe HFMD.Although EV71 virus vaccine has been widely vaccinated across the country since 2015,the number of severe HFMD cases is still very large every year.The construction of EV71 HFMD animal model is of significance for the development of vaccines and the mechanism of EV71 virus infection induced severe disease.In recent years,more and more attention has been paid to the study of EV71 virus at home and abroad.The construction of EV71-infected animal models has also been actively explored.At present,EV71 virus is inoculated in 1 to 7-day-old suckling mice.However,most of the viruses used in these studies are mouse-adapted strains.Mutations occur during the passage of the virus in mice.Although the infection may be increased in mice,the transmission route,pathogenic mechanism,and pathological damage are inevitably changed.Therefore,the direct construction of animal models using clinically isolated EV71 virus can better reflect the true estrus of the human body.In this study,intracranial inoculation of EV71 virus was used to infect C57 neonatal mice.The survival and clinical scores of the neonatal mice were recorded.The pathological changes of different organs after infection were observed by multiple staining.Immunohistochemistry and immunofluorescence were used to detectthe tissue.Expression of EV71 Antigen and Inflammatory Cytokines Objective1.The use of clinically isolated EV71 virus to construct a neonatal C57 mouse model and simulate severe complications such as encephalitis and pulmonary edema in mice.2.To detect pathological features and inflammatory reactions of mouse tissues and organs infected with EV71 virus.Methods1.The EV71 strain was isolated from feces of a severe hand,foot and mouth disease patient with central nervous system infection in Zhengzhou Children’s Hospital and purified and enriched in Vero and RD cells.2.One-day-old C57 mice were infected by intracranial injection and the control group was injected with the same dose of saline.Mouse body weight changes,survival,and clinical scores were observed and recorded.3.On day 7 after the mice were infected with the virus,the brain,spinal cord,skeletal muscle,heart,lung,small intestine,spleen and skin of the mice were anesthetized and examined by HE staining to observe the pathological changes of various parts of tissues;immunohistochemical detection of distribution of viral antigens in brain and muscle was carried out;Transmission electron microscopy was used to observe the destruction of brain tissue in ultrastructure;the damage of nerve cells in brain and spinal cord was observed by LFB staining;Masson staining was performed to observe pulmonary edema;Expression of iNOS in brain,spinal cord and skeletal muscle was evaluated.Results1.EV71 virus infection causes CPE in Vero and RD cells.2.In the infected mice,activity decreased and apathy began 3 days after exposure.Subsequently,the symptoms gradually worsened,and symptoms such as ataxia,lethargy,convulsions,and hind limb paralysis began to appear,and some mice died.3.HE staining revealed that there were serious injuries and inflammatory reaction in multiple organ tissues of mice at the late stage of infection: "sleeves" changes in brain and spinal cord tissues;skeletal muscle fibers were broken,inflammatory cells infiltrated;inflammatory cells appeared in heart tissue Infiltration;inflammatory cell infiltration in the lungs and red blood cell leakage;visible small villi in the villi,concomitant with erosion;spleen visible congestion;skin hair follicle atrophy,reduced number.4.Immunohistochemical staining indicated that there were a large number of EV71 virus antigens in brain and skeletal muscle.Transmission electron microscopy revealed that the neurons in the brain tissue of mice were obviously shrinking and the cytoplasm was vacuolized.LFB staining revealed significant structural damage in the brain and spinal cord of mice,and the number of neurons was also significantly reduced.Masson staining showed that there was mucus in the lung;the expression of IL-6 and iNOS in the brain tissue,spinal cord tissue,and muscle tissue was increased after immunofluorescence staining.Conclusion1.The use of clinically isolated EV71 virus to infect 1-day-old C57 mice is feasible and can successfully simulate central nervous system damage and pulmonary edema.2.EV71 virus has neurological and musculoskeletal properties and can cause destruction of nerve cells and muscle cells.Skeletal muscle is the main site of virus replication.3.The elevated levels of interleukin 6(IL-6)and inducible nitric oxide synthase(iNOS)are associated with EV71 infection.