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The content of hepatitis B virus (HBV) replicative forms and HBV core protein in the liver of HBV transgenic mice is transiently reduced during massive liver regeneration following partial hepatectomy while the steady-state content of viral RNA is unchanged. This antiviral effect is triggered by interferon and tumor necrosis factor that are induced in the liver following hepatectomy and either prevent the formation or accelerate the degradation of viral nucleocapsids in the cytoplasm of the hepatocyte. Despite massive hepatocellular turnover, this effect is independent of liver cell division, indicating that HBV replicates efficiently in resting and dividing hepatocytes.

We have compared the in vitro and in vivo behaviors of a set of isogenic E1- and E1/E4-defective adenoviruses expressing the lacZ gene of Escherichia coli from the Rous sarcoma virus long terminal repeat. Infection of tumor-derived established cell lines of human origin with the doubly defective adenoviruses resulted in (i) a lower replication of the viral backbone that correlated with reduced levels of E2A-specific RNA and protein, (ii) a significant shutoff of late gene and protein expression, and (iii) no apparent virus-induced cytotoxicity. Independently of the extent of the deletion, the additional inactivation of E4 from the viral backbone therefore drastically disabled the virus in vitro, with no apparent effect on transgene expression. A lacZ-transgenic model was used to compare the different recombinant adenoviruses in the livers of C57BL/6 mice. The immune response to the virally encoded beta-galactosidase was minimal in this model, as infusion of the E1-defective adenovirus resulted in a time course of transgene expression that mimicked that in immunodeficient (nu/nu) mice, with very little inflammation and necrosis in the liver. Administration of a doubly defective adenovirus to the transgenic animals led to long-term extrachromosomal persistence of viral DNA in the liver, with no detectable methylation of CpG dinucleotides. However, transient transgene expression was observed independently of the extent of the E4 deletion, suggesting that the choice of the promoter may be critical to maintain transgene expression from these attenuated adenovirus vectors.

VP60, the unique component of rabbit hemorrhagic disease virus capsid, was expressed in the baculovirus system. The recombinant VP60, released in the supernatant of infected insect cells, assembled without the need of any other viral component to form viruslike particles (VLPs), structurally and immunologically indistinguishable from the rabbit hemorrhagic disease virion. Intramuscular vaccination of rabbits with the VLPs conferred complete protection in 15 days; this protection was found to be effective from the fifth day after VLP injection and was accompanied by a strong humoral response.

Circulating leukocytes are important in dissemination of cytomegalovirus (CMV) infection in humans. In the mouse model of murine CMV infection (MCMV), it has been shown that infection peaks on days 5 to 7 after experimental infection, when 0.01 to 0.1% of the circulating leukocytes contain viral DNA. In our laboratory, MCMV DNA was detected by in situ hybridization predominantly in the mononuclear cells on day 6 after acute infection. Infectious virus was recovered from day 6 mononuclear fractions in 16 of 16 mice compared with that from day 6 polymorphonuclear fractions in 4 of 16 mice. An eclipse phenomenon was noted in the blood leukocytes by quantitative blot hybridization: the amount of MCMV DNA present was small on day 2, diminished on days 3 and 4, and then increased markedly on days 5 and 6 in both the mononuclear and polymorphonuclear fractions immediately following viral augmentation in the liver and spleen. MCMV immediate-early and glycoprotein B (late) transcripts were present in pooled mononuclear fractions only on day 6 of acute infection but not in pooled polymorphonuclear fractions. Collectively, these data demonstrate that (i) circulating leukocytes, predominantly mononuclear, are involved in dissemination of MCMV; (ii) a primary viremia with dissemination of MCMV to reticuloendothelial organs (liver and spleen) occurs and is followed by viral amplification and a subsequent, more intense secondary viremia; and (iii) immediate-early viral mRNA and glycoprotein B mRNA transcripts are detectable only during peak infection on day 6 in mononuclear leukocytes but not in polymorphonuclear leukocytes.

A panel of serotype 3 (T3) reovirus strains was screened to determine their relative capacities to cause lethal infection and hepatobiliary disease following peroral inoculation in newborn mice. A wide range of 50% lethal doses (LD50s) was apparent after peroral inoculation of the different virus strains. Two of the strains, T3 Abney and T3 clone 31, caused mice to develop the oily fur syndrome associated with biliary atresia. The capacity to cause biliary atresia was not related to the capacity to cause lethal infection, however, because the LD50s of T3 Abney and T3 clone 31 were grossly disparate. Examination of liver and bile duct tissues revealed histopathologic evidence of biliary atresia and hepatic necrosis in T3 Abney-infected mice but not in mice inoculated with a T3 strain of similar virulence or with the hepatotropic T1 Lang strain. The consistency with which T3 Abney-infected mice developed biliary atresia-associated oily fur syndrome permitted us to determine the viral genetic basis of reovirus-induced biliary atresia. Analysis of reassortant viruses isolated from an in vitro coinfection with T3 Abney and T1 Lang indicated a strong association of the hepatobiliary disease-producing phenotype with the T3 Abney S1 gene, which encodes the viral cell attachment protein, sigma 1. Amino acid residues within the sigma 1 protein that were unique to disease-producing T3 strains were identified by comparative sequence analysis. Specific changes exist within two regions of the protein, one of which is thought to be involved in binding to host cell receptors. We hypothesize that changes within this region of the protein are important in determining the tropism of this virus for bile-ductular epithelium.

Cytomegalovirus is transmitted with blood and organs from seropositive individuals, although the particular leukocyte population harboring latent or persistent virus remains poorly characterized. Murine cytomegalovirus, tagged with the Escherichia coli lacZ gene, was used to identify cells in which virus replicates during acute infection of immunocompetent mice. Recombinant murine cytomegaloviruses, RM461, RM460, and RM427, were constructed to express beta-galactosidase under control of the human cytomegalovirus ie1/ie2 promoter/enhancer. The lacZ gene was inserted between the ie2 and sgg1 genes in RM461 and RM460, disrupting a 0.85-kb late transcript that was found to be dispensable for replication in cultured cells as well as for infection of mice. In BALB/c mice, lacZ-tagged and wild-type viruses exhibited a similar 50% lethal dose and all had the capacity to latently infect the spleen. Peripheral blood mononuclear phagocytes were the major infected leukocyte cell type, as demonstrated by the ability of infected cells to adhere to glass and to phagocytize latex beads; however, these cells did not exhibit typical monocyte markers. Plaque assay for virus and 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal) staining of frozen sections of organs from infected mice revealed that the major target organs included the spleen, adrenal glands, liver, and salivary glands, although tissues as diverse as brown fat and lungs were also involved. Individual blue-staining cells were readily identified in all infected tissues. These studies identified a mononuclear phagocyte, possibly a macrophage or dendritic cell precursor, as the vehicle of virus dissemination during acute infection, and demonstrate the utility of using lacZ-tagged murine cytomegalovirus for tropism, pathogenesis, and latency studies.

Transgenic mice that express the hepatitis B virus core protein were used to examine factors that influence the intracellular localization of nucleocapsid particles in the primary hepatocyte in vivo. In this model, viral nucleocapsid particles are strictly localized to the nucleus of the hepatocyte except when the nuclear membrane dissolves during cell division, at which time they enter the cytoplasm. The cytoplasmic nucleocapsid particles do not reenter the nucleus, however, when the nuclear membrane re-forms after cell division. The data support the notion that nucleocapsid particles can form de novo within the nucleus, and they suggest that performed nucleocapsid particles cannot be transported across the intact nuclear membrane in either direction. The results imply that nucleocapsid disassembly is probably required for entry of the hepadnaviral genome into the nucleus, and they question the role of the intranuclear viral nucleocapsid particle during the viral life cycle.

Hepatitis delta virus (HDV) is a subviral agent of humans which is dependent upon hepatitis B virus as a helper for transmission. HDV can be experimentally transmitted to woodchucks by using woodchuck hepatitis virus (WHV) as the helper. We used this model system to study two types of HDV infections: those of animals already chronically infected with WHV and those of animals without any evidence of prior exposure to WHV. At 5 to 10 days after infection with HDV, liver biopsies of these two groups of animals indicated that around 1% of the hepatocytes were infected (HDV antigen positive). Moreover, similar amounts of replicative forms of HDV RNA were detected. In contrast, by 20 days postinfection, the two groups of animals were quite different in the extent of the HDV infection. The animals chronically infected with WHV showed spread of the infection within the liver and the release of high titers of HDV into the serum. In contrast, the animals not previously exposed to WHV showed a progressive reduction in liver involvement, and at no time up to 165 days postinfection could we detect HDV particles in the serum. However, if these animals were inoculated with a relatively high titer of WHV at either 7 or even 33 days after the HDV infection, HDV viremia was observed. Our data support the interpretation that in these animals, hepatocytes were initially infected in the absence of helper virus, HDV genome replication took place, and ultimately these replicating genomes were rescued by the secondary WHV infection. The observation that HDV can survive in the liver for at least 33 days in the absence of coinfecting helper virus may be relevant to the reemergence of HDV infection following liver transplantation.

Earlier studies have suggested that transient hepadnavirus infections in mammals are associated with virus replication in a large fraction of hepatocytes. Although the viremia that occurred during transient infections in some individuals would presumably lead to virus replication in all hepatocytes, these studies did not reveal if this was the case. The question of the extent of hepatocyte infection was therefore reinvestigated because of the implications of the results for the mechanisms of virus clearance. Woodchucks were inoculated with woodchuck hepatitis virus, and the course of hepatic infection was determined. These studies indicated that essentially 100% of the hepatocytes became infected in the majority of woodchucks. In 7 of 10 woodchucks, the viral infection was then rapidly cleared from the liver, generally in less than 4 weeks. In another three woodchucks, though productive infection was just as rapidly cleared, viral covalently closed circular DNA remained for weeks to months after other indicators of virus infection had disappeared from the liver. Bromodeoxyuridine labeling and anti-proliferating cell nuclear antigen staining to detect hepatocytes passing through S phase indicated an increase in hepatocyte proliferation during the recovery phase of infection. The rate of cell division appeared to be sufficient to replace no more than 2 to 3% of the hepatocytes per day, at the times at which the biopsies were performed. Histopathologic evaluation of the biopsy samples did not provide evidence for a massive amount of liver regeneration. Models to explain virus clearance, with or without massive immune system-mediated destruction of infected hepatocytes, are reviewed.

Hepadnaviruses cause persistent noncytopathic infections of hepatocytes in humans and other animals. Virus replication depends on the pool of viral covalently closed circular DNA (cccDNA) molecules, which serve as transcriptional templates in the nuclei of infected cells. The size of this pool of cccDNA molecules is regulated by the ability of the large envelope protein of the virus to direct newly synthesized viral DNAs into a pathway for viral secretion and thereby inhibit their utilization for viral cccDNA synthesis. In this study, we showed that single amino acid changes in the large envelope protein could cause profound changes in cccDNA levels in transfected permissive cells or in infected cultured hepatocytes. While defects in cccDNA regulation were accompanied by a decrease of enveloped virus production in transfected cells, primary hepatocytes infected by such mutant viruses transiently produced wild-type or higher levels of enveloped virus. Moreover, high levels of cccDNA were always associated with cytopathic effects in the infected hepatocytes. The results demonstrate that the large envelope protein promotes persistent noncytopathic infection of hepatocytes by acting as an overall repressor of virus replication.

The hepatitis delta virus (HDV) envelope contains the large (L), middle (M), and small (S) surface proteins encoded by coinfecting hepatitis B virus. Although HDV-like particles can be assembled with only the S protein in the envelope, the L protein is essential for infectivity in vitro (C. Sureau, B. Guerra, and R. Lanford, J. Virol. 67:366-372, 1993). Here, we demonstrate that the M protein, previously described as carrying a site for binding to polymerized human albumin, is not necessary for infectivity. HDV-like particles coated with the S plus L or the S plus M plus L proteins are infectious in primary cultures of chimpanzee hepatocytes. We conclude that the S and L proteins serve two essential functions in the HDV replication cycle; the S protein ensures the export of the HDV genome from an infected cell by forming a particle, and the L protein ensures its import into a human hepatocyte.

A viral deletion mutant (delta UL21) that lacked the sequences encoding 484 of the predicted first 535 amino acids of the UL21 open reading frame was genetically engineered and studied with respect to its phenotype in cells in culture. We report the following. (i) The replication of delta UL21 was identical to that of the parent herpes simplex virus 1 (HSV-1) strain F in Vero cells, but the yields were three- to fivefold lower than those of the parent virus in human embryonic lung cells. (ii) To characterize the UL21 protein, we immunized rabbits against a purified bacterial fusion protein consisting of glutathione S-transferase fused to the majority of the coding domain of the UL21 gene. Rabbit antiserum directed against the fusion protein recognized a broad band with an apparent M(r) of 62,000 to 64,000 in lysates of cells infected with HSV-1 strain F and in virions purified from the infected cell cytoplasm. This band was absent from lysates of mock-infected cells or cells infected with the delta UL21 virus. The band was significantly reduced in intensity in lysates of cells infected in the presence of phosphonoacetic acid, indicating that it is expressed as a late (gamma 1) gene. (iii) Immunofluorescence studies localized the UL21 antigen primarily in brightly staining granules in the cytoplasms of infected cells. Taken together, the data indicate that the UL21 protein is a virion component dispensable for all aspects of replication of HSV-1 in the cells tested. The electrophoretic mobility of the UL21 protein suggests that it is extensively modified posttranslationally.

The role of gamma interferon (IFN-gamma) in the resolution of rat cytomegalovirus (RCMV) infection was investigated. In the spleen, IFN-gamma-producing cells reached maximum numbers on day 7 after infection. Prophylactic treatment with high doses of recombinant rat IFN-gamma exerted antiviral activity in fibroblasts and protected immunosuppressed rats against a lethal RCMV challenge. Remarkably, in immunocompetent rats, neutralization of endogenous IFN-gamma activity significantly reduced the numbers of RCMV antigen-expressing cells in the spleen, the predominant site of viral replication. Moreover, protection of radiation-immunosuppressed infected rats by transferred immune T cells was enhanced by coinjection of IFN-gamma neutralizing antibodies. The observations were paralleled by in vitro findings: low concentrations of IFN-gamma enhanced viral replication in both macrophages and fibroblasts. These data suggest that IFN-gamma can play different and even opposite roles in the regulation of RCMV replication in vivo; T lymphocytes may contribute to the progression of RCMV infection by secreting IFN-gamma.

We investigated the role of tumor necrosis factor alpha (TNF-alpha) in the pathogenesis of rat cytomegalovirus (RCMV) infection. TNF-alpha levels found in the sera of radiation-immunosuppressed rats in the course of infection (> 350 pg/ml) correlated with the development of RCMV disease. Administration of anti-TNF-alpha antibodies strongly reduced the severity of pneumonia and led to a reduction in virus titers. In immunocompetent rats, anti-TNF-alpha antibodies also significantly suppressed viral replication. Conversely, administration of TNF-alpha augmented RCMV replication and aggravated the disease signs. In vitro, TNF-alpha enhanced RCMV replication in the macrophage, whereas a reduction of viral replication was observed in fibroblasts, indicating that the effect on viral replication is cell type specific. Besides activation of viral replication and exacerbation of RCMV disease, TNF-alpha also favored lymphoid and hematopoietic tissue reconstitution after irradiation, which may contribute to antiviral resistance and survival. This finding demonstrates the protean nature of TNF-alpha, with both beneficial and adverse effects for the host. Our results suggest that TNF-alpha plays an important role in modulating the pathogenesis of RCMV infection.

To test the hypothesis that in vivo resistance to hepadnavirus infection was due to resistance of host hepatocytes, we isolated hepatocytes from Muscovy ducklings and chickens, birds that have been shown to be resistant to duck hepatitis B virus (DHBV) infection, and attempted to infect them in vitro with virus from congenitally infected Pekin ducks. Chicken hepatocytes were resistant to infection, but we were able to infect approximately 1% of Muscovy duck hepatocytes in culture. Infection requires prolonged incubation with virus at 37 degrees C. Virus spread occurs in the Muscovy cultures, resulting in 5 to 10% DHBV-infected hepatocytes by 3 weeks after infection. The relatively low rate of accumulation of DHBV DNA in infected Muscovy hepatocyte cultures is most likely due to inefficient spread of virus infection; in the absence of virus spread, the rates of DHBV replication in Pekin and Muscovy hepatocyte cultures are similar. 5-Azacytidine treatment can induce susceptibility to DHBV infection in resistant primary Pekin hepatocytes but appears to have no similar effect in Muscovy cultures. The relatively inefficient infection of Muscovy duck hepatocytes that we have described may account for the absence of a detectable viremia in Muscovy ducklings experimentally infected with DHBV.

Hepatitis B viruses (hepadnaviruses) can cause chronic, productive infections of hepatocytes. Analyses of the enhancers and promoters of these viruses in cell lines have suggested a requirement of these elements for liver-enriched transcription factors. In this study, a minimum of seven factor-binding sites on the duck hepatitis B virus enhancer were detected by DNase I footprinting using duck liver nuclear extracts. Among the sites that were tentatively identified were one C/EBP-, one HNF1-, and two HNF3-binding sites. Mutations of the HNF1- and HNF3-like sites, which eliminated factor binding, as assessed by both DNase I footprinting and competitive gel shift assays, were evaluated for their effects on enhancer activity. Using a construct in which human growth hormone was expressed from the viral enhancer and core gene promoter, we found that all of the mutations, either alone or in combination, reduced expression two- to fourfold in LMH chicken hepatoma cells. The mutations in the HNF1 site and one of the HNF3 sites, when inserted into the intact viral genome, also suppressed virus RNA synthesis in primary hepatocyte cultures. Virus carrying the latter HNF3 mutation was also examined for its ability to infect and replicate in ducks. No significant inhibition of virus replication was observed in a short-term assay; however, virus with the HNF3 mutation was apparently unable to grow in the pancreas, a second site of duck hepatitis B virus replication in the duck.

The X gene of the mammalian hepadnaviruses is believed to encode a protein of 17 kDa which has been shown to transactivate a wide range of viral and cellular promoters. The necessity for X gene expression during the viral life cycle in vivo has recently been suggested (H.-S. Chen, S. Kaneko, R. Girones, R. W. Anderson, W. E. Hornbuckle, B. C. Tennant, P. J. Cote, J. L. Gerin, R. H. Purcell, and R. H. Miller, J. Virol. 67:1218-1226, 1993). We have independently constructed two variants of woodchuck hepatitis virus (WHV) with mutations in the X coding region. Transient transfection of two different hepatoma cell lines showed that these WHV X gene mutants were competent for virus replication in vitro. To determine whether X expression was required for viral replication in vivo, we injected mutant and wild-type genomes into the livers of susceptible woodchucks. While the wild-type WHV genomes were infectious in all animals examined, the mutant genomes did not initiate a WHV infection in woodchucks. These results indicate that the X gene of the hepadnaviruses plays a major role in viral replication in vivo.

Enhancer II of human hepatitis B virus has dual functions in vivo. Located at nucleotides (nt) 1646 to 1741, it can stimulate the surface and X promoters from a downstream position. Moreover, the same sequence can also function as upstream regulatory element that activates the core promoter in a position- and orientation-dependent manner. In this study, we report the identification and characterization of a negative regulatory element (NRE) upstream of enhancer II (nt 1613 to 1636) which can repress both the enhancer and upstream stimulatory function of the enhancer II sequence in differentiated liver cells. This NRE has marginal inhibitory effect by itself but a strong repressive function in the presence of a functional enhancer II. Mutational analysis reveals that sequence from nt 1616 to 1621 is required for repression of enhancer activity by the NRE. Gel shift analysis reveals that this negative regulatory region can be recognized by a specific protein factor(s) present at the 0.4 M NaCl fraction of HepG2 nuclear extracts. The discovery of the NRE indicates that HBV gene transcription is controlled by combined effects of both positive and negative regulation. It also provides a unique system with which to study the mechanism of negative regulation of gene expression.

Hepatitis B virus (HBV) mutants unable to synthesize HBV e antigen have been described in association with fulminant hepatitis. We have cloned and sequenced the entire viral genome of an HBV strain associated with an epidemic of fulminant hepatitis. This strain contained, in addition to two G-to-A mutations in the precore region (nucleotides 1898 and 1901), numerous other mutations in conserved nucleotide positions resulting in significant amino acid substitutions in HBV gene products. We introduced either or both of the two G-to-A mutations into wild-type HBV by oligonucleotide-directed mutagenesis and generated replication-competent constructs of these mutants as well as the fulminant strain. Viral antigen synthesis, transcription, and replication were analyzed after transfection into human hepatoma cells. All viral constructs produced and secreted similar levels of envelope proteins (HBV surface antigen). Analysis of cellular lysate for core-specific immunoreactivity demonstrated a much higher level of core-associated antigens in cells transfected with the fulminant strain. While cells transfected with mutant and wild-type HBV DNAs synthesized similar levels of viral RNAs, the fulminant strain directed the synthesis of a much higher level of core-associated replicative intermediates (as well as virion particles) than the wild type and mutants with either or both of the precore mutations. Increase in the encapsidation of pregenomic RNA into core particles likely the basis for the enhanced replication associated with the fulminant strain. Our study suggests that an HBV mutant with enhanced viral replication may be important in the pathogenesis of fulminant hepatic failure, and mutations other than the precore mutations may be responsible for this variant behavior.

The carbocyclic analog of 2'-deoxyguanosine (2'-CDG) is a strong inhibitor of hepatitis B virus (HBV) DNA synthesis in HepG2 cells (P.M. Price, R. Banerjee, and G. Acs, Proc. Natl. Acad. USA 86:8543-8544, 1989). We now report that 2'-CDG inhibited duck hepatitis B virus (DHBV) DNA synthesis in primary cultures of duck hepatocytes and in experimentally infected ducks. Like foscarnet (phosphonoformic acid [PFA]) and 2'-,3'-dideoxycytidine (ddC), 2'-CDG blocked viral DNA replication in primary hepatocyte cultures when present during an infection but failed to inhibit the DNA repair reaction that occurs during the initiation of infection to convert virion relaxed circular DNA to covalently closed circular DNA, the template for viral mRNA transcription. Moreover, as for PFA and ddC, viral RNA synthesis was detected when infection was initiated in the presence 2'-CDG. In another respect, however, 2'-CDG exhibited antiviral activity unlike that of ddC or PFA: a single 1-day treatment of hepatocytes with 2'-CDG blocked initiation of viral DNA synthesis for at least 8 days, irrespective of whether DHBV infection was carried out at the time of drug treatment or several days later. Furthermore, orally administered 2'-CDG was long-acting against DHBV in experimentally infected ducklings. Virus replication was delayed by up to 4 days in ducklings infected after administration of 2'-CDG. These observations of long-lasting efficacy in vitro and in vivo even after oral administration suggest that this inhibitor or a nucleoside with similar pharmacological properties may be ideal for reducing virus replication in patients with chronic HBV infection.

To better define the molecules involved in the initial interaction between hepadnaviruses and hepatocytes, we performed binding and infectivity studies with the duck hepatitis B virus (DHBV) and cultured primary duck hepatocytes. In competition experiments with naturally occurring subviral particles containing DHBV surface proteins, these DNA-free particles were found to interfere with viral infectivity if used at sufficiently high concentrations. In direct binding saturation experiments with radiolabelled subviral particles, a biphasic titration curve containing a saturable component was obtained. Quantitative evaluation of both the binding and the infectivity data indicates that the duck hepatocyte presents about 10(4) high-affinity binding sites for viral and subviral particles. Binding to these productive sites may be preceded by reversible virus attachment to a large number of less specific, nonsaturable primary binding sites. To identify which of the viral envelope proteins is responsible for hepatocyte-specific attachment, subviral particles containing only one of the two DHBV surface proteins were produced in Saccharomyces cerevisiae. In infectivity competition experiments, only particles containing the large pre-S/S protein were found to markedly reduce the efficiency of DHBV infection, while particles containing the small S protein had only a minor effect. Similarly, physical binding of radiolabelled serum-derived subviral particles to primary duck hepatocytes was inhibited well only by the yeast-derived pre-S/S particles. Together, these results strongly support the notion that hepadnaviral infection is initiated by specific attachment of the pre-S domain of the large DHBV envelope protein to a limited number of hepatocellular binding sites.

The mechanisms of endothelial cell damage that lead to cerebral hemorrhage are not completely understood. In this study, a cloned murine retrovirus, TR1.3, that uniformly induced stroke in neonatal BALB/c mice is described. Restriction digest mapping suggests that TR1.3 is part of the Friend murine leukemia virus (FMuLV) family. However, unlike mice exposed to other FMuLVs, mice infected with TR1.3 virus developed tremors and seizures within 8 to 18 days postinoculation. This was uniformly followed by paralysis and death within 1 to 2 days. Postmortem examination of TR1.3-inoculated mice revealed edematous brain tissue with large areas of intracerebral hemorrhage. Histologic analysis revealed prominent small vessel pathology including syncytium formation of endothelial cells. Immunohistochemical analysis of frozen brain sections using double fluorescence staining demonstrated that TR1.3 virus specifically infected small vessel endothelial cells. Although infection of vessel endothelial cells was detected in several organs, only brain endothelial cells displayed viral infection associated with hemorrhage. The primary determinant of TR1.3-induced neuropathogenicity was found to reside within a 3.0-kb fragment containing the 3' end of the pol gene, the env gene, and the U3 region of the long terminal repeat. The restricted tropism and acute pathogenicity of this cloned murine retrovirus provide a model for studying virus-induced stroke and for elucidating the mechanisms involved in syncytium formation by retroviruses in vivo.

Hepadnaviruses contain a relaxed circular DNA genome (RC-DNA) with discontinuities in both strands. Upon infectious entry into a host cell, this genome is converted into a covalently closed superhelical form (CCC-DNA), which later serves as the template for transcription. Here we examined whether the viral polymerase activity is required for this repair reaction. Primary hepatocytes prepared from embryonated duck eggs were infected with the duck hepatitis B virus. Conversion of the RC-DNA into the CCC-DNA was then analyzed by a newly developed polymerase chain reaction technique. This method allows the efficient discrimination between the two DNA forms and is sensitive enough to monitor repair of the infecting viral DNA in the absence of replication and amplification. Thus, we were able to monitor this process in the presence of a potent inhibitor of the viral polymerase, the nucleoside analog 2',3'-dideoxyguanosine. The data show that inhibition of the viral polymerase activity has no influence on genome repair, suggesting that this enzymatic function is not required for conversion of the RC-DNA into the CCC-DNA. Consequently, antiviral drugs blocking the polymerase activity cannot prevent the infectious entry of the virus into a host cell.

A hepatitis B virus (HBV) binding factor (HBV-BF) was identified in normal human serum interacting with the pre-S1 and pre-S2 epitopes of the viral envelope located within the protein domains involved in recognition of hepatocyte receptor(s). This molecule was characterized as a 50-kDa glycoprotein showing an isoelectric point of 7.13 with a biological activity depending on its native molecular conformation and on intact sulfhydryl bonds. Monoclonal antibodies to HBV-BF recognized a membrane component of the normal human liver whereas they were unreactive with hepatocyte membranes of other species and with those of the HepG2 cell line. These results suggest that the HBV-BF represents a soluble fragment of the membrane component and can be related to the HBV receptor mediating attachment of HBV to human liver cells.

Human hepatitis delta virus (HDV), obtained from the serum of an experimentally infected woodchuck, was injected into either the peritoneal cavity or the tail vein of both adult CB17 mice and mice with a severe combined immunodeficiency (CB17-scid mice). Three lines of evidence indicated that the virus was able to reach the liver and infect hepatocytes: (i) the amount of HDV genomic RNA detected in the liver by Northern (RNA) analysis increased during the first 5 to 10 days postinoculation, reaching a peak that was about threefold the amount in the original inoculum; (ii) also detected in the liver was the viral antigenomic RNA, which is complementary to the genomic RNA found in virions, and is diagnostic for virus replication; and (iii) by immunoperoxidase staining of liver sections, the delta antigen was detected in the nuclei of scattered cells identifiable as hepatocytes. In all of the mice, clearance of the infection occurred between 10 and 20 days after inoculation. The half-life for clearance was about 3 days in CB17-scid mice, indicating that clearance of infection did not involve a T- and B-cell-dependent immune response. Cell-to-cell spread of the initial infection was not detected. One possible interpretation of our results is that HDV infection of hepatocytes is directly cytopathic. Also, the results imply that chronic infection of the liver in humans may require continuous spread of virus within the liver. Alternatively, HDV in the absence of helper virus may be unable to cause a chronic infection of hepatocytes in vivo.

The susceptibility of rodent hepatocytes to infection by mouse type C retroviruses was examined in vivo and in vitro and compared with the expression of two membrane proteins that function as transporters for the cationic amino acids CAT-1 and CAT-2. CAT-1 expression in rodents determines susceptibility to ecotropic retrovirus infection by serving as the virus receptor. Recently, it has been suggested that CAT-2 may be a receptor for amphotropic murine leukemia virus. In the present study, CAT-1 expression was observed in Hepa1, a cell line derived from a murine hepatoma, and in rat hepatocytes propagated on collagen monolayers in vitro but not in intact or regenerating rat liver in vivo. The expression of CAT-1 correlated with susceptibility to infection by an ecotropic retrovirus encoding beta-galactosidase. CAT-2 expression was observed in hepatocytes in vitro and in vivo, consistent with reports of infection of regenerating and cultured hepatocytes by amphotropic retroviruses. However, introduction of murine CAT-2 into nonpermissive Chinese hamster cells was not sufficient to confer susceptibility to amphotropic retrovirus infection, using a protocol that could easily demonstrate CAT-1-dependent infection by an ecotropic virus. Our data establish CAT-1 as a major determinant of ecotropic retrovirus infection in rodent hepatocytes and suggest that CAT-2 is not a receptor for viruses in the amphotropic subgroup.

The buoyant density of hepatitis C virus (HCV), with high in vivo infectivity (strain H) or low in vivo infectivity (strain F), was determined by sucrose gradient equilibrium centrifugation. Viral RNA of strain H was detected in fractions with densities of < or = 1.09 g/ml (principally approximately 1.06 g/ml), while that of strain F was found in fractions with densities of approximately 1.06 and approximately 1.17 g/ml. The observed difference was confirmed by differential flotation centrifugation; in NaCl solution with a density of 1.063 g/ml, most of the HCV RNA of strain H was detected in the top fraction, while that of strain F appeared in the bottom. The same relationship between buoyant density and infectivity was observed in flotation centrifugation experiments with other HCV strains. In immunoprecipitation experiments with anti-human immunoglobulin, HCV (as measured by HCV RNA) was precipitated from the samples with low infectivity and high density but not from those with high infectivity and low density. Examination of serial sera from a chimpanzee infected with HCV revealed parallel changes in the buoyant density and immunoprecipitability of HCV-associated RNA during the course of infection. These data suggest that HCV is bound to anti-HCV antibodies as antigen-antibody complexes in chronic hepatitis C.

All mammalian hepadnaviruses possess a gene, termed X, that encodes a protein capable of transactivating virus gene expression. The X gene overlaps the polymerase and precore genes as well as two newly identified open reading frames (ORFs) termed ORF5 and ORF6. In this investigation, we examined whether ORF5, ORF6, and the X gene were important for the replication of woodchuck hepatitis virus (WHV) in susceptible woodchucks. First, we investigated whether proteins were produced from ORF5 and ORF6 by in vitro translation of appropriate viral transcripts, searched for antibodies against the putative proteins in the sera of animals infected with wild-type virus, and looked for an antisense WHV transcript, necessary for expression of a protein from ORF6, in the livers of acutely or chronically infected woodchucks. All such experiments yielded negative results. Next, we used oligonucleotide-directed mutagenesis to introduce termination codons into ORF5 and ORF6 at two locations within each ORF. Adult woodchucks in groups of three were transfected with one of the four mutant genomes. All of these woodchucks developed WHV infections that were indistinguishable from those of animals transfected with the wild-type WHV recombinant. Polymerase chain reaction amplification and direct DNA sequencing confirmed that reversion of the mutants to a wild-type genotype did not occur. Taken together, these data indicate that ORF5 and ORF6 are not essential for virus replication and are unlikely to represent authentic genes. Finally, we generated five WHV X-gene mutants that either removed the initiation codon for protein synthesis or truncated the carboxyl terminus of the protein by 3, 16, 31, or 52 amino acids. Groups of three adult woodchucks were transfected with one of the five X-gene mutants. Only the mutant that possessed an X gene lacking 3 amino acids from the carboxyl terminus was capable of replication within the 6-month time frame of the experiment. In contrast, all seven woodchucks transfected with wild-type WHV DNA developed markers consistent with viral infection. Thus, it is likely (P < 0.01) that the WHV X gene is important for virus replication in the natural host.

Hepatitis B virus is a hepatotropic virus. Its replication and gene expression are mainly restricted to hepatocytes in the infective process. The viral gene expression thus provides a unique system with which to study the control of tissue-specific gene expression. We have previously reported the identification and characterization of the second enhancer (enhancer II) of hepatitis B virus. In this report, we further demonstrate that the minimal functional constituents of the second enhancer, box alpha and box beta, display liver cell and differentiation state specificity. Moreover, box alpha exhibits the same liver cell and differentiation state specificity when functioning as an upstream regulator for the basal core promoter. Gel shift experiments reveal a unique box alpha-binding protein, protein a, which is present only in differentiated liver cells, where enhancer II is functional. The converse is true for another box alpha-binding protein, protein f, which is present only in poorly differentiated liver cells and nonliver cells. The simplest hypothesis that explains these results is that protein a activates and/or protein f suppresses the enhancer and upstream regulator functions. Although C/EBP is a candidate for a transcription factor that interacts with box alpha or box beta, none of the binding factors identified in the gel shift assays, including protein a and protein f, is likely to be C/EBP because they differ from C/EBP in heat lability and sequence preference.

Liver specificity of hepatitis B virus (HBV) replication has been attributed to the action of its second enhancer (EII). We report here the characterization of EII and the subsequent isolation of a novel liver-specific DNA-binding protein which binds to and activates EII. The cDNA clone of the protein, designated E2BP, was isolated from a lambda gt11 expression library constructed from the hepatoma cell line HuH-6 which was screened with a binding site probe derived from EII. Sequence analysis of E2BP revealed 86.6% homology with a rat heterogeneous nuclear ribonucleoprotein C protein sequence, while conformational studies suggest a helix-loop-helix motif as a DNA-binding site. Cloned E2BP expressed in human fibroblasts by transient transfection displayed EII binding and activating characteristics similar to those of native E2BP in hepatocytes.

The SJL mouse strain is resistant to infection by some strains of the murine coronavirus mouse hepatitis virus (MHV), such as JHM and A59. The block to virus infection has been variously attributed to defects in virus receptors or virus spread. Since the cellular receptors for MHV, mmCGM1 and mmCGM2, have recently been identified as members of the carcinoembryonic antigen family, we reexamined the possible defectiveness of the MHV receptors in SJL mouse strain. Cloning and sequencing of the cDNAs of both mmCGMs RNAs from SJL mice revealed that they were identical in size to those of the susceptible C57BL/6 (B6) mouse. There was some sequence divergence in the N terminus of the mmCGM molecules between the two mouse strains, resulting in a different number of potential glycosylation sites. This was confirmed by in vitro translation of the mmCGM RNAs, which showed that the glycosylated mmCGM2 of SJL was smaller than that of B6 mice. However, transfection of either mmCGM1 or mmCGM2 from SJL mice into MHV-resistant Cos 7 cells rendered the cells susceptible to MHV infection. The ability of the SJL mmCGM molecules to serve as MHV receptors was comparable to that of those from B6. These molecules are expressed in SJL mouse brain and liver in a similar ratio and in amounts equivalent to those in the B6 mouse. Furthermore, we demonstrated that an SJL-derived cell line was susceptible to A59 but resistant to JHM infection. We concluded that the MHV receptor molecules in the SJL mouse are functional and that the resistance of SJL mice to infection by some MHV strains most likely results from some other factor(s) required for virus entry or some other step(s) in virus replication.

Two regions of the hepatitis B virus (HBV) genome have been shown to display properties of a transcriptional enhancer. Enhancer 1 is active in most hepatoma lines examined as well as in some non-hepatocyte-derived cell lines. In contrast, enhancer 2 activity is strictly liver specific. In this study, we show that adenovirus E1A expression in the highly differentiated human hepatoma line Huh6 strongly inhibits HBV enhancer 2-stimulated transcription while having no effect on HBV enhancer 1 activity. A sequence motif in HBV enhancer 2 which is essential for its enhancer function is the target for E1A-mediated repression. The repression of HBV enhancer 2 activity is mediated through the N-terminal region of the E1A proteins known to bind a 300-kDa cellular protein. Our results suggest that HBV enhancer function may be modulated by a cellular mechanism similar to E1A-mediated transcriptional repression.

Direct inoculation of marmoset livers with an in vitro transcription mixture containing cDNA and full-length genomic RNA transcripts of hepatitis A virus resulted in acute viral hepatitis. Elevations in serum levels of liver enzymes were correlated with appearance of antibody to hepatitis A virus. Genomes of infectious hepatitis A virus isolated from the feces of transfected marmosets contained the same mutation as the cDNA template used for transfection. Liver biopsies confirmed that the virus encoded by the cDNA clone induced histopathological changes equivalent to those caused by virulent wild-type virus.

A number of naturally occurring hepatitis B virus mutants that cannot synthesize the virus precore protein have been identified. Such mutants have been associated with more severe forms of hepatitis, including fulminant hepatitis. The most common mutation observed is a substitution of G to A in the distal precore gene that converts a codon specifying Trp (TGG) to a termination codon (TAG). Using oligonucleotide-directed mutagenesis, we have produced the same point mutation in the precore gene of an infectious clone of woodchuck hepatitis virus (WHV). Transfection of mutant WHV DNA into the livers of adult woodchucks resulted in replication of the mutant in three of three susceptible animals. Levels of virus replication and transient elevations in liver enzymes in serum were similar to those of adult animals infected with wild-type WHV. Virions, found to possess mutant precore genes by polymerase chain reaction amplification and DNA sequencing, were recovered from the serum of one of the animals and inoculated subcutaneously into neonatal woodchucks. They produced infection in all five animals studied. The level of virus replication in neonatal animals infected with this mutant virus was comparable to that found in neonatal woodchucks infected with wild-type WHV, but none of five woodchucks infected with the precore mutant virus as neonates became chronic virus carriers. It was concluded that the precore gene of the WHV genome is not essential for virus replication in the natural host but may be important for chronic infection.

The pH dependency for initiation of infection by the hepadnavirus duck hepatitis B virus (DHBV) was investigated in primary duck hepatocytes. First, an infection assay was developed using a radioimmunoblot to measure DHBV e antigen secreted into tissue culture fluid from infected hepatocytes. The quantity of this viral marker was proportional to the duration of inoculation and the amount of DHBV used as inoculum. The role of pH in initiation of DHBV infection was investigated by using this assay, but no dependence on low pH was found. DHBV was able to infect hepatocytes in the presence of NH4Cl and monensin, agents that raise the pH in intracellular vesicles and prevent penetration of viruses dependent on low pH in endosomes. In control experiments, infection by Semliki Forest virus, which is low pH dependent, was inhibited, whereas herpes simplex virus type 1 infection, which is pH independent, occurred. Attempts to trigger DHBV-cell fusion by exposure of DHBV prebound to hepatocytes to mildly acidic pH were unsuccessful. In these experiments, it was also observed that internalization of DHBV occurred only between pH 6.8 and 8.0. Additionally, in the absence of cells, infectivity of DHBV was stable at pH 4.6 to 4.8, which is lower than the pH encountered in endosomes (pH 5 to 6.6). Thus, no evidence for a role for mildly acidic pH in the initiation of DHBV infection was found. Therefore, we propose that the infection route followed by DHBV resembles that of the group of enveloped viruses, including herpesviruses, that fuse with their host cells at neutral pH.

A study was carried out to determine some of the factors that might distinguish transient from chronic hepadnavirus infection. First, to better characterize chronic infection, Pekin ducks, congenitally infected with the duck hepatitis B virus (DHBV), were used to assess age-dependent variations in viremia, percentage of DHBV-infected hepatocytes, and average levels of DNA replication intermediates in the cytoplasm and of covalently closed circular DNA in the nuclei of infected hepatocytes. Levels of viremia and viral DNA were found to peak at about the time of hatching but persisted at relatively constant levels in chronically infected birds up to 2 years of age. The percentage of infected hepatocytes was also constant, with DHBV replication in virtually 100% of hepatocytes in all birds. Next, we found that adolescent ducks inoculated intravenously with a large dose of DHBV also developed massive infection of hepatocytes with an early but low-level viremia, followed by rapid development of a neutralizing antibody response. No obvious quantitative or qualitative differences between transiently and chronically infected liver tissue were detected in the intracellular markers of viral replication examined. However, in the adolescent duck experiment, DHBV infection was rapidly cleared from the liver even when up to 80% of hepatocytes were initially infected. In all of these ducks, clearance of infection was accompanied by only a mild hepatitis, with no evidence that massive cell death contributed to the clearance. This finding suggested that mechanisms in addition to immune-mediated destruction of hepatocytes might make major contributions to clearance of infections, including physiological turnover of hepatocytes in the presence of a neutralizing antibody response and/or spontaneous loss of the capacity of hepatocytes to support virus replication.

Hepatitis delta virus (HDV) particles were produced in Huh7 human hepatoma cells by transfection with cloned hepatitis B virus (HBV) DNA and HDV cDNA. The particles were characterized by their buoyant density, the presence of encapsidated viral RNA, and their ability to infect primary cultures of chimpanzee hepatocytes. Successful infection was evidenced by the appearance of increasing amounts of intracellular HDV RNA after exposure to particles. Infection was prevented when particles were incubated with antibodies directed against synthetic peptides specific for epitopes of the pre-S1 or pre-S2 domains of the HBV envelope proteins before exposure to hepatocytes. These data demonstrate that HDV particles produced in vitro are infectious and indicate (i) that infectious particles are coated with HBV envelope proteins that contain the pre-S1 and pre-S2 regions, (ii) that epitopes of the pre-S1 and pre-S2 domains of HBV envelope proteins are exposed at the surface of HDV particles, and (iii) that antibodies directed against those epitopes have neutralizing activity against HDV.

Many diverse tumors contain cells that select for mutations at the p53 gene locus. This appears to be the case because the p53 gene product can act as a negative regulator of cell division or a tumor suppressor. These mutations then eliminate this activity of the p53 gene product. The simian virus 40 (SV40) large T antigen binds to p53 and acts as an oncogene to promote cellular transformation and initiate tumors. If the binding of T antigen to the p53 protein inactivated its tumor suppressor activity, there would be no selection pressure for p53 mutants to appear in tumors. To test this idea, transgenic mice that carried and expressed the SV40 large T-antigen gene were created. Expression of the T antigen was directed to the liver, using the albumin promoter, and the choroid plexus, using the SV40 enhancer-promoter. A large number of papillomas (indicated in parentheses) of the choroid plexus (14), hepatocellular carcinomas (5), liver adenomas (10), and tumors of clear-cell foci (5) were examined for mutant and wild-type p53 genes and gene products. In all cases, the tumor extracts contained readily detectable T-antigen-p53 protein complexes. A monoclonal antibody specifically recognizing the wild-type p53 protein (PAb246) reacted with p53 in every tumor extract. A monoclonal antibody specifically recognizing mutant forms of the p53 protein (PAb240) failed to detect p53 antigens in these extracts. Finally, p53 partial cDNAs were sequenced across the regions of common mutations in this gene, and in every case only the wild-type sequence was detected. These results strongly support the hypothesis that T antigen inactivates the wild-type p53 tumor-suppressing activity and there is no need to select for mutations at the p53 locus.

Large epidemic outbreaks of enterically transmitted non-A, non-B viral hepatitis (ET-NANBH) have been documented in developing countries. A molecular clone derived from the causative agent, the hepatitis E virus (HEV), has recently been described (G.R. Reyes, M.A. Purdy, J.P. Kim, K.-C. Luk, L.M. Young, K.E. Fry, and D. Bradley, Science 247:1335-1339, 1990). We now report the isolation, by serologic screening, of two cDNA clones derived from a fecal sample collected during a 1986 outbreak of ET-NANBH in Telixtac, Mexico. The cDNA clones encode epitopes that specifically reacted with acute- and convalescent-phase sera collected during five different ET-NANBH epidemics and represent the initial cloning of the Mexico strain of HEV. Recombinant fusion proteins expressed from these clones were also recognized by antibodies from cynomolgus macaques experimentally infected with HEV. The cDNA clones were shown to be derived from HEV by their specific hybridization to the previously recognized full-length genomic RNA transcript of approximately 7.5 kb. In addition, however, subgenomic polyadenylated transcripts of approximately 2.0 and approximately 3.7 kb were also identified in HEV-infected cynomolgus monkey liver. Sequences homologous to the epitope clones were isolated from the Burma strain of the virus, and these demonstrated reactivity comparable to that seen with the Mexico strain epitopes. When compared with the available full-length sequence of the Burma strain of HEV, it was discovered that the cDNA clones were encoded in different open reading frames (ORFs). The comparison between Mexico and Burma HEV strains indicated amino acid homologies of 90.5 and 73.5% for these epitope-encoding clones derived from ORF2 and ORF3, respectively. The identification of these clones not only has provided insight into the expression strategy of HEV but has also resulted in a source of recombinant protein useful in the diagnosis of HEV-induced hepatitis.

Generation of replicative defective viruses is frequently observed during viral infections. We now report that encapsidation and reverse transcription of spliced viral RNA is an additional mechanism for synthesis of defective viral particles. We have investigated the in vivo synthesis of a spliced hepatitis B virus (HBV) RNA. By using the polymerase chain reaction with different sets of primers on DNA purified from infected livers and the HepG2 HBV cell line, we detected a subgenomic HBV DNA complementary to the spliced viral RNA. Its nucleotide sequence was found to be identical to that previously described for the spliced RNA. This HBV RNA is packaged and reverse transcribed in vivo, the cDNA being incorporated into circulating particles. This finding establishes the synthesis of spliced HBV RNA in vivo and indicates that its reverse transcription can give rise to defective viruses.

Callitrichid hepatitis (CH) is an acute, often fatal viral infection of New World primates from the family Callitrichidae. The etiologic agent of CH is unknown. We report here the isolation of an arenavirus from a common marmoset (Callithrix jacchus) with CH by using in vitro cultures of marmoset hepatocytes and Vero-E6 cells. Enveloped virions 67 to 133 nm in diameter with ribosomelike internal structures were seen in infected cultures. Immunofluorescence and Western immunoblot analysis using CH-specific antisera (principally from animals exposed to CH during zoo outbreaks) revealed three antigens in cells infected with this CH-associated virus (CHV). These antigens had the same electrophoretic mobilities on sodium dodecyl sulfate-polyacrylamide gels as did the nucleocapsid, GP2, and GPC proteins of lymphocytic choriomeningitis virus (LCMV). Monoclonal antibodies specific for these arenavirus proteins also reacted with the three CHV antigens. Conversely, the CH-specific antisera reacted with the nucleocapsid, GP2, and GPC proteins of LCMV. CHV thus appears to be a close antigenic relative of LCMV. The serologic association of CHV with several CH outbreaks implicate it as the etiologic agent of this disease.

The transcriptional map of hepatitis B virus (HBV) has been expanded recently by the discovery of a singly spliced transcript in hepatoma cell lines transfected with cloned viral DNA and a doubly spliced one in naturally infected human liver tissues. By the use of reverse transcription and a subsequent polymerase chain reaction, the two spliced HBV RNAs were shown to be present in both types of cells. As further evidence, an HBV mutant was constructed and found to exclusively express the singly spliced RNA. This mutant was also used to quantitate the two spliced species in transfected HepG2 cells; they were found to be equally abundant, and each represented about 30% of the pregenomic RNA. The HBV mutant could still produce replication-competent HBV virions when transfected into HepG2 cells, indicating that the doubly spliced transcript, just like the singly spliced one, was not essential for HBV replication. However, the two abundant spliced HBV transcripts were detected in most naturally infected human liver tissues, suggesting that they may have biologic functions in vivo.

In adult rats infected with Borna disease virus, the virus was found exclusively in the brain, whereas in cyclosporine A-treated rats, infectious virus was also detected in peripheral nerve fibers and, unexpectedly, in adjacent organ-specific cells. In contrast to untreated virus-infected rats, no major histocompatibility complex class II expression was found in the brain of cyclosporine A-treated animals.

The hepatitis B virus X protein acts as a transcriptional transactivator in vitro. To elucidate possible biological effects of X protein on liver cells in vivo, we generated four lines of transgenic mice carrying the X gene open reading frame under the control of the human alpha-1-antitrypsin regulatory region. The plasmid construct used to introduce the transgene was shown to encode a 16-kDa X protein with transactivating capability. The expression of X protein was detectable in liver tissue of transgenic animals of three of the lines by immunoblot analysis; levels of expression were highest in the first month after birth of the animals. Over 80 animals from the expressing lines were examined histologically. Most transgenic mice, some of which were observed for up to 2 years, remained normal. However, a few transgenic animals developed mild focal hepatitis, nuclear pleomorphism, focal necrosis, and/or nodular hyperplasia in the liver. Increased mitotic activity of hepatocytes also was observed. We conclude that, at the level of expression achieved in these transgenic mice, the hepatitis B virus transcriptional transactivator X protein alone does not appear to mediate the development of serious liver damage or hepatocellular carcinomas.

Callitrichid hepatitis (CH) is an acute, frequently fatal viral hepatitis which affects members of the primate family Callitrichidae (R. J. Montali, E. C. Ramsay, C. B. Stephensen, M. Worley, J. A. Davis, and K. V. Holmes, J. Infect. Dis. 160:759-765, 1989; E. C. Ramsay, R. J. Montali, M. Worley, C. B. Stephensen, and K. V. Holmes, J. Zoo Wildlife Med. 20:178-183, 1989). Outbreaks of the disease occur in zoos and animal parks. In this study, CH-specific antigens were identified in the livers of infected animals by using immune sera from primates with CH and CH-exposed asymptomatic animals. Three CH-specific antigens with apparent molecular masses of 34, 54, and 65 kDa were identified. A polyclonal antiserum was raised against the 54-kDa antigen. These antigens were not found in the livers of uninfected animals and may be viral proteins. Our results suggest that at least five of the six outbreaks of CH considered here were caused by the same virus or by an antigenically related virus.

Six plaque-purified virus isolates were obtained from liver and heart tissues of a DBA/2 mouse infected 7 days earlier with 10(4) PFU of coxsackievirus group B type 3. Each virus isolate was assayed in vitro for infectivity to vascular endothelial cells (VEC) of the liver, lungs, and heart. Both the percentage of VEC infected and the mean progeny PFU produced per infected VEC were determined. Virus isolates from the heart showed greater infectivity and replication in heart VEC than in VEC derived from either the liver or lungs. Similarly, virus isolated from the liver preferentially infected liver VEC. Virus receptor expression varied between VEC populations, as demonstrated by binding studies with a [35S]methionine-radiolabeled heart virus and by enzyme-linked immunoadsorption assay studies with a monoclonal antibody to the coxsackievirus group B type 3 receptor on heart tissue. Finally, the heart and liver virus isolates were injected (10(4) PFU) intraperitoneally into BALB/c mice. After 7 days, the animals were sacrificed, and the hearts, livers, and lungs were evaluated for tissue injury and virus concentrations. Viruses originally isolated from the heart preferentially infected the heart when reinjected into animals and caused severe myocarditis. Viruses originally derived from the liver most consistently reinfected the liver, although significant virus concentrations were also detected in the heart. The liver virus isolates, however, were incapable of causing myocarditis. Thus, selective tropism of viruses for particular organs in vivo corresponds to the ability of these isolates to infect VEC in vitro.

Reovirus type 1 strain Lang is restricted from replicating in adult murine livers. In noninjured livers, approximately 1% of hepatocytes express reovirus antigen during infection. However, hepatocytes can be induced to express reovirus antigen if challenged with either toxins or trauma. We used selective hepatotoxins or surgical trauma to demonstrate that reovirus antigen localization in liver is determined by the site of hepatocellular insult and the timing of the virus inoculum.

The receptor for mouse hepatitis virus strain A59 (MHV-A59) is a 110- to 120-kilodalton (kDa) glycoprotein which is expressed in MHV-susceptible mouse strains on the membranes of hepatocytes, intestinal epithelial cells, and macrophages. SJL/J mice, which are highly resistant to MHV-A59, were previously shown to lack detectable levels of receptor by using either solid-phase virus receptor assays or binding of a monoclonal anti-receptor antibody (MAb) which blocks infection of MHV-susceptible mouse cells. This MAb was used for affinity purification of the receptor glycoprotein from livers of MHV-susceptible Swiss Webster mice. The MHV receptor and an antigenically related protein of 48 to 58 kDa were copurified and then separated by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The first 15 amino acids of the receptor were sequenced, and a synthetic peptide of this amino acid sequence was prepared. Rabbit antiserum made against this peptide bound to the MHV receptor glycoprotein and the 48- to 58-kDa protein from livers of MHV-susceptible BALB/c mice and Swiss Webster mice and from the intestinal brush border of BALB/c mice. In immunoblots of intestinal brush border and hepatocyte membranes of MHV-resistant SJL/J mice, the antibody against the amino terminus of the receptor identified proteins that are 5 to 10 kDa smaller than the MHV receptor and the 48- to 58-kDa related protein from Swiss Webster or BALB/c mice. Thus, SJL/J mice express a protein which shares some sequence homology with the MHV receptor but which lacks virus-binding activity and is not recognized by the blocking anti-receptor MAb. These results suggest that resistance of SJL/J mice to MHV-A59 may be due to absence or mutation of the virus-binding domain in the nonfunctional receptor homolog in SJL/J mice.

Liver tissue from animals that died of rabbit hemorrhagic disease (RHD) was used to identify the causative agent. After extraction of liver homogenates and sucrose density gradient ultracentrifugation, distinct bands were obtained. The respective gradient fractions reacted positively in an enzyme-linked immunosorbent assay as well as in hemagglutination assays and were infective for rabbits. These fractions contained virions which had a diameter of 40 nm and resembled morphologically those of the family Caliciviridae. By immunoblotting, a major structural protein with a molecular weight of 60,000 was identified. Highly pure RNA of about 8 kilobases was isolated from virions. Labeled cDNA synthesized from virion RNA detected two RNAs of 8 and 2 kilobases in Northern (RNA) blots of liver RNA from animals infected with RHD virus. Finally, isolated virion RNA injected into the liver of rabbits produced a disease with clinical symptoms and pathological findings typical of RHD. We conclude that a calicivirus represents the causative agent of RHD.

Primary duck hepatocytes were infected with a mutant duck hepatitis B virus defective in envelope protein but competent for viral DNA synthesis. Cells infected by this mutant accumulated higher levels of viral covalently closed, circular DNA (cccDNA) than those infected by wild-type virus. The accumulation of high levels of cccDNA was due to a failure of the mutant-infected cells to suppress de novo cccDNA synthesis compared with suppression by cells infected by the wild type. The envelope-defective virus failed to establish a persistent infection in vitro, possibly because of a virus-mediated cell death. Therefore, one or both viral envelope proteins are required for regulation of cccDNA synthesis and for maintenance of persistent infection in vitro.