Detection of hepatitis B virus DNA in mononuclear blood cells.

The Southern transfer hybridisation technique was used to test mononuclear blood cells for hepatitis B virus DNA. Viral DNA sequences were detected in mononuclear cells of 10 out of 16 patients with hepatitis B virus infection and in none of 21 normal controls. Blood contamination was excluded by the absence of hepatitis B virus DNA in the corresponding serum samples in all cases. Free monomeric hepatitis B virus DNA was found in three patients positive for hepatitis Be antigen (HBeAg) and one positive for anti-HBe, and integrated hepatitis B virus DNA was present in four patients positive for anti-HBe. In two other patients the small size of the samples did not allow a distinction between free and integrated viral DNA. The state of the virus in the mononuclear cells seemed to correlate with the HBeAg or anti-HBe state, as has been noted in the liver. These results indicate that hepatitis B virus may infect mononuclear blood cells, thereby expanding the tissue specificity of this agent beyond the liver, as has been reported for pancreatic, kidney, and skin tissue. They also suggest that hepatitis B virus infection of mononuclear cells might be related to immunological abnormalities observed in carriers of the virus.     

Human lymphocyte responses to hepatitis A virus-infected cells: interferon production and lysis of infected cells

Peripheral blood mononuclear cells (PBMC) from nonimmune healthy donors who did not have antibody to hepatitis A virus lysed hepatitis A virus-infected BS-C-1 cells to a greater degree than uninfected BS-C-1 cells. The predominant effector cells were contained in the nonadherent peripheral blood lymphocyte (PBL) fraction, although some lytic activity was associated with adherent cells. Characterization of the PBL with monoclonal antibodies showed that the responsible effector lymphocytes were contained in Leu-11+ and M1+ subsets, but not in the T3+ or T4+ subsets. The phenotypes of the effector cells active in the lysis of hepatitis A virus-infected cells are similar to those of human natural killer cells that lyse K562 cells. Human PBL produced high titers of interferon-alpha (IFN-alpha) when exposed to hepatitis A virus-infected cells. These results imply that hepatitis A virus infection may be controlled by lymphocyte responses in the liver, i.e., by lymphocyte-mediated lysis of the hepatitis A virus-infected cells, and by the production of high titers of IFN-alpha by lymphocytes exposed to hepatitis A virus-infected cells. Furthermore, these results, along with the observations that hepatitis A virus infection results in a persistent noncytocidal infection in vitro, support the hypothesis that lysis of hepatocytes infected with hepatitis A virus is by lymphocyte-mediated cytotoxicity and not by virus-induced destruction of the liver cell.     

Production of 22 nm HBsAG particles by human lymphocytes infected with a recombinant vaccinia virus containing the coding sequence for hepatitis B virus surface antigen.

To examine the possible role of lymphocytes in the course of hepatitis B virus infection, we studied peripheral blood lymphocytes and Epstein-Barr virus transformed B-cells for their capability to produce hepatitis B virus gene products. Infection of these cells with a recombinant vaccinia virus containing the hepatitis B virus surface antigen resulted in the production and secretion of hepatitis B virus surface antigen shortly after infection reaching a peak after three days. When the supernatants of the cells were analyzed on density gradients, a peak of reactivity for hepatitis B virus surface antigen was reached at 1.21 g/cm3. Electron microscopy revealed the presence of spherical and filamentous HBsAg particles. These findings show that human lymphocytes are capable of producing hepatitis B virus surface antigen.     

Antiviral activity of interferon-alpha against hepatitis B virus can be studied in non-hepatic cells and Is independent of MxA.

It is well established that interferon-alpha can induce non-cytotoxic intracellular suppression of hepatitis B virus replication, but the mechanisms involved are unclear. Cell culture studies to characterize these mechanisms are restricted, in part because hepatitis B virus replicates almost exclusively in liver-derived cells. To overcome this limitation we used a cytomegalovirus promoter-controlled hepatitis B virus expression system, which leads to intracellular viral replication even in non-hepatic cell lines. In this experimental system interferon-alpha treatment specifically suppressed viral replication demonstrating that antiviral activities against hepatitis B virus are not restricted to hepatic cells. Furthermore, the interferon-inducible MxA protein was recently reported to play a key role in the antiviral action of interferon-alpha against hepatitis B virus. Our data demonstrate that interferon-alpha also suppresses hepatitis B virus replication in MxA-deficient HEp2 cells, indicating that MxA is not essential for these activities. Taken together, our data imply that the experimental approach presented can also be adapted to established cell lines which are deficient in parts of the signal transduction pathway or other elements located further downstream, providing important insights into mechanisms specifically suppressing hepatitis B virus.     

Restricted replication of human hepatitis A virus in cell culture: intracellular biochemical studies.

When hepatitis A virus was inoculated into Vero cells, virus-specified protein and RNA synthesis was detected. Production of viral protein was detected by electrophoretic analysis in polyacrylamide gels by using a double-label coelectrophoresis and subtraction method which eliminated the contribution of host protein components from the profiles of virus-infected cytoplasm. Eleven virus-specified proteins were detected in the net electrophoretic profiles of hepatitis A virus-infected cells. The molecular weights of these proteins were very similar to those detected in cells infected with poliovirus type 1. Virus-specified protein synthesis could be detected at 3 to 6 h and continued for at least 48 h postinfection, but no significant effect on host-cell macromolecular synthesis was observed. Limited viral RNA replication occurred between 2 and 6 h postinfection. The genomic RNA of hepatitis A virus was extracted and shown to be capable of infecting cells and inducing the same set of proteins as intact virus, indicating that the RNA genome is positive stranded. Progeny virus was never detected in the supernatant fluids of infected cell cultures, and the cells showed no observable cytopathology, even though hepatitis A virus-specific proteins and antigens were being produced. The nature of the defect in the replicative cycle of hepatitis A virus in this system remains unknown.     

An immunofluorescence test for detection of serum antibody to rodent coronaviruses

An indirect immunofluorescence test, using cultured cells infected with two strains of mouse hepatitis virus, was developed for detection of antibody to rodent coronaviruses. The immunofluorescence test detected serum antibody to mouse hepatitis virus and rat sialodacryoadenitis virus earlier than the neutralization test. In the case of mouse hepatitis virus, the results of the immunofluorescence test closely paralleled those obtained with a commercially available enzyme-linked immunosorbent assay.     

Production of cytopathology in FRhK-4 cells by BS-C-1-passaged hepatitis A virus.

Cytopathic effects were produced in fetal rhesus monkey kidney (FRhK-4) cells 7 days postinfection by a serially BS-C-1-passaged strain of hepatitis A virus. Typical enterovirus cytopathology was produced by the HM-175 strain after 15 passages at 7-day intervals in BS-C-1 cells. No cytopathic effects were obtained after neutralization of virus with human anti-hepatitis A virus immunoglobulin G. Normal human serum had no effect on development of cytopathology. Maximum antigen and cDNA probe-based hybridization activity were associated with a CsCl gradient fraction having a density of 1.34 g/cm3. Large quantities of 27- to 30-nm virions typical of hepatitis A virus were associated with the same fraction. These data led to the conclusion that the observed cytopathology was caused by hepatitis A virus.     

Production of cytopathology in FRhK-4 cells by BS-C-1-passaged hepatitis A virus

Cytopathic effects were produced in fetal rhesus monkey kidney (FRhK-4) cells 7 days postinfection by a serially BS-C-1-passaged strain of hepatitis A virus. Typical enterovirus cytopathology was produced by the HM-175 strain after 15 passages at 7-day intervals in BS-C-1 cells. No cytopathic effects were obtained after neutralization of virus with human anti-hepatitis A virus immunoglobulin G. Normal human serum had no effect on development of cytopathology. Maximum antigen and cDNA probe-based hybridization activity were associated with a CsCl gradient fraction having a density of 1.34 g/cm3. Large quantities of 27- to 30-nm virions typical of hepatitis A virus were associated with the same fraction. These data led to the conclusion that the observed cytopathology was caused by hepatitis A virus.     

Ani immunomodulator Hepon inhibits hepatitis C virus replication in human cell cultures in vitro]

Antiviral activity of immunomodulator "Hepon" was evaluated in human cells culture infected with hepatitis C virus. "Hepon" presence protected human cells SW-13 from cytopathogenic effect of hepatitis C virus. Maximum antiviral effect was demonstrated by "Hepon" at concentration 1 mcg/mL. Control antiviral agent reaferon (interferon alfa-2a) was more potent as vitality protecting agent in the case of SW-13 human cells culture. "Hepon" activity is based on changes of cytokins and interferons spectrum so this immunomodulator is expected to be effective against different viruses including herpes virus and encephalocarditis virus.     

HBV prec/c shRNA慢病毒真核表达载体的构建和鉴定

目的构建针对HBV prec/c区的shRNA真核表达载体psiHBV,感染HepG2 2.15细胞后观察其对HBeAg表达的抑制作用,为探讨防治HBV感染的新措施提供实验依据。方法针对HBV prec/c基因序列,构建shRNA表达载体psiHBV1、psiHBV2和无关序列psiHBVc。psiHBV与慢病毒辅助系统质粒共转染293T细胞组装慢病毒颗粒后,感染HepG2 2.15细胞,RT-PCR检测prec/c mRNA的转录,微粒子化学发光分析仪(MEIA)检测细胞上清和细胞裂解液中HBeAg表达。结果重组质粒双酶切和测序鉴定与预期结果相符合;组装慢病毒颗粒感染HepG2 2.15细胞后,prec/c mRNA转录降低;与对照组比较,HBeAg的表达水平也显著降低,病毒颗粒对HBeAg表达的抑制作用差异有统计学意义(P<0.01)。结论成功构建针对HBVprec/c的慢病毒载体psiHBV1、siHBV2,慢病毒介导的RNA i能抑制HBV表达,为应用RNA干扰技术治疗乙型肝炎提供了实验依据。     

Antibody is required for clearance of infectious murine hepatitis virus A59 from the central nervous system, but not the liver.

Intracerebral inoculation with mouse hepatitis virus strain A59 results in viral replication in the CNS and liver. To investigate whether B cells are important for controlling mouse hepatitis virus strain A59 infection, we infected muMT mice who lack membrane-bound IgM and therefore mature B lymphocytes. Infectious virus peaked and was cleared from the livers of muMT and wild-type mice. However, while virus was cleared from the CNS of wild-type mice, virus persisted in the CNS of muMT mice. To determine how B cells mediate viral clearance, we first assessed CD4(+) T cell activation in the absence of B cells as APC. CD4(+) T cells express wild-type levels of CD69 after infection in muMT mice. IFN-gamma production in response to viral Ag in muMT mice was also normal during acute infection, but was decreased 31 days postinfection compared with that in wild-type mice. The role of Ab in viral clearance was also assessed. In wild-type mice plasma cells appeared in the CNS around the time that virus is cleared. The muMT mice that received A59-specific Ab had decreased virus, while mice with B cells deficient in Ab secretion did not clear virus from the CNS. Viral persistence was not detected in FcR or complement knockout mice. These data suggest that clearance of infectious mouse hepatitis virus strain A59 from the CNS requires Ab production and perhaps B cell support of T cells; however, virus is cleared from the liver without the involvement of Abs or B cells.     

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 (approximately 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.     

Aggravation of viral hepatitis by platelet-derived serotonin

More than 500 million people worldwide are persistently infected with hepatitis B virus or hepatitis C virus. Although both viruses are poorly cytopathic, persistence of either virus carries a risk of chronic liver inflammation, potentially resulting in liver steatosis, liver cirrhosis, end-stage liver failure or hepatocellular carcinoma. Virus-specific T cells are a major determinant of the outcome of hepatitis, as they contribute to the early control of chronic hepatitis viruses, but they also mediate immunopathology during persistent virus infection. We have analyzed the role of platelet-derived vasoactive serotonin during virus-induced CD8(+) T cell-dependent immunopathological hepatitis in mice infected with the noncytopathic lymphocytic choriomeningitis virus. After virus infection, platelets were recruited to the liver, and their activation correlated with severely reduced sinusoidal microcirculation, delayed virus elimination and increased immunopathological liver cell damage. Lack of platelet-derived serotonin in serotonin-deficient mice normalized hepatic microcirculatory dysfunction, accelerated virus clearance in the liver and reduced CD8(+) T cell-dependent liver cell damage. In keeping with these observations, serotonin treatment of infected mice delayed entry of activated CD8(+) T cells into the liver, delayed virus control and aggravated immunopathological hepatitis. Thus, vasoactive serotonin supports virus persistence in the liver and aggravates virus-induced immunopathology.     

Adaptation of hepatitis A virus strain (JaM-55) originally pathogenic for monkeys to a cell culture

The results of adaptation of hepatitis A viral strain JaM-55 to the culture of embryo kidney cells FRhk-4 from macaque Rhesus are presented. The viral strain was isolated from a M. fascicularis suffering from spontaneous hepatitis. Before inoculating the cell culture the virus was passaged twice in the M. arctoides capable of reproducing hepatitis. FRhk-4 cell line inoculation by the monkey liver extract, containing the strain HAV-YaM-55, resulted in isolation of single viral particles of hepatitis A in the preparations obtained at the first 3 passages by the 28-31 day of cultivation. Beginning from the fourth passage the abrupt increase in the number of viral particles and hepatitis A antigen was registered. There were no traces of cytopathogenic effect at any level of viral passages in the inoculated cell culture. The adapted virus contains hepatitis A viral RNA identified by spot hybridization with the cloned cDNA of hepatitis A virus.     

Characteristics of immune response regulation in viral hepatitis A and B

In 272 patients with virus hepatitis A and B the content of theophylline-sensitive lymphocytes (T-suppressors) and theophylline-resistant lymphocytes (T-helpers) in the peripheral blood was determined. Differences in the content of T-suppressors in cases of acute virus hepatitis A and B with an equal degree of severity were revealed: at the peak of hepatitis A infection in the mild form of the disease the number of the cells was decreased, while at the peak of hepatitis B infection an increase in their number was observed in the mild and moderate forms of the disease and a decrease, in the severe form of the disease. In chronic persistent hepatitis a decrease in the content of T-suppressors and an increase in the content of T-helpers were observed, and in chronic active hepatitis (at the period of remission) and increase in the T-helpers occurred. Changes in the content of the cells of both types are not characteristic of HBsAg carriership.