ICR Mice Infected With Enterovirus71of Different Virulence Phenotype

Enterovirus71(EV71), a small RNA viruses in the family of Picornaviridae, can cause hand, foot and mouth disease (HFMD). EV71is one of the most common pathogens that lead to HFMD. It usually cause rash and herpes angina, and the prognosis is well. Occasionally EV71lead to diseases of central nervous system such as aseptic meningitis, brain stem encephalitis, poliomyelitis-like paralysis, with high morbidity and mortality mainly in infants and young children under5years old.EV71’s popularity in Asia-pacific countries and regions is on the rise in recent years. However, no drugs or vaccines of specific for the prevention and treatment of EV71infection are commercially available at present, and the pathogenic mechanism is also not clear. A suitable animal model is an important means for studying the pathogenic mechanism, the characteristics of EV71infection and evaluation of virus vaccines and drugs, but we still have no suitable animal models. Therefore, for the lack of animal model research with virus EV71and the needs of molecular mechanism research, we use the SDLY107and SDLY11isolated from patients with HFMD in Linyi, Shandong, China in this experiment, so as to establish a different virulence phenotype isolates of EV71infection animal model, to explore the reason of different virulence EV71phenotypic isolates cause the different clinical manifestations.SDLY107and SDLY were used to infect ICR mice of1day-old and two weeks-old through the intraperitoneal infection,in the meantime, we do the research of the clinical manifestation, etiology, molecular biology and pathology of the infected mice. The establishment of the different virulence EV71phenotype isolates infection mouse model has a great significance for the further research of pathogenesis mechanism related to virulence.Objective1. To make a comparison of the infection characteristics of the ICR mice infected with the different virulence isolated strains to provide a basis for further study of different symptoms caused by EV71infection.2. To develop the Real-time fluorescent quantitative RT-PCR technique with SYBR Green I and to detect the viral load of EV71RNA copies of muscle tissue for evaluating the situation of the viral multiplication in mice.3. To use ELISA to detect EV71antibody titer in the1-day-old and2-weeks-old mice everyday and evaluate the elimination of the virus in the host after then, for the further study of the reason of different virulence in mice after infected the SDLY107and SDLY11.Methods1. The ICR mice of1-day-old and2-weeks-old were infected through the intraperitoneal infection with SDLY107(the strain isolated from the dead patient) and SDLY11(the strain isolated from the patient manifested as rash and fever).2. The mice were grouped by age and treatment as following:1-day-old SDLY107strains as experimental group A,1-day-old SDLY107strains experimental group B,1-day-old SDLY11strains experimental group C,1-day-old SDLY11strains experimental group D,1-day-old mock control group E,2-weeks-old SDLY107strains experimental group F,2-weeks-old SDLY107strains experimental group G,2-weeks-old SDLY11strains experimental group H,2-weeks-old SDLY11strains experimental group I and2-weeks-old mock control group J. There were5-10mice in each group. Each mouse was, infected with virus through the intraperitoneal infection, and the mice in control group were injected with normal saline (NS).3. To observe the clinic manifestation after EV71infection and collect the lung, brain, intestine and muscle tissue of1-day-old SDLY107strains of experimental group A,1-day-old SDLY11strains of experimental group C,2-weeks-old SDLY107strains of experimental group F,2-weeks-old SDLY107strains of experimental group H at3d,4d,5d,6d,7d after EV71infection.4. We picked off the eyeballs of the mouse got blood samples of1-day-old SDLY107strains of experimental group B,1-day-old SDLY11strains of experimental group D,2-weeks-old SDLY107strains of experimental group G,2-weeks-old SDLY11strains of experimental group I,1-day-old control group E,2-weeks-old SDLY107strains of experimental group F in two weeks every day, measured the serum antibody titers of mice with ELISA test, and took muscle tissue to detect the RNA copies.5. To establish the method of Real-time fluorescent quantitative RT-PCR technique with SYBR Green I. To prepare the standard curve with the purpose sequence RNA standard, evaluate the specificity, sensitivity, repeatability of this method meanwhile, and make the accurate quantitative of RNA copies of mice tissue.Results1. The1-day-old and2-weeks-old ICR mice are injected SDLY107and SDLY11separately. The mice infected by SDLY107showed obvious symptoms, including piloerection, arched back, loss weight, limb paralysis, while the mice infected by SDLY11had lighter symptoms, there was no significant alteration for the control group (normal saline). We detect the histopathology examination, and found that tissues of both mice infected by SDLY107and SDLY11shows pathological changes.2. To establish the method of Real-time fluorescent quantitative RT-PCR technique with SYBR Green I.The method of high sensitivity and good stability can be used for the quantitative determination of EV71RNA copies. The results showed that the viral load of SDLY107in each group was higher than that of SDLY11, and the highest copies in muscle tissue. The copies of muscle tissue in the1-day-old mice infected with the SDLY107reached the maximum(6.02×104copies/μl)5days postinfection, and the copies of muscle tissues in the2-weeks-old mice infected with the SDLY11reached the maximum (3.27×104copies/μl)7days postinfection.3. The results of ELISA assay showed that the A value of serum antibodies in mice increased as the infected days. The highest A value of1-day-old mice serum antibody appears in7d. The highest A value of the2-week-old mice appears in the8d. The serum antibody levels of2-week-old mice were higher than that of1-day-old mice. The serum antibody levels of the similar days old mice have the same trend.Conclusion1. The strains of different virulence showed different pathogenicity.2. The strains of different virulence showed different replication ability. The different replication ability might be related to the different clinical manifestations that might depend on key amino acid mutations in the virus itself associated with replication of area, and also related to the elimination of the virus by the antibodies produced by the host itself.3. To establish the method of Real-time fluorescent quantitative RT-PCR technique with SYBR Green I, the method of high sensitivity and good stability can be used for the quantitative determination of EV71RNA copies, and it’s the basis for the evaluation of viral load, establishment of the animal model and research of the vaccines and drugs. The replication capacity of EV71may be a main reason for different virulence of EV71.