Duration of mouse hepatitis virus infection: studies in immunocompetent and chemically immunosuppressed mice

The duration of mouse hepatitis virus (MHV) infection was examined in mice inoculated intranasally with selected strains of MHV. Following inoculation with virulent MHV-JHM, genetically susceptible BALB/c mice and resistant CD1 mice had detectable virus in the brain at 1 month, but not later intervals up to 12 months. BALB/c mice infected with avirulent MHV-S or MHV-1 had no detectable virus in brains at 1 month or thereafter. Immunosuppression of BALB/c mice with treatment regimens of hydrocortisone acetate or cyclophosphamide at 1 and 2 months after infection with MHV-JHM did not activate detectable virus in liver or increase the prevalence or degree of brain infection. Immunosuppression with these drugs during the acute phase of MHV-JHM infection influenced MHV infection, based on virus quantification in livers, but timing of drug treatment relative to MHV infection was critical. Mice infected with MHV developed IgG serum antibody titers that persisted without decline for up to 1 year after infection. Antibody titers varied with mouse genotype and infecting virus. These studies, using intranasal inoculation, support the conclusions of others, using other routes of inoculation, that MHV infection is not persistent in adult, immunocompetent mice.     

Persistence of inoculated hepatitis A virus in mixed human and animal wastes.

The persistence of hepatitis A virus (HAV) was determined both in mixtures of septic tank effluent (STE) with dairy cattle manure slurry (DCMS) and in mixtures of STE with swine manure slurry (SMS). HAV was consistently inactivated more rapidly in the two types of mixed wastes than in STE alone or in the control Dulbecco's phosphate-buffered saline (PBS). At 5 degrees C, the D values (time, in days, for a 90% reduction of virus titer) were 34.6 for the mixed STE and DCMS, 48.5 for the mixed STE and SMS, 58.5 for STE, and 217.4 for the Dulbecco's PBS control. At 22 degrees C, the D values were 23.0, 17.1, 35.1, and 90.1 for the four suspension media, respectively. A comparison of HAV inactivation in mixed wastes subjected to different treatments at the same pH and temperatures showed that the virus inactivation in the mixed wastes was related, at least in part, to microbial activity. In mixed STE and DCMS, the D values at 25 degrees C were 8.3 for raw mixed wastes, 15.1 for autoclaved mixed wastes, and 9.6 for bacterium-free filtrate of raw mixed wastes; D values at 37 degrees C were 6.8, 10.1, and 7.0 for these three suspension media, respectively. In mixed STE and SMS, the D values at 25 degrees C were 8.1 for raw mixed wastes, 14.3 for autoclaved mixed wastes, and 9.1 for bacterium-free filtrate of raw mixed wastes; the D values at 37 degrees C were 6.8, 9.4, and 6.9 for the three suspensions, respectively.     

High-level hepatitis B virus replication in transgenic mice.

Hepatitis B virus (HBV) transgenic mice whose hepatocytes replicate the virus at levels comparable to that in the infected livers of patients with chronic hepatitis have been produced, without any evidence of cytopathology. High-level viral gene expression was obtained in the liver and kidney tissues in three independent lineages. These animals were produced with a terminally redundant viral DNA construct (HBV 1.3) that starts just upstream of HBV enhancer I, extends completely around the circular viral genome, and ends just downstream of the unique polyadenylation site in HBV. In these animals, the viral mRNA is more abundant in centrilobular hepatocytes than elsewhere in the hepatic lobule. High-level viral DNA replication occurs inside viral nucleocapsid particles that preferentially form in the cytoplasm of these centrilobular hepatocytes, suggesting that an expression threshold must be reached for nucleocapsid assembly and viral replication to occur. Despite the restricted distribution of the viral replication machinery in centrilobular cytoplasmic nucleocapsids, nucleocapsid particles are detectable in the vast majority of hepatocyte nuclei throughout the hepatic lobule. The intranuclear nucleocapsid particles are empty, however, suggesting that viral nucleocapsid particle assembly occurs independently in the nucleus and the cytoplasm of the hepatocyte and implying that cytoplasmic nucleocapsid particles do not transport the viral genome across the nuclear membrane into the nucleus during the viral life cycle. This model creates the opportunity to examine the influence of viral and host factors on HBV pathogenesis and replication and to assess the antiviral potential of pharmacological agents and physiological processes, including the immune response.     

Serological relationship of woodchuck hepatitis virus to human hepatitis B virus.

Two antigenic systems of the woodchuck hepatitis virus have been identified. The relationship between viral antigens of the woodchuck hepatitis virus and the human hepatitis B virus was determined by using immunoprecipitation, hemagglutination, and immune electron microscopy techniques. Antigens found on the cores of the two viruses were cross-reactive. Lack of cross-reactivity between the surface antigens of the two viruses in immunodiffusion experiments suggested that the major antigenic determinants of the viral surfaces are different; however, results of passive hemagglutination tests indicated that there are common minor determinants. Nucleic acid homology, as measured by liquid hybridization, was found to be 3 to 5% of the viral genomes. The results of this study provide further evidence that woodchuck hepatitis virus is the second member of a new class of viruses represented by human hepatitis B virus. Since virus-infected woodchucks may acquire chronic hepatitis and hepatocellular carcinoma, these antigens and their respective antibodies will be useful markers for following the course of virus infection in investigations of the oncogenic potential of this class of viruses. The nucleocapsid antigen described may be a class-specific antigen of these viruses and, thus, may be useful in discovering new members of the group.     

Infectivity period of mice inoculated with human adenoviruses

Due to non-productive infections, mice are not a good model to study some human adenoviruses. However, mice provide an excellent model to study the metabolic effects of human adenovirus, Ad36. Research interest in Ad36 is increasing rapidly, and consequently an increase in the use of mice as a model is anticipated. However, little is known about the transmission potential of Ad36 from infected mice to other laboratory animals or personnel. While underestimating the infectivity could promote inadvertent spread of Ad36, overstating it could drain valuable laboratory resources and animals. Therefore, we determined the duration of infectivity in female C57BL/6J mice that were experimentally infected with human adenoviruses Ad36 or Ad2. Other uninfected mice were co-housed for one week with the experimentally-infected animals, four or eight weeks postinfection. Additionally, uninfected mice were housed in the cages of mice that were infected with Ad36, 12 weeks earlier. The presence of viral DNA in tissues was used to indicate infection of mice. Although experimentally-infected mice harboured viral DNA at least up to 12 weeks, the horizontal transmission of infection was observed in co-housed mice only up to four weeks postinfection. Thus, Ad36-infected mice should be considered potentially infective for eight weeks and appropriate handling and barrier containment should be used. After eight week postinfection, horizontal transmission appears unlikely. This information may provide guidelines for animal handling, and experimental design using Ad36, which may increase safety for laboratory personnel and reduce the number of mice required for experiments.     

Autophagy required for hepatitis B virus replication in transgenic mice

Recent studies indicate that hepatitis B virus (HBV) may induce autophagy to enhance its replication in cell cultures. To understand whether autophagy can indeed enhance HBV replication in vivo, we generated HBV transgenic mice with liver-specific knockout of the Atg5 gene, a gene critical for the initiation of autophagy. Immunoblot analyses confirmed the inhibition of autophagy in the livers of Atg5 knockout mice. This inhibition of autophagy slightly reduced HBV gene expression and affected nuclear localization of the HBV core protein. It also reduced the HBV DNA level in sera by more than 90% and the level of the HBV DNA replicative intermediate in the mouse liver to an almost undetectable level. Our results thus demonstrate that autophagy is important for HBV replication in vivo and raise the possibility of targeting this pathway to treat HBV patients.     

Inhibition of hepatitis B virus in mice by RNA interference

Hepatitis B virus (HBV) infection substantially increases the risk of chronic liver disease and hepatocellular carcinoma in humans. RNA interference (RNAi) of virus-specific genes has emerged as a potential antiviral mechanism. Here we show that RNAi can be applied to inhibit production of HBV replicative intermediates in cell culture and in immunocompetent and immunodeficient mice transfected with an HBV plasmid. Cotransfection with plasmids expressing short hairpin RNAs (shRNAs) homologous to HBV mRNAs induced an RNAi response. Northern and Southern analyses of mouse liver RNA and DNA showed substantially reduced levels of HBV RNAs and replicated HBV genomes upon RNAi treatment. Secreted HBV surface antigen (HBsAg) was reduced by 94.2% in cell culture and 84.5% in mouse serum, whereas immunohistochemical detection of HBV core antigen (HBcAg) revealed >99% reduction in stained hepatocytes upon RNAi treatment. Thus, RNAi effectively inhibited replication initiation in cultured cells and mammalian liver, showing that such an approach could be useful in the treatment of viral diseases.     

Overcoming T cell tolerance to the hepatitis B virus surface antigen in hepatitis B virus-transgenic mice

The sequence of the hepatitis B virus (HBV) major envelope (Env) protein (ayw subtype) was scanned for the presence of H-2(d,b) motifs. Following binding and immunogenicity testing, two new H-2(d)-restricted epitopes (Env.362 and Env.364) were identified. These epitopes induced CTLs capable of recognizing naturally processed HBV-Env, but were apparently generated with lower efficiency than the previously defined dominant Env.28 epitope. Next, HBV-transgenic mice that express all of the HBV proteins and produce fully infectious particles were immunized with a mixture of lipopeptides encompassing the Env.28, Env.362, and Env.364 epitopes. Significant CTL responses were obtained, but they had no effect on viral replication in the liver, nor did they induce an inflammatory liver disease. However, in adoptive transfer experiments, CTL lines generated from the HBV-transgenic mice following immunization were able to inhibit viral replication in vivo without causing hepatitis. This is in contrast to CTL lines derived from nontransgenic mice that displayed both antiviral and cytopathic effects, presumably because they displayed higher avidity for the viral epitopes than the transgenic CTLs. These results suggest that T cell tolerance to HBV can be broken with appropriate immunization but the magnitude and characteristics of the resultant T cell response are significantly different from the response in HBV-naive individuals since their antiviral potential is stronger than their cytotoxic potential. This has obvious implications for immunotherapy of chronic HBV infection.     

Inhibition of replication of human hepatitis B virus

Several nucleoside 5'-triphosphate analogs were investigated as inhibitors of human hepatitis B virus replication. Different analogs inhibited DNA synthesis differently, 3'-azido-2',3'-dideoxythymidine 5'triphosphate being the most active compound. This inhibitor blocked DNA synthesis by 50% at inhibitor: substrate molar ratio 1:8, and by 80% - at 1:1. The hypothesis is formulated that 3'-azido-2',3'-dideoxythymidine 5'-triphosphate inhibits RNA directed viral DNA replication due to incorporation of this compound into 3'-termini of newly synthesized DNA chains. The phenomenon observed opens new possibilities for chemotherapy of acute and chronic human hepatitis B.     

Integrated structures of duck hepatitis B virus DNA in hepatocellular carcinoma.

The structure of integrated viral DNA in a hepatocellular carcinoma of a duck from Chi-tung county in China was analyzed. Three different clones of integrated viral DNA, lambda DHS 6-1, lambda DHS 6-2, and lambda DHE 6-2, were obtained from the neoplastic portion of the liver by molecular cloning. One of the three clones, lambda DHS 6-1, showed inverted repetition of integrated viral DNA with chromosomal flanking sequences. Another clone, lambda DHS 6-2, showed a head-to-head configuration of the core and surface gene regions of duck hepatitis B virus (DHBV) DNA. The virus-chromosome junctions were often located near direct repeat 1 or 2 of DHBV DNA in three independent clones. Nucleotide sequences at the virus-virus junctions in two clones, lambda DHS 6-1 and 6-2, indicated the possible rearrangement of chromosomal DNA and recombination of viral DNA. DHBV DNA appears to be integrated into the genome of hepatocytes in a manner similar to that of human and woodchuck hepatitis viruses. Thus, the duck system may serve as a useful animal model for the study of human hepatocarcinogenesis.     

Interleukin-12 inhibits hepatitis B virus replication in transgenic mice.

Interleukin-12 (IL-12) is a heterodimeric cytokine produced by antigen-presenting cells that has the ability to induce gamma interferon (IFN-gamma) secretion by T and natural killer cells and to generate normal Th1 responses. These properties suggest that IL-12 may play an important role in the immune response to many viruses, including hepatitis B virus (HBV). Recently, we have shown that HBV-specific cytotoxic T lymphocytes inhibit HBV replication in the livers of transgenic mice by a noncytolytic process that is mediated in part by IFN-gamma. In the current study, we demonstrated that the same antiviral response can be initiated by recombinant murine IL-12 and we showed that the antiviral effect of IL-12 extends to extrahepatic sites such as the kidney. Southern blot analyses revealed the complete disappearance of HBV replicative intermediates from liver and kidney tissues at IL-12 doses that induce little or no inflammation in these tissues. In addition, immunohistochemical analysis demonstrated the disappearance of cytoplasmic hepatitis B core antigen from both tissues after IL-12 treatment, suggesting that IL-12 either prevents the assembly or triggers the degradation of the nucleocapsid particles within which HBV replication occurs. Importantly, we demonstrated that although IFN-gamma, tumor necrosis factor alpha, and IFN-alpha/beta mRNA are induced in the liver and kidney after IL-12 administration, the antiviral effect of IL-12 is mediated principally by its ability to induce IFN-gamma production in this model. These results suggest that IL-12, through its ability to induce IFN-gamma, probably plays an important role in the antiviral immune response to HBV during natural infection. Further, since relatively nontoxic doses of recombinant IL-12 profoundly inhibit HBV replication in the liver and extrahepatic sites in this model, IL-12 may have therapeutic value as an antiviral agent for the treatment of chronic HBV infection.     

Interferon-regulated pathways that control hepatitis B virus replication in transgenic mice

We previously showed that the intrahepatic induction of cytokines such as alpha/beta interferon (IFN-alpha/beta) and gamma interferon (IFN-gamma) inhibits hepatitis B virus (HBV) replication noncytopathically in the livers of transgenic mice. The intracellular pathway(s) responsible for this effect is still poorly understood. To identify interferon (IFN)-inducible intracellular genes that could play a role in our system, we crossed HBV transgenic mice with mice deficient in IFN regulatory factor 1 (IRF-1), the double-stranded RNA-activated protein kinase (PKR), or RNase L (RNase L) (IRF-1(-/-), PKR(-/-), or RNase L(-/-) mice, respectively), three well-characterized IFN-inducible genes that mediate antiviral activity. We showed that unmanipulated IRF-1(-/-) or PKR(-/-) transgenic mice replicate HBV in the liver at slightly higher levels than the respective controls, suggesting that both IRF-1 and PKR individually appear to mediate signals that modulate HBV replication under basal conditions. These same animals were responsive to the antiviral effects of the IFN-alpha/beta inducer poly(I-C) or recombinant murine IFN-gamma, suggesting that under these conditions, either the IRF-1 or the PKR genes can mediate the antiviral activity of the IFNs or other IFN-inducible genes mediate the antiviral effects. Finally, RNase L(-/-) transgenic mice were undistinguishable from controls under basal conditions and after poly(I-C) or IFN-gamma administration, suggesting that RNase L does not modulate HBV replication in this model.