Establishment Of Enterovirus 71(ev71)ICR Neonatal Mice Model

HFMD is an acute infectious disease caused by a variety of small RNA enteroviruses.In most cases,it occurs in children under the age of three.It’s mainly characterized by mild flu-like symptoms and rashes and herpes in hand,foot,mouth and other parts of the body.Usually the disease is self-limiting.But it is highly contagious,usually being popular in the kindergartens and nursery schools.Generally the prognosis is fine,but individual critically ill patients will have serious neurological complications and even die.The virus which causes HFMD belongs to virus RNA family enterovirus genus.It mainly includes: Coxsackie virus group A type 4,5,7,9,10,16;Coxsackie virus group B type 2,5;Enterovirus type 71(EV71)and echovirus,of which CA16 and EV71 are the major pathogens.Since 1969 when EV71 virus was isolated for the first time and a child with HFMD suffered from meningitis in the California United States,HFMD has had scores of outbreaks around the world,especially in the Asia-Pacific region.From 2008 to 2012,there have been 7,200,092 cases of HFMD and 2,457 deaths due to HFMD in the Mainland of China.In 2008,387 cases of severe cases caused by EV71 were reported in Taiwan,of which 14 died finally.At present,except for poliovirus vaccination,the remaining enterovirus infections include EV71 virus infection which has no special treatment.One of the most important reasons is the lack of a stable animal model to effectively assess the vaccine,so the establishment of the stable and effective animal model is very important.Currently,the most commonly-used models are the simian model and the suckling mouse model.Although the simian model is considered to be a good animal model at an early stage,it is often expensive and lack the performance of heart failure and pulmonary edema,which are the important causes of the death of human with HFMD.Its application is limited.With its low price,simple manufacture and symptom of nervous system infection similar to that of human disease,it is suitable for understanding the pathogenesis of EV71 and evaluating the vaccine titer.At present,Chinese HFMD’s outbreak of the popular virus subtype strain mainly belongsto the C4 subtype.In the previous study,most animal models are not sensitive to C4 subtype of EV71 virus strains,leading to no typical symptoms of HFMD after infection,especially the symptom of nervous system infection.In this research,four strains of EV71 clinical isolated strains from Jiangsu Provincial Center for Disease Control and Prevention were used in the poison attack experiment in mice.Finally a clinical strain is chosen to induce the hind limb paralysis of mice.Then a stable G03 EV71 virus strain was established by cell culture,passage and cloning.The measurement and analysis of the whole genome sequence of the strain showed that the strain was the EV71 strain of C4 subtype.The researcher used the 1-day-old ICR mice infected by the isolated G03 EV71 virus strain through intracranial routes to make animal models.Then the clinical manifestations of the animals were observed daily to understand the regularity and pathogenesis of EV71 infection.At the same time,regular euthanizing animals and various organs and tissues are used to carry out semi-quantitative analysis of the virus in the animal.In addition,the research carries out pathological observation and immunologic tissue chemical analysis and the Western blot were used to identify the expression of viral protein.Pathological observation found that after infection,the myocardium,lung tissue,skeletal muscle,small intestine,brain tissue and other organs of mice will have specific pathological changes,such as bleeding,inflammatory cell infiltration and so on.Immunohistochemistry showed that the five tissues of the mice infected all had EV71 virus-specific distribution.Western blot analysis showed that EV71 VP1 protein was expressed in all five tissues.The results above all showed that the EV71 infected mice model was established successfully,which provided a practical animal evaluation tool for the relationship between virus and host,the virus vaccine development and other researches.At the same time,it has laid a good foundation for the following intervention study in the body.To sum up,this research has selected appropriate clinical virus strains through the clinical sensitive strain experiment for screening EV71.Then,it has cultured them.At the same time,the suckling mouse model of one-day-old is established by usingthis virus strain and corresponding test methods,which has provided research tools for the EV71 pathogenesis,the development of virus vaccine and other researches.