In Vitro and In Vivo Infectivity and Pathogenicity of the Lymphoid Cell-Derived Woodchuck Hepatitis Virus

Woodchuck hepatitis virus (WHV) and human hepatitis B virus are closely related, highly hepatotropic mammalian DNA viruses that also replicate in the lymphatic system. The infectivity and pathogenicity of hepadnaviruses propagating in lymphoid cells are under debate. In this study, hepato- and lymphotropism of WHV produced by naturally infected lymphoid cells was examined in specifically established woodchuck hepatocyte and lymphoid cell cultures and coculture systems, and virus pathogenicity was tested in susceptible animals. Applying PCR-based assays discriminating between the total pool of WHV genomes and covalently closed circular DNA (cccDNA), combined with enzymatic elimination of extracellular viral sequences potentially associated with the cell surface, our study documents that virus replicating in woodchuck lymphoid cells is infectious to homologous hepatocytes and lymphoid cells in vitro. The productive replication of WHV from lymphoid cells in cultured hepatocytes was evidenced by the appearance of virus-specific DNA, cccDNA, and antigens, transmissibility of the virus through multiple passages in hepatocyte cultures, and the ability of the passaged virus to infect virus-naive animals. The data also revealed that WHV from lymphoid cells can initiate classical acute viral hepatitis in susceptible animals, albeit small quantities (∼10(3) virions) caused immunovirologically undetectable (occult) WHV infection that engaged the lymphatic system but not the liver. Our results provide direct in vitro and in vivo evidence that lymphoid cells in the infected host support propagation of infectious hepadnavirus that has the potential to induce hepatitis. They also emphasize a principal role of the lymphatic system in the maintenance and dissemination of hepadnavirus infection, particularly when infection is induced by low virus doses.

土拨鼠肝炎病毒（Woodchuck hepatitis virus, WHV）与人类乙型肝炎病毒亲缘关系密切，二者均为高度嗜肝性的哺乳动物DNA病毒，且均可在淋巴系统中复制。目前，淋巴样细胞中增殖的嗜肝DNA病毒（hepadnavirus）的感染性与致病性仍存在学术争议。本研究通过特异性构建的土拨鼠肝细胞与淋巴样细胞单培养及共培养体系，检测了天然感染的淋巴样细胞所产生的WHV的嗜肝性与嗜淋巴性，并在易感动物中测试了该病毒的致病性。本研究采用可区分WHV全基因组与共价闭合环状DNA（covalently closed circular DNA, cccDNA）的基于聚合酶链式反应（PCR）的检测方法，结合酶解法去除可能附着于细胞表面的细胞外病毒序列，证实了在土拨鼠淋巴样细胞中复制的病毒可在体外感染同源肝细胞与淋巴样细胞。淋巴样细胞来源的WHV在培养肝细胞中发生的增殖性复制，可通过以下三方面证据得到证实：病毒特异性DNA、cccDNA与抗原的出现；该病毒可在肝细胞培养中经多次传代仍保持传播能力；传代后的病毒可感染未接触过该病毒的动物。研究数据同时显示，淋巴样细胞来源的WHV可在易感动物中引发经典急性病毒性肝炎；即便仅使用少量病毒（约10³个病毒粒子），也可引发免疫病毒学检测无法检出的隐匿性WHV感染，该感染仅累及淋巴系统而不波及肝脏。本研究结果提供了直接的体外与体内实验证据，证实感染宿主内的淋巴样细胞可支持具有感染性的嗜肝DNA病毒的增殖，这类病毒具备引发肝炎的潜力。同时，本研究也强调了淋巴系统在嗜肝DNA病毒感染的维持与播散中发挥的核心作用，尤其是在低病毒剂量诱导感染的场景下。