The Role Of TLR7 In The Process Of EV71 Infection And The Exploration Of The Establishment Of HBV Animal Models Using Mouse And Ferrets

Background Enterovirus 71(EV71)is a positive-strand RNA,neurotropic virus,which was first described in 1969 in the United States,but the pathogenesis was undefined.EV71 is the mianly virus to cause the hand,foot,and mouth disease,which infections are generally mild,but occasionally lead to severe diseases,such as aseptic meningitis,fatal encephalitis in neonates.The first outbreak of EV71 in Malaysia in 1997 killed 41 children,and since then EV71 infection has become endemic in china,Singapore,Australia,Vietnam,Cambodia and Asia-Pacific countries,which caused serious consequences.There is no effective clinical treatment,although there are two types of vaccine have been approval to production,the prevention effect do not experience test of actual combat.Toll-like receptor co?ld lead to the antiviral,and the activity TLR7 co?ld lead to induce inflammatory cytokines,such as type I interferon,and help the host rapidly mount an immune response to infection with an invasive pathogen.MethodsEV71 infection mouse model is established,and oral TLR7 agonist(GS-9620)to comparison the experimental groups of the survival rates and clinical symptoms.The experiment to detect the given medicine the best way and dose,the GS-9620 co?ld obviously improve the survival rates and clinical symptoms,then we use the CBA,real-time PCR,HE staining and immunofluorescence to detect the cytokines/chemokine in the blood,viral load,muscle pathological damage and EV71 antigens distribution expressed in the muscle of the experiment groups.Using the inhibitors or agonist of NF-?B and PI3K to the mice,observed the viral load,the pathological damage and the immune factors expression of the NF-?B and PI3K signaling pathways,which study the mechanism of the NF-?B and PI3K to antiviral.IFN-AR knockdown mice infected with EV71 observed mortality and viral load.Res?lts MP10 is the mouse-adapted EV71 strain,inoc?lated 10-day-old ICR mice by intraperitoneal(i.p.)injection with more than half of fatalityrate dose(100?l 109TCID50/ml),oral the TLR7 agonist(GS-9620)4.5mg/kg in 2,26 and 50 h.i.p.which can obviously improve the survival rates and clinical symptoms.Flow cytometric analysis of blood revealed that GS-9620 treatment of EV71-infected mice ameliorated tissue damage by significantly inhibiting the cytokine storm,inhibited EV71 replication by increasing the levels of IL-1?,IL-5,IL-6,IL-12,IFN-a,IFN-?,IFN-?,TNF-?,MIP-la and MCP-1.When EV71-infected mice were treated with drugs that inhibit or activate NF-?B or PI3K,individually or sim?ltaneously,before GS-9620 treatment,viral replication was restored,and the levels of secretion of certain cytokines and chemokines changed.IFN-AR knockout mice infected with EV71 co?ld promote viral replication and increase the mortality rates.Conclution the TLR7 inhibited EV71 replication and relieved virus-induced tissue damage through increasing the levels of IL-1?,IL-5,IL-6,IL-12,IFN-?,IFNO ?,IFN-y,TNF-a,MIP-la and MCP-1through activation the NF-?B and PI3K-AKT signaling pathways.Background Hepatitis B virus(HBV)infection is a major public health problem in the world.Serological evidence shows that about 30%of the pop?lation in the world now or ago has been infected with HBV,and 240 million people are chronic HBV infection,of which 15%40%is likely to develop into a serious liver disease,such as:cirrhosis and Hepatocell?lar carcinoma.When the body infected with HBV,in the liver cell replication,secretion of virus particles can be up to several decades,to the patient life and family great pain,but also to the community has brought a lot of public health problems.At present,the clinical treatment,there is no effective drug to eradicate HBV infection.In view of the limitations of HBV infection,in addition to infected people,the current study shows that HBV can still infect chimpanzees and tree shrew,but its limitations and limitations of the reasons,as the prospects for HBV animal model is still not optimistic,colleagues can not be carried out Cell c?lture,but also limited its mechanism.So the establishment of a new,easy to breed and experimental operation,gene-specific HBV animal model,imminent.Method To establish the HBV animal model with the serum of HBV in chinchilla and ferrets.Real-time PCR was used to determine the viral copy number with HBV sera using the standard curve set by HBV plasmid.Infected ad?lt chinchillas were injected the HBV with tail vein,subcutaneously,and 3-day-old hairs and 3-day-old ferrets were injected with subcutaneous.Serology was used to detect HBV DNA and hepatitis B(HBsAg,anti-HBs,HBeAg,anti-HBe and anti-HBc)and liver function.HBV DNA was detected in liver tissue,HE pathological examination,immunohistochemical detection of HBsAg antigen.Res?lts Ferrites were observed for 6 months,HBV DNA was not detected in the blood;HBsAb antibody was positive in serum;youth and ad?lt chinchillas had a certain percentage of serum HBV DNA detection every month HBV DNA;H&E slices showed infection with HBV virus,the liver will have a slight inflammatory response;At the same time serum liver biochemical ALT also have some changes.Conclusion The ferrets can not infect HBV,and HBV DNA of the chinchillas can still be detected for 12 months.However,the detection rate of HBV DNA was not high,and wether can develop into liver cirrhosis,liver cancer still need to continue to observe.