Replication and gene expression of mutant hepatitis B virus in a transgenic mouse containing the complete viral genome with mutant s gene

Abstract

Aim: To establish the transgenic mouse line harbouring complete hepatitis B virus (HBV) genome with mutant s gene (adr subtype).

Methods: Transgenic mice were generated by microinjecting HBV genome into fertilized eggs. Integration, expression, replication of HBV gene and histological changes in transgenic mice were estimated by genomic DNA PCR, serum DNA PCR, Southern blot, ELISA, HE staining, immunohistochemistry and transmission electron microscopy. Transgenic mice with HBsAg positive in serum were bred and analyzed.

Results: A total of 288 eggs survived from microinjections were transplanted into the oviducts of 13 pseudopregnant mice and 49 pups were produced. Twenty-six mice were identified to have the integrated HBV gene. Serum HBsAg and HBeAg were detected in 2 of 43 mice. HBsAg and HBcAg in cytoplasm or nuclei of hepatocytes were detected in 10 mice. Founders with HBsAg in serum were named lineages G145R-15 and G145R-18. Of the 16 F1 offsprings generated by G145R-15 founder, 12 were positive for HBV genome with PCR, 10 were positive for HBsAg and HBcAg with immunohistochemistry and 7 were positive for HBsAg and HBeAg with ELISA. Only 1 of 8 F1 offsprings generated by G145R-18 founder was survived and it was detected positive for HBV genome, HBsAg, HBcAg and HBeAg. Both of the two lineages had some pathological characteristics of mild chronic hepatitis B in the liver, such as swelling of hepatocytes and focal hepatocellular necrosis and parenchymal lymphomononuclear cell infiltrate.

Conclusion: Transgenic mice harbouring HBV with mutant s gene can be generated. The HBV genes are integrated in the transgenic mice genome and can be expressed, replicated, packaged and excreted. HBV DNA can be stably transmitted in the transgenic mice.

Figure 1

Southern blot analysis of 20 μg of total DNA ex-tracted from transgenic mouse tail tissues of lineages G145R-15 and G145R-18. Lane 0: Positive control; Lane 1: Lineage G145R-15; Lane 2: Lineage G145R-15; Lane 3: F₁ transgenic mice of lineage G145R-15; Lane 4: F₁ transgenic mice of lineage G145R-18; Lane 5: Lineage of transgenic mice harbouring pro-totype HBV genome; Lane 6: Non-transgenic mice as negative control. All DNA samples were RNase treated before gel electrophoresis. The filters were hybridized with a digoxigeninlabeled HBV specific DNA probe.

Figure 2

Immunohistochemical analysis of HBsAg and HBcAg in the liver. A: Presence of cytoplasmic HBsAg in almost all of hepatocytes in immunohistochemical analysis of the liver of an 6-week-old male mouse. Magnification, × 100. B: Cytoplasmic HBcAg in centrilobular region hepatocytes surrounding the central veins. Magnification, × 200. C: Nuclear HBcAg scattering widely throughout the hepatic lobule. Magnification, × 100.

Figure 3

Ultrastructural analysis of HBcAg in the liver.

Figure 4

Analysis of pathological changes in the liver of transgenic mice by HE staining. Magnification × 100. A: Mild swollen hepatocytes around the central veins (CV) in the centrilobular region. B: “Ballooning degeneration” around the central veins or between the two central veins in the liver. C: Predominantly lymphocytic cell infiltrates in portal tract area and hepatic lobules. D: Focal necrosis and predominantly lymphocytic cell infiltrates in hepatic lobule. E: Liver of normal mice.