Replication And Persistence Of Wild Type And Chimeric Genomes Of Woodchuck Hepatitis Virus And Hepatitis B Virus In The Mouse Model Based On Hydrodynamic Injection

Objective1. To replace the homologous sequences in the woodchuck hepatitis virus genome with the coding sequences of HBsAg, respectively. Construct the chimeric plasmids WHV-HBV-Sa. WHV-HBV-SS,WHV-HBV-MS, test the expression level and secretion level of the HBsAg in eukaryotic cellss.2. To establish a viral persistence mice model by hydrodynamic injection these chimeric plasmids into tail vein. To test the immune response and the mechanism of viral persistence in these mouse models.3. To inspect the replication and expression of WHV-HBV-Sa,WHV-HBV-SS, WHV-HBV-MS in the preimmunized mice.Methods1. To construct the chimeric plasmids WHV-HBV-Sa, WHV-HBV-SS and WHV-HBV-MS with molecular cloning technology, transfect them into Huh7and HepG2. The HBsAg in culture supernatant was test.2. By hydrodynamic injection, inject the chimeric plasmids into tail vein of BALB/c mice. At indicated time point after injection, mice sera and liver tissues were collected. The samples were detected in ELISA, real-time PCR, IHC, Southern Blot.Separate the spleen lymphocytes from the WHV-HBV infected mice, stimulate with WHcAg/HBsAg and peptides, detect the IFNg expression of different cell subset (CD4+and CD8+cell populations) by FACS, detect IFN-gamma producing cells by using ELISPOT.3. pcDNA3.1-HBsAg immunized mice chimeric plasmids and pBS-HBV1.3hydrodynamic injection, test the HBsAg, HBsAb in the serum with ELISA, test the virus DNA in the serum with PCR.Results1. Three plasmids named WHV-HBV-Sa,WHV-HBV-SS,WHV-HBV-MS were successfully constructed. Transfection with WHV-HBV-SS, WHV-HBV-MS led to the production of detectable HBsAg in both hepatoma cell lines, though at very different levels. Rather unexpectedly, transfection with WHV-HBV-Sa did not result in the production of chimeric WHsAg detectable in HBsAg immunoassay.2. Serum WHV DNA became weakly positive in2of9mice at day1after HI. Thereafter, all mice were positive for serum WHV DNA at days10and18after HI. WHV DNA remained to be detectable up to45weeks after HI. WHsAg and WHcAg expression were detectable in mouse liver sections by IHC. Serum samples from4mice with WHV-HBV-Sa were highly positive in HBsAg ELISA up to week45. PCR results with serum samples from mice after HI with Sa confirmed the persistence of WHV DNA at low levels in mice. IHC of liver sections with anti-WHc antibody showed that WHcAg was expressed in mouse livers after HI with Sa, similar to the findings in mice received pBS-WHV1.3. The mice received SS and MS showed significantly prolonged antigenemia than that with pBS-HBV1.3and lost HBsAg positivity gradually. Notably, two mice with MS were continuously HBsAg positive at high levels until week36after HI. Similar to the previous experiments, viral DNA at low levels was detected in mouse sera after HI. WHcAg was expressed in all mice received SS and MS. failed to demonstrate the presence of specific T cells to WHcAg and HBsAg in mouse splenocytes after HI from both HBsAg-positive or-negative mice received SS or MS.3. After immunization, HI with WHV-HBV-SS and WHV-HBV-MS resulted in early clearance of serum HBsAg and viral DNA.Conclusions1. Successfully construct WHV-HBV chimeric plasmids:WHV-HBV-Sa WHV-HBV-SS,WHV-HBV-MS.2. WHV genomes had the ability to persist and maintain the gene expression in mouse liver tissue for prolonged time. One major factor allowed the long term persistence of WHV genomes was the lack of immune activation against WHV and HBV proteins after HI in mice.3. Pre-existing immunity against HBsAg could effectively prevent the gene expression and persistence of chimeric WHV genomes after HI in mice.