Establishment Of Neonatal Mouse Models For Enterovirus A71 And Virus Replication Analysis

Enterovirus 71(EV-A71)was one of the important pathogens causing HFMD through fecal-oral route,respiratory droplet or direct contact.Hand-foot-mouth disease is a common acute infectious disease,mostly in infants under 5 years of age,the main symptoms include fever,rash or mucous herpes.A small number of patients may be complicated with aseptic meningitis,brainstem encephalitis,acute delayed paralysis and myocarditis,causing serious neurological complications and even death,which has become an important public health problem seriously threatening the health and life safety of children.Compared with other enteroviruses,EV-A71 can breach the bloodbrain barrier and infect the central nervous system,causing more severe neurological symptoms.Therefore,to explore the pathogenesis of EV-A71 has been a hot and difficult research topic,and the establishment of an appropriate animal infection model is an important means to study EV-A71.Therefore,the EV-A71 infection model of five commonly used strains of mice was established in this study,and the susceptibility of different strains of mice to EV-A71 was comprehensively evaluated,so as to select the most susceptible strain of mice to EV-A71.By detecting the viral load in different tissues and organs at different time points after EV-A71 infection,the temporal and spatial distribution of EV-A71 in the whole body tissues and organs were analyzed,and the pathological injuries in various tissues and organs were evaluated.The effects of EV-A71 infection on the expression of SCARB2 receptor in different tissues and organs and the role of SCARB2 in EV-A71 infection were further discussed.Five commonly used lines,including BALB/c,C57BL/6,ICR,Kunming and NIH,were selected.One-day-old lactating mice were injected intraperitoneally and infected with low and high doses of EV-A71 virus,respectively.Body weight,survival data and the number of paralyzed mice were observed and recorded daily.Compared with the control group,it was found that the mice infected with low-dose EV-A71 showed a slight decrease in body weight and symptoms of limb weakness,tremor and paralysis,among which the survival rates of BALB/c and C57BL/6 mice were 72.73% and 73.34%,respectively,while the survival rates of ICR,Kunming and NIH mice were 100%.In the high-dose infection group,the weight loss was more significant,both hind limb paralysis and the number of paralysis increased significantly.The survival rate of all strains of mice further decreased,and the survival rate of BALB/c and C57BL/6 mice was only 63.64% and 50%.The survival rates of ICR,Kunming and NIH mice were85%,90% and 92%,respectively.These results suggest that newly born ICR,Kunming,and NIH mice are not susceptible to EV-A71,while BALB/c and C57BL/6 mice are more susceptible to low and high doses of EV-A71,which can not only cause similar clinical symptoms of EV-A71 infection,but also cause a severe neuroinfection phenotype.It provides a good infection model for studying EV-A71.In order to study the temporal and spatial distribution characteristics of EV-A71 infection,one-day-old BALB/c and C57BL/6 neonatal mice were infected with low doses of EV-A71 at 1,3,5,7 and 13 days after infection.The expression of EV-A71 viral load was detected in brain,skeletal muscle,lung,spinal cord,intestine,liver,spleen,heart,kidney and stomach tissues.Viral RNA was detected in all tissues on day1 after infection,and viral load in tissues of the two strains increased significantly and reached its maximum value on day 3-5 after infection.However,the viral load began to decrease with the extension of infection time until the virus was detected only in the spinal cord of BALB/c mice and skeletal muscle of C57BL/6 mice on day 13 after infection.Of all the tissues tested in BALB/c and C57BL/6 mice,viral load was highest in skeletal muscle and lowest in intestinal tract.We also found that the viral load in tissues of C57BL/6 mice was generally higher than that of BALB/c mice.These results indicate that EV-A71 has a strong tissue tropism to the nervous system and skeletal muscle,and the sensitivity of different strains of mice to EV-A71 is significantly different,with C57BL/6 mice more susceptible to EV-71.Since the viral load was the highest in all tissues on the 5th day after infection,we further analyzed the pathological damage of each tissue on the 5th day after infection.A large amount of VP1 protein was detected by immunofluorescence staining in brain and lung tissues.HE staining showed neuronal degeneration and extensive proliferation of glial cells in brain tissue.Moderate myelitis with gray matter necrosis was found in the spinal cord.Alveolar enlargement,mucus production and the filling of the alveolar cavity with homogeneous red serous fluid in the lung tissue;The skeletal muscle showed mild myositis and muscle fiber disorder.Local tissue erosion of intestine and stomach,partial cell edema and necrosis;Myocardial cell necrosis and inflammatory infiltration;Lymphocyte edema and necrosis are seen in the spleen.Hepatocyte steatosis;There was a small amount of inflammatory cell infiltration in renal interstitium.LFB staining showed more neuron damage in brain tissue.Masson staining showed that the content of collagen fibers in lung tissue increased significantly.These results suggest that EV-A71 can infect multiple tissue types and cause pathological damage,as well as invade the central nervous system and disrupt brain structures and cell biological functions.SCARB2 is the only cell surface receptor that has been shown to mediate EV-A71 attachment,internalization,and unhulling.The expression of SCARB2 in brain,skeletal muscle,lung,spinal cord,intestine,liver,spleen,heart,kidney and stomach tissues of newborn rats on the 5th day was detected.It was found that the expression of SCARB2 was different in different tissues,with higher expression in brain,lung,spinal cord,intestine,liver and stomach,but lower expression in skeletal muscle,spleen,heart and kidney.After EV-A71 infection,SCARB2 expression was generally increased in all tissues,with significant differences in brain,skeletal muscle,lung,spinal cord,spleen,heart,kidney and stomach.In addition,WB results further verified that EV-A71 infection can cause increased expression of SCARB2 protein.These results suggest that EV-A71 may further enhance the infective ability of the virus itself by promoting the expression of SCARB2.In summary,EV-A71 infection models have been established for BALB/c,C57BL/6,ICR,Kunming and NIH mice,and it is found that BALB/c and C57BL/6 mice have higher susceptibility to EV-A71.On the 3-5 days after infection,the viral load in all tissues reached the highest value,and the viral load in skeletal muscle was the highest,indicating that EV-A71 had strong skeletal muscle tropism.Although EV-A71 loads were not the highest in the brain,both HE and LFB staining indicated that EV-A71 could invade the central nervous system and cause nerve cell damage.It was also found that EV-A71 infection affected the expression of SCARB2 receptor,and the increased expression of SCARB2 receptor may promote the effective infection of EV-A71.These results comprehensively evaluated the commonly used EV-A71-infected mouse model and established a new EV-A71-infected mouse model,which can be used as a more effective means for the study of virus infection mechanism,vaccine evaluation and drug screening.