The Application Of STAT1 Knockout Mouse Model In EV71 Infection Research And The Establishment And Application Of Woodchuck Hepatitis Virus Persistent Infection Model

Objective To analysis the Sensitivity of STAT1 knockout mice after EV71 infection.Provide the database for EV71 animal model optimization.Methods Use cytometric bead assay(CBA)to quantitative the secretion level of serum immune factors after LPS stimμLation;Observe the symptoms and pathology,and use Real-time PCR to quantitative the virus RNA level of muscle after EV71 infection.Results A variety of immune-related cytokines secretion levels decreased from STAT1 knockout mice after LPS stimμLation;In STAT1 knockout mice,the pathological damage increased and high-level virus load in muscle are observed,even show higher mortality and more severe symptoms after EV71 infection.Conclusions STAT1 plays an important role in the immune process and suggesting that STAT1 knockout can increase the sensitivity of mice to EV71 infection.Objective The purpose of this study was to establish a woodchuck hepatitis B virus(WHV)persistent infection animal model with 3-days-old woodchuck,and to evaluate the immunomodulatory effect of Ganoderma lucidum spore powder drugs,thus provide an animal model for the research on the chronic hepatitis virus infection and the evaluation of anti-hepatitis virus drugs.Method WHV DNA detection:primers were designed according to the coding sequence of woodchuck hepatitis virus surface antigen(WHsAg).The primers demonstrated high sensitivity and specificity were used for Real-time PCR detection of WHV DNA in woodchuck serum.Then,the method was applied for detection of WHV DNA in the serum of woodchuck.Using the polyclonal antibody which prepared and purified by the laboratory in rabbits and eggs,to detect the WHsAg and the woodchuck core antibody(WHcAb).The Trizol method was used to extract the liver mRNA of woodchuck,then,reverse transcribed into cDNA and detected by Real-time PCR method.Result The detection sensitivity of Primers WHV N3 F and WHV N3 R in PCR nearly 1×104 copies/mL,and no obvious nonspecific bands and primer dimers in electrophoresis.The R2 value of the standard curve of viral copy number and the Real-time PCR Ct-value was 0.992,and the melting curves of WHV DNA genome plasmids were consistent.3 month after infection,the level of viral load can be detected and to achieve the peak of the serum viral load,and maintain at 104copies/ml until 8 months later.The serum WHV load in female woodchuck is higher than in the male.Under the experimental conditions,we verify that the mixed drug of Ganoderma lucidum spore powder and Ganoderma lucidum spore oil had an effect on the serological indicators and the pathology diagnosis.The mRNA which coding CDS in the administration group was higher than that in the control group,and it was about 8 times higher than the blank group.Conclusions In this study,we established the animal model of woodchuck which have a persistent infection of the woodchuck hepatitis virus.After analyzed the viral load,the serological indicators,the liver function index and the pathology,we provided the database for the establishment of the woodchuck hepatitis model and the cure of hepatitis B virus.Then we used this model to evaluate the effect of the mixed drug of Ganoderma lucidum spore powder and Ganoderma lucidum spore oil in antivirus function and the mRNA level,and find Ganoderma lucidum spore powder and Ganoderma lucidum spore oil can make positive affection on woodchuck hepatitis.We also found that Ganoderma lucidum may increasing the number of cytotoxic T cells in animal models.