Yokoyama Yoshinobu, Miyagi Takuya, Hikita Hayato, Yoshioka Teppei, Mukai Kaori, Nawa Takatoshi, Sakamori Ryotaro, Ohkawa Kazuyoshi, Hiramatsu Naoki, Takahashi Takeshi, Suemizu Hiroshi, Ryo Akihide, Tatsumi Tomohide, Takehara Tetsuo
Department of Gastroenterology and Hepatology, Osaka University Graduate School of Medicine, Suita, Osaka, Japan.
Laboratory Animal Research Department, Central Institute for Experimental Animals, Kawasaki, Kanagawa, Japan.
PLoS One. 2015 Dec 14;10(12):e0144775. doi: 10.1371/journal.pone.0144775. eCollection 2015.
BACKGROUND & AIMS: At 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.
Immunodeficient 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.
One 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.
Immunodeficient 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.
已鉴定出至少八种乙型肝炎病毒（HBV）基因型。HBV基因型C是日本最常见的基因型，不过HBV基因型A的发病率正在上升。HBV各基因型病毒增殖差异的潜在原因尚不清楚，尤其是在体内。本研究的目的是阐明基因型A和C在体内HBV载量及病毒血症持续性方面的差异。
通过流体动力学注射，将分别含有HBV基因型A或C基因组超长（1.2倍体）拷贝的HBV表达质粒pHBA1.2或pHBC1.2转染至免疫缺陷的NOG小鼠。
转染后一天，用pHBA1.2和pHBC1.2转染的小鼠之间，HBcAg阳性肝细胞数量和血清HBV DNA水平相似。用pHBA1.2转染的小鼠血清中HBV DNA、HBsAg和HBeAg水平在5个月内保持稳定。相比之下，用pHBC1.2转染的小鼠血清中这些指标随时间逐渐下降，并在转染后3个月内降至检测不到的水平。转染后5个月，在pHBA1.2转染的小鼠中检测到HBcAg染色的肝细胞，而在pHBC1.2转染的小鼠中未检测到。双重免疫组化染色显示，转染后3天，pHBC1.2转染小鼠中裂解的caspase3染色且HBcAg阳性的肝细胞数量高于pHBA1.2转染的小鼠。此外，pHBC1.2转染小鼠肝脏中的质粒DNA和共价闭合环状DNA水平降低。这些结果表明，在没有免疫细胞的情况下，表达HBV基因型C的肝细胞比表达HBV基因型A的肝细胞更常通过凋亡被清除。
用HBV基因型A转染的免疫缺陷小鼠会出现持续性病毒血症，而用HBV基因型C转染的小鼠则表现为短暂性病毒血症，并伴有表达HBV的肝细胞凋亡。这种差异可能会影响感染HBV基因型A和C的患者的临床病程。
