Plasmid.

The plasmid of HBV is perhaps the crucial factor determining the expression level and persistence of HBV in mice. Common plasmids include pAAV, ¹¹ pBluescript II KS(+) (pBS), ^19,20 pcDNA3.1(+), and ²⁷ pSM2. ^28,43 Among these vectors, pAAV is probably the most commonly used. AAV is a single-stranded linear DNA parvovirus that promotes high infection efficiency, low pathogenicity, low immunogenicity, and the ability to express foreign genes for extended periods. Delivery of HBV genes via pAAV can lead to long-term expression of HBsAg and a tolerant phenotype with the minimal liver inflammation. ⁴⁴ pBS is derived from the pUC19 plasmid vector and is a nonviral vector with multiple cloning sites; it is commonly used for cloning and sequencing. pcDNA3.1(+) is a versatile nonviral vector used for both high-level stable and transient expression in mammalian hosts. pSM2 is a lentiviral/retroviral vector with high transfection efficiency (Table 1). pAAV and pcDNA3.1(+) carrying the HBV genome can achieve high and sustained expression in mice, making them suitable for establishing HBV infection models.

Dose.

The persistence of HBV in mice is notably influenced by the dose of the administered plasmid. A study of varying doses of the pAAV-HBV1.2 plasmid in mice (a 1.2-fold HBV genome) ² revealed that a plasmid dose of 6 μg induces immune tolerance, thereby sustaining continuous HBV infection. In contrast, 20 μg of plasmid induced antigen recognition receptors and triggered an anti-HBV immune response that cleared HBV in a short period of time. ⁴⁰ Consequently, implementation of the HBV HDI model requires careful consideration of both the plasmid structure and the injected dose. Using high-dose HBV plasmids tends to expedite the clearance of HBV in mice. Therefore, using a large dose of HBV DNA plasmid is generally not advisable for establishing a chronic HBV infection model.

Genetic background.

The genetic background of mice plays a significant role in their HBV phenotypes. Currently, numerous mouse strains can be used; the appropriate strain should be selected based on the research objectives. Acute HBV infection using HDI in C57BL/6 mice is the classic HBV model. In 2006, one group established an HDI model using pAAV-HBV in C57BL/6 mice; 40% of the mice exhibited sustained HBsAg expression for over 6 mo, simulating long-term HBV expression and providing new approaches to studying chronic HBV infection. ¹¹ Another group found that C3H/HeN mice showed higher and more persistent HBV expression than C57BL/6 mice after HDI. Differences in HBV infection related to genetic background have been described in different mouse strains and strains (Table 2). ³⁰ C57BL/6 substrains have significant phenotypic differences in physiology, biochemistry, and immunocompetence, which result in differential susceptibility to virus-induced inflammatory disease. ²⁹ Viral clearance differs among different C57BL/6 substrains created using HBV HDI. Around 40% of C57BL/6J mice had detectable HBsAg and HBV DNA in their serum at week 26, while HBsAg and HBV DNA dropped below detection limits by week 8 in C57BL/6N mice. ³⁸ The use of HDI to establish the HBV infection in transgenic mice via the human AAVS1 site element transgenic mice resulted in high-level and sustained expression of HBV genes. ⁴⁶ C57BL/6J, CBA/CaJ, and C3H/He mice exhibit higher and more persistent HBV expression as compared with NOD/ShiLtJ mice and are therefore better choices for establishing HBV mouse models.

Age.

Another crucial factor that affects HBV clearance is age. In adult humans, some proportion of infected individuals can clear the virus from their bodies, whereas newborns and children tend to have high rates of virus replication after HBV infection, ^8,25,34 often leading to chronic infection. Juvenile mice (approximately 6 to 8 wk) ⁸ show age-dependent HBV clearance (Table 3), partly because HBV can overcome the immature liver immune functions to establish a chronic infection. As mice age, their immune systems gradually mature and become more efficient at HBV clearance. Adult mice (approximately 8 to 12 wk) ⁷ have a mature liver immune system and exhibit strong immune responses to HBV. ^6,12,42

Gut microbiota.

The ability of mice to clear intrahepatic HBV is closely related to their gut microbiota. As compared with juvenile mice, the gut microbiota in adult mice is relatively abundant and complex and can stimulate liver immunity and facilitate rapid HBV clearance. The addition of combinations of antibiotics to the drinking water of 6- to 8-wk-old C57BL/6 mice with established HBV HDI resulted in lower HBV clearance by mice in the antibiotic group as compared with the control group; this difference occurs mainly because antibiotics disrupt the composition and function of gut microbiota, leading to elevated levels of serum lipopolysaccharide, which in turn induce endotoxemia and inflammatory factor production, which subsequently accelerates HBV clearance. ⁵¹ Mice treated with antibiotics and subsequent fecal microbiota transplantation were less susceptible to HBV infection. ³⁷ This type of evidence highlights the crucial role of gut microbiota in the ability of mice to clear HBV. Therefore, the use of juvenile mice for about 6 to 8 wk is recommended when establishing the HBV HDI model.

Sex.

Susceptibility to HBV infection is also related to sex. Sex hormone receptor signaling drives sexual dimorphism in gene expression throughout liver development. This sexual dimorphism is evident in the different infection rates of male and female mice after inoculation with HBV. ⁵⁰ Human men tend to have a higher rate of chronic HBV infection and higher viral loads than do women. Women have stronger innate and adaptive immune responses than men. Sex hormones such as testosterone and estrogen influence systemic immune responses through hormone receptors expressed in immune cell lineages. Hormones also contribute to hepatic epigenetics and genetic alternations. ²⁴ Mice show sex differences in HBV-related immune responses, particularly with regard to immune tolerance and clearance. Testosterone can enhance the synthesis of HBV mRNA, ³⁵ resulting in higher HBV gene expression and viral replication levels in males as compared with females (Table 4). ³⁹ However, the mechanisms underlying the sex hormone-regulated HBV-specific immune response remain unclear.

Advantages.

The HDI method is widely applicable for the study of HBV, including areas such as recombinant covalently closed circular DNA (rcccDNA) persistence and the development of new intervention strategies. Except for its wide range of applications, HDI is also highly efficient. A study that used site-specific DNA recombination technology and HDI to establish HBV infection in mice showed that HBsAg and rcccDNA persisted for more than 62 wk after inoculation, providing the opportunity to study many aspects of HBV pathobiology. ¹⁸

In addition to HBV, the HDI method has been used to create various other disease models, including nonalcoholic fatty liver disease, ⁴⁹ hemophilia B, ²⁶ and enterovirus 71 infection. ³¹ Furthermore, the HDI transfection method, originally used in mice, has also been used in rats ⁴⁸ and pigs. ³

Another advantage of the HDI model is its utility for comparing different HBV genotypes and virus strains in mice, enabling rapid in vivo analysis and the basis of persistent HBV expression and potential pathogenic mechanisms. ³²

Limitations and challenges.

Constructing the HDI model involves injecting a substantial volume of liquid into the mouse’s tail vein, which can potentially cause short-term liver damage, ¹ Hepatocyte swelling, formation of hemorrhagic necrotic foci, and elevation of alanine aminotransferase have been observed. Hepatic injury is most severe 30 min after injection and is then repaired naturally by cell proliferation in about 1 wk. ²¹ However, these effects could complicate the interpretation of experimental results.

The process of HDI may be painful for mice, so anesthesia or analgesia may be appropriate before HDI. The effect of anesthetics or analgesics on the efficiency of transforming rcDNA into cccDNA in mouse liver cells has not been studied. The efficiency of transformation in mouse liver cells is normally low, and anesthetics or analgesics could reduce the transfer of cccDNA into the cells, perhaps limiting the study of some HBV biologic processes.

Mice are not natural hosts for HBV, and their infection rate is considerably lower than that of human HBV patients. Consequently, the HDI model cannot fully replicate the entire course of human HBV infection and has limitations as a model of natural infection.