The Hepatitis B Virus Genotype Affects the Persistence of Viral Replication in Immunodeficient NOG Mice

Background & AimsAt least eight genotypes of Hepatitis B virus (HBV) have been identified. HBV genotype C is the most common genotype in Japan, although the incidence of HBV genotype A is increasing. The reason underlying the differences in viral multiplication of the HBV genotypes is unclear, especially in vivo. The purpose of this study was to elucidate the differences in HBV load and the persistence of viremia in vivo between genotypes A and C.MethodsImmunodeficient NOG mice were transfected by hydrodynamic injection with the HBV expression plasmids pHBA1.2 or pHBC1.2, which contain overlength (1.2-mer) copies of the genomes of HBV genotype A or C, respectively.ResultsOne day after transfection, the number of HBcAg-positive hepatocytes and serum HBV DNA levels were similar between mice transfected with pHBA1.2 and pHBC1.2. Serum levels of HBV DNA, HBsAg and HBeAg in mice transfected with pHBA1.2 were maintained over 5 months. In contrast, those in mice with pHBC1.2 gradually decreased over time and reached undetectable levels within 3 months after transfection. HBcAg-stained hepatocytes were detected in mice transfected with pHBA1.2, but not pHBC1.2, 5 months post-transfection. Double-staining immunohistochemistry revealed that the number of cleaved caspase3-stained, HBcAg-positive hepatocytes in the pHBC1.2-transfected mice was higher than in the pHBA1.2-transfected mice 3 days post-transfection. Moreover, the plasmid DNA and covalently closed circular DNA levels were decreased in the livers of pHBC1.2-transfected mice. These results suggested that hepatocytes expressing HBV genotype C were eliminated by apoptosis in the absence of immune cells more often than in hepatocytes expressing HBV genotype A.ConclusionsImmunodeficient mice transfected with HBV genotype A develop persistent viremia, whereas those transfected with HBV genotype C exhibit transient viremia accompanied by apoptosis of HBV-expressing hepatocytes. This differences may affect the clinical courses of patients infected with HBV genotypes A and C.

研究背景与目的：目前已鉴定出至少8种乙型肝炎病毒（HBV）基因型。基因型C是日本最常见的HBV基因型，不过基因型A的感染发生率正逐步升高。HBV不同基因型的病毒增殖差异背后的机制尚不明确，尤其在活体环境中。本研究旨在阐明基因型A与基因型C型HBV在活体中的病毒载量差异及病毒血症持续情况的差异。

实验方法：将分别携带HBV基因型A、C型1.2倍全长基因组的HBV表达质粒pHBA1.2与pHBC1.2，通过水动力转染法导入免疫缺陷NOG小鼠体内。

实验结果：转染后1天，pHBA1.2转染组与pHBC1.2转染组小鼠的乙型肝炎核心抗原（HBcAg）阳性肝细胞数量及血清HBV DNA水平均无显著差异。pHBA1.2转染组小鼠的血清HBV DNA、乙型肝炎表面抗原（HBsAg）及乙型肝炎e抗原（HBeAg）水平可维持5个月以上。与之相反，pHBC1.2转染组小鼠的上述血清标志物水平随时间逐渐下降，并于转染后3个月内降至检测下限以下。转染后5个月，仅在pHBA1.2转染组小鼠中可检测到HBcAg阳性肝细胞，pHBC1.2转染组未检出。免疫组织化学双染色结果显示，转染后3天，pHBC1.2转染组小鼠中裂解型半胱氨酸天冬氨酸蛋白酶3（cleaved caspase3）阳性且HBcAg阳性的肝细胞数量显著高于pHBA1.2转染组。此外，pHBC1.2转染组小鼠肝脏内的质粒DNA及共价闭合环状DNA（cccDNA）水平均有所降低。上述结果表明，在无免疫细胞的条件下，表达基因型C型HBV的肝细胞相较于表达基因型A型HBV的肝细胞更易通过凋亡途径被清除。

研究结论：免疫缺陷小鼠转染基因型A型HBV后可出现持续性病毒血症，而转染基因型C型HBV的小鼠则表现为一过性病毒血症，并伴随表达HBV的肝细胞凋亡。该差异可能会影响感染不同基因型HBV患者的临床病程。