Establishment Of A Mouse Model With Hepatitis B Virus Infection

Persistent infection with hepatitis B virus (HBV) is a major worldwide health problem and chronically infected individuals are at high risk for cirrhosis and hepatocellular carcinoma. Despite the availability of an HBV vaccine, there are still more than 350 million chronically infected people worldwide, approximately 5% of the world population. Hepatitis B virus has narrow host. In natural condition, HBV could not infect small animals, which are commonly used in medical scientific research. The lack of suitable in vitro infection systems and convenient small animal models have greatly hampered the progress of HBV research.We adapted hydrodynamic-base procedure and/or phage integrase system for the establishment of simple mouse models for HBV infection and showed its strength in the assessment of anti-viral drugs.1. Construction and identification of eukaryotic expression plasmid1.3-fold overlength genome of HBV derived from pcDNA3.1-HBV1.3, was cloned into the eukaryotic expression vectors. We constructed the recombinant plasmids pCI-neo-AerApoEAATP-HBV1.3 and pCI-neo-CMV-HBV1.3.2. Acute hepatitis B virus infection mouse modelsBalb/c mice (immunocompetent mice) and Balb/c nude mice(immunodeficient mice) were hydrodynamically injected with a plasmid having 1.3-fold overlength of HBV DNA .We analyzed HBV replication and expression in vivo.HBV mRNA was detected in mouse liver tissue. Expression and secretion of virus proteins were estimated by immunohistochemistry and ELISA . With ELISA, it was suggested that HBsAg was secreted into serum in 100% mice on day 3. The HBV-DNA copy number in murine sera was detected by real-time PCR. The levels of HBV DNA were as high as 10~7 copies/ml after the injection on 3 day and gradually decreased to 10~3 copies/ml over 30 days in Balb/c mice and 45 days in Balb/c nude mice.Hepatitis B virus gene expression was transient both in Balb/c mice and Balb/c nude mice by hydrodynamic transfection. Then we considered to construct a long-term expression mouse model by hydrodynamic transfection coupling with phage integrase system.3. A long-term HBV expression mouse modelΦC31 phage integrase can recognize both the site attP of the bacteriophage and the site attB of host genome, which in fact are homologous sequences.ΦC31 integrase mediated site-specific recombination between the attP and attB. Recent studies showed that two new homologous sequences (designated as site mpsL1 and mpsL2) in the chromosome of mouse liver cells were identified, both of which are phage integrase recognizable site. So the phage integrase mediated exogenous DNA into the mouse chromosome by inserting the specific sequence site attB.In the present study, a HBV long-term expression mouse model was established by hydrodynamic transfection coupling with phage integrase system. pCI-neo- attB -AerApoEAATP-HBV1.3 or pCI-neo- attB -CMV-HBV1.3 were co-transfected with pCMV-Int into the Balb/c mouse liver cells respectively. The level and time of HBV expression in mouse were observed.HBcAg was detected in mouse liver tissue on day 45. HBsAg was secreted into serum and persistently expressed over 30 days. The HBV-DNA copy number in murine sera was detected by real-time PCR. The levels of HBV DNA were as high as 10~7 copies/ml after the injection and gradually decreased to 10~3 copies/ml over 100 days in Balb/c mice.We specifically detected intergration site in mouse liver by a nest-PCR. The cccDNA (covalently closed circular DNA ,cccDNA) was detected by PCR. The current study indicated that integrase mediated efficient integration of HBV into the host genome.We assessed this model with anti-viral agents (lamivudine and entecavir),the result showed that drugs against HBV could inhibit viral gene expression and replication in mouse.In summary , we established a HBV long-term expression mouse model. This model would contribute to the research of HBV and the evaluation of anti-viral drugs in vivo.