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

Date Published: December 14, 2015

Publisher: Public Library of Science

Author(s): Yoshinobu Yokoyama, Takuya Miyagi, Hayato Hikita, Teppei Yoshioka, Kaori Mukai, Takatoshi Nawa, Ryotaro Sakamori, Kazuyoshi Ohkawa, Naoki Hiramatsu, Takeshi Takahashi, Hiroshi Suemizu, Akihide Ryo, Tomohide Tatsumi, Tetsuo Takehara, Tatsuo Kanda.


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.

Partial Text

Hepatitis B virus (HBV) infection is one of the most common viral infections and is a worldwide health problem [1]. At least eight genotypes of HBV have been classified, with the proportion of genotypes varying depending on the region [2]. HBV genotype C was the most common genotype in Japan [3–5], whereas HBV genotype A was rare. However, the proportion of HBV genotype A (especially genotype A2) is increasing in Japan, mainly via sexual transmission [6–9]. HBV genotype A develops into a persistent infection more often than genotype C [9, 10]. Ito K et al. [9] reported that the maximum serum HBV DNA levels were higher among patients with acute HBV genotype A infection compared to those infected with HBV genotype C. However, the mechanisms underlying the differences in the persistence and viral loads of HBV genotypes A and C are unclear. Many studies have shown that immune cells are involved in HBV infection and that immunosuppressive or immunotolerable responses may differ among HBV genotypes [11–14]. Sugiyama M et al. [15] reported that HBV replication speed was slower and the level of hepatitis B surface antigen (HBsAg) in cultured medium was higher for genotype A compared to genotype C in vitro; these differences may contribute to differences in HBV DNA level among patients infected with HBV. Ito K et al. [16] proposed that differences in the hepatitis B e antigen (HBeAg) component of genotype A would induce immune tolerance to HBV infection. Based on these reports, the differences in HBV DNA levels between genotypes might be explained by different immune system responses. However, whether immune system-independent mechanisms affect HBV DNA levels in vivo is unclear.

In this study, we demonstrated that HBV genotype A-transfected NOG mice developed persistent viremia, whereas the HBV DNA levels in HBV genotype C-transfected NOG mice decreased to undetectable levels within 3 months. This was somehow surprising because many past studies which examined HBV replication in immunodeficient mice transfected by hydrodynamic injection always showed viral persistence [23, 24]. These reports employed an HBV genotype A or genotype D construct. The present study, for the first time, examined HBV replication in immunodeficient mice using plasmid of HBV genotype C and revealed that NOG mice transfected with HBV genotype C by hydrodynamic injection failed to exhibit persistent viremia. Because we used only one strain of HBV genotype C in this study, we cannot exclude the possibility that the observed difference in HBV clearance may be due to the HBV strain used rather than the genotypes. However, this study clearly demonstrates the existence of a mechanism by which HBV-expressing cells were eliminated, even in an immunodeficient context.

Immune cell-independent hepatocyte apoptosis is more frequently induced by the expression of HBV genotype C than genotype A. This leads to the failure of persistent viremia for the HBV genotype C but not for genotype A. This difference may contribute to a lower viral load and reduced persistence in patients with acute hepatitis caused by infection with HBV genotype C compared to HBV genotype A.