Ribavirin induces error-prone replication of GB virus B in primary tamarin hepatocytes.

GB virus B (GBV-B) is the closest relative of hepatitis C virus (HCV) and is an attractive surrogate model for HCV antiviral studies. GBV-B induces an acute, resolving hepatitis in tamarins. Utilizing primary cultures of tamarin hepatocytes, we have previously developed a tissue culture system that exhibits high levels of GBV-B replication. In this report, we have extended the utility of this system for testing antiviral compounds. Treatment with human interferon provided only a marginal antiviral effect, while poly(I-C) yielded >3 and 4 log units of reduction of cell-associated and secreted viral RNA, respectively. Interestingly, treatment of GBV-B-infected hepatocytes with ribavirin resulted in an approximately 4-log decrease in viral RNA levels. Guanosine blocked the antiviral effect of ribavirin, suggesting that inhibition of IMP dehydrogenase (IMPDH) and reduction of intracellular GTP levels were essential for the antiviral effect. However, mycophenolic acid, another IMPDH inhibitor, had no antiviral effect. Virions harvested from ribavirin-treated cultures exhibited a dramatically reduced specific infectivity. These data suggest that incorporation of ribavirin triphosphate induces error-prone replication with concomitant reduction in infectivity and that reduction of GTP pools may be required for incorporation of ribavirin triphosphate. In contrast to the in vitro studies, no significant reduction in viremia was observed in vivo following treatment of tamarins with ribavirin during acute infection with GBV-B. These findings are consistent with the observation that ribavirin monotherapy for HCV infection decreases liver disease without a significant reduction in viremia. Our data suggest that nucleoside analogues that induce error-prone replication could be an attractive approach for the treatment of HCV infection if administered at sufficient levels to result in efficient incorporation by the viral polymerase.

DNA microarray analysis of chimpanzee liver during acute resolving hepatitis C virus infection.

Hepatitis C virus (HCV) poses a worldwide health problem in that the majority of individuals exposed to HCV become chronically infected and are predisposed for developing significant liver disease. DNA microarray technology provides an opportunity to survey transcription modulation in the context of an infectious disease and is a particularly attractive approach in characterizing HCV-host interactions, since the mechanisms underlying viral persistence and disease progression are not understood and are difficult to study. Here, we describe the changes in liver gene expression during the course of an acute-resolving HCV infection in a chimpanzee. Clearance of viremia in this animal occurred between weeks 6 and 8, while clearance of residual infected hepatocytes did not occur until 14 weeks postinfection. The most notable changes in gene expression occurred in numerous interferon response genes (including all three classical interferon antiviral pathways) that increased dramatically, some as early as day 2 postinfection. The data suggest a biphasic mechanism of viral clearance dependent on both the innate and adaptive immune responses and provide insight into the response of the liver to a hepatotropic viral infection.

The chimpanzee model of hepatitis C virus infections.

The chimpanzee (Pan troglodytes) is the only experimental animal susceptible to infection with hepatitis C virus (HCV). The chimpanzee model of HCV infection was instrumental in the initial studies on non-A, non-B hepatitis, including observations on the clinical course of infection, determination of the physical properties of the virus, and eventual cloning of the HCV nucleic acid. This review focuses on more recent aspects of the use of the chimpanzee in HCV research. The chimpanzee model has been critical for the analysis of early events in HCV infection because it represents a population for which samples are available from the time of exposure and all exposed animals are examined. For this reason, the chimpanzee represents a truly nonselected population. In contrast, human cohorts are often selected for disease status or antibody reactivity and typically include individuals that have been infected for decades. The chimpanzee model is essential to an improved understanding of the factors involved in viral clearance, analysis of the immune response to infection, and the development of vaccines. The development of infectious cDNA clones of HCV was dependent on the use of chimpanzees, and they will continue to be needed in the use of reverse genetics to evaluate critical sequences for viral replication. In addition, chimpanzees have been used in conjunction with DNA microarray technology to probe the entire spectrum of changes in liver gene expression during the course of HCV infection. The chimpanzee will continue to provide a critical aspect to the understanding of HCV disease and the development of therapeutic modalities.

GB virus B as a model for hepatitis C virus.

GB viruses A and B (GBV-A and GBV-B) are members of the Flaviviridae family and are isolated from tamarins injected with serum from a human hepatitis patient. Along with a related human virus, GB virus C, or alternatively, hepatitis G virus (GBV-C/HGV), the three viruses represent the GB agents. Of the three viruses, GBV-B has been proposed as a potential surrogate model for the study of hepatitis C virus (HCV) infections of humans. GBV-B is phylogenetically most closely related to HCV and causes an acute, self-resolving hepatitis in tamarins as indicated by an increase in alanine aminotransferase and changes in liver histology. Similarities between GBV-B and HCV are found at the nucleotide sequence level with the two viruses sharing 28% amino acid homology over the lengths of their open reading frames. Short regions have even higher levels of homology that are functionally significant as shown by the ability of the GBV-B NS3 protease to cleave recombinant HCV polyprotein substrates. The shared protease substrate specificities suggest that GBV-B may be useful in testing antiviral compounds for activity against HCV. Although there are numerous similarities between GBV-B and HCV, there are important differences in that HCV frequently causes chronic infections in people, whereas GBV-B appears to cause only acute infections. The acute versus chronic course of infection may point to important differences between the two viruses that, along with the numerous similarities, will make GBV-B in tamarins a good surrogate model for HCV.

Protective immune response to hepatitis C virus in chimpanzees rechallenged following clearance of primary infection.

Hepatitis C virus (HCV) infections were evaluated in chimpanzees that had previously cleared HCV and were rechallenged. Animals that had previously cleared HCV infection rapidly cleared homologous and heterologous virus upon rechallenge, indicative of a strong protective immunity. In one animal, sterilizing immunity was observed with regard to viremia, although viral RNA was transiently detected in the liver. Accelerated viral clearance following rechallenge with HCV was observed in animals that had not been exposed to HCV for over 16 years, suggesting that long-lasting protective immunity may be possible. The ability of peripheral blood mononuclear cells (PBMC) to recognize HCV proteins was evaluated during the course of the rechallenge experiments. A very early and strong in vitro recall response to HCV nonstructural proteins appeared to be associated with viral clearance. In contrast, proliferative responses to HCV proteins were not observed in 4 persistently infected chimpanzees, and a weak proliferative response was observed in 1 of 2 animals during acute resolving infection. The results suggest that a strong T-cell proliferative response is induced upon rechallenge of chimpanzees with HCV and that this response is associated with rapid viral clearance. The antibody response to HCV proteins increased by over 1,000-fold in all animals following rechallenge as well. A more complete understanding of the role of the cellular immune response in the clearance of HCV and the nature of the protective immune response following viral clearance may aid in the generation of therapies and vaccines.

Interaction between hepatitis B virus core protein and reverse transcriptase.

Previous mutagenesis studies with hepatitis B virus (HBV) suggest that continued interactions with core are required for several steps in genomic replication. To examine core-polymerase (Pol) interactions, insect cells were coinfected with baculovirus constructs that independently expressed core and Pol. The results demonstrated several features with implications that core plays an interactive role with HBV Pol: (i) core coprecipitated with constructs expressing full-length Pol as well as the terminal protein (TP), reverse transcriptase (RT) and RNase H domains of Pol, independently; (ii) coprecipitation of core was not dependent on the presence of an epsilon stem-loop sequence; and (iii) core-Pol complexes migrated as intact capsid particles, as detected by sucrose gradient analysis. To analyze the structural and sequence requirements of core in recognition of Pol, a series of core mutants with two- to four-amino-acid insertions or carboxy-terminal deletions were assessed for Pol interaction. The results indicated that capsid formation is required but not sufficient for interaction with Pol and that the TP and RT domains of Pol have different requirements for interaction with core. To map the core binding sites on Pol, a panel of amino- and carboxy-terminal deletion mutants of the TP and RT domains of Pol were analyzed for interaction with core. At least three separate core binding sites on Pol were detected. This analysis begins to define basic requirements for core-Pol interactions, but further study is necessary to delineate the effects of these interactions on encapsidation and genome replication.

Development of a primary tamarin hepatocyte culture system for GB virus-B: a surrogate model for hepatitis C virus.

GB virus-B (GBV-B) causes an acute hepatitis in tamarins characterized by increased alanine transaminase levels that quickly return to normal as the virus is cleared. Phylogenetically, GBV-B is the closest relative to hepatitis C virus (HCV), and thus GBV-B infection of tamarins represents a powerful surrogate model system for the study of HCV. In this study, the course of infection of GBV-B in tamarins was followed using a real-time 5' exonuclease (TaqMan) reverse transcription-PCR assay to determine the level of GBV-B in the serum. Peak viremia levels exceeded 10(9) genome equivalents/ml, followed by viral clearance within 14 to 16 weeks. Rechallenge of animals that had cleared infection resulted in viremia that was limited to 1 week, suggestive of a strong protective immune response. A robust tissue culture system for GBV-B was developed using primary cultures of tamarin hepatocytes. Hepatocytes obtained from a GBV-B-infected animal maintained high levels of cell-associated viral RNA and virion secretion for 42 days of culture. In vitro infection of normal hepatocytes resulted in rapid amplification of cell-associated viral RNA and secretion of up to 10(7) genome equivalents/ml of culture supernatant. In addition, infection could be monitored by immunofluorescence staining for GBV-B nonstructural NS3 protein. This model system overcomes many of the current obstacles to HCV research, including low levels of viral replication, lack of a small primate animal model, and lack of a reproducible tissue culture system.

Human monoclonal antibodies that inhibit binding of hepatitis C virus E2 protein to CD81 and recognize conserved conformational epitopes.

The intrinsic variability of hepatitis C virus (HCV) envelope proteins E1 and E2 complicates the identification of protective antibodies. In an attempt to identify antibodies to E2 proteins from divergent HCV isolates, we produced HCV E2 recombinant proteins from individuals infected with HCV genotypes 1a, 1b, 2a, and 2b. These proteins were then used to characterize 10 human monoclonal antibodies (HMAbs) produced from peripheral B cells isolated from an individual infected with HCV genotype 1b. Nine of the antibodies recognize conformational epitopes within HCV E2. Six HMAbs identify epitopes shared among HCV genotypes 1a, 1b, 2a, and 2b. Six, including five broadly reactive HMAbs, could inhibit binding of HCV E2 of genotypes 1a, 1b, 2a, and 2b to human CD81 when E2 and the antibody were simultaneously exposed to CD81. Surprisingly, all of the antibodies that inhibited the binding of E2 to CD81 retained the ability to recognize preformed CD81-E2 complexes generated with some of the same recombinant E2 proteins. Two antibodies that did not recognize preformed complexes of HCV 1a E2 and CD81 also inhibited binding of HCV 1a virions to CD81. Thus, HCV-infected individuals can produce antibodies that recognize conserved conformational epitopes and inhibit the binding of HCV to CD81. The inhibition is mediated via antibody binding to epitopes outside of the CD81 binding site in E2, possibly by preventing conformational changes in E2 that are required for CD81 binding.

Hypervariable region 1 sequence stability during hepatitis C virus replication in chimpanzees.

The putative envelope 2 (E2) gene of hepatitis C virus (HCV) contains a highly variable region referred to as hypervariable region 1 (HVR1). We hypothesized that this genetic variability is driven by immune selection pressure, rather than representing the accumulation of random mutations in a region with relatively little functional constraint. To test this hypothesis, we examined the E2 sequence of a human inoculum that was passaged through eight chimpanzees, which appear to have a replicative rate (opportunity for chance mutation) similar to that of humans. Acute-phase plasma samples from a human (the inoculum) and six of eight serially infected chimpanzees were studied. For each, 33 cloned cDNAs were examined by a combined heteroduplex-single-stranded conformational polymorphism assay to assess quasispecies complexity and optimize selection of clones with unique gel shift patterns (clonotypes) for sequencing. The sequence diversity of HCV was significantly lower in the chimpanzees than in the humans, and during eight serial passages there was no change in the sequence of the majority clonotype from each animal examined. Similarly, the rates of protein sequence altering (nonsynonymous) substitution were lower in the chimpanzees than in the humans. These findings demonstrate that nonsynonymous mutations indicate selection pressure rather than being an incidental result of HCV replication.

Hepadnavirus infection in captive gibbons.

The recent isolation of a nonhuman primate hepadnavirus from woolly monkeys prompted an examination of other primates for potentially new hepadnaviruses. A serological analysis of 30 captive gibbons revealed that 47% were positive for at least one marker of ongoing or previous infection with a hepatitis B virus (HBV). The amino acid sequences of the core and surface genes of human and gibbon virus isolates were very similar. Phylogenetic analysis indicated that the gibbon isolates lie within the human HBV family, indicating that these HBV isolates most likely stem from infection of gibbons from a human source.

Molecular quantification of cell cycle-related gene expression at the protein level.

BACKGROUND: Immunofluorescence cytometry of antigen and DNA content provides relative measurements of the cell cycle phase distribution of a specific epitope. Measurement of correlated expression of epitopes on signaling and regulatory proteins will be useful in the study of the complex pathways involved in cell cycle regulation and carcinogenesis. However, to formulate regulatory pathway models, measurements of molecules per cell would be more useful than relative measurements of intensity. Here, we report on a system in which the relationship between molecules and fluorescence is determined for a reference set of cell lines that are then used to directly calculate the number of molecules for unknowns. To demonstrate the process, we calculated the cell cycle phase distribution of SV40 large T antigen (Tag) in the reference cells. METHODS: A set of cell line clones expressing different levels of Tag were isolated. Quantitative Western blots of these cells and purified, recombinant Tag were performed. Cells from the same sample were stained and analyzed by flow cytometry for Tag and DNA. The relationship between molecules and fluorescence was established and calculations were performed for the phase distributions of Tag. RESULTS: The five cell lines had 0.11, 0.27, 1.06, 2.44, and 2.63 x 10(6) molecules of Tag per cell, determined by Western blot. The average coefficient of variation was 10.6%. The relationship of molecules to fluorescence fit a linear equation (r(2) = 0.96) over the range, 0.11 - 2.63 x 10(6) molecules, however, the same equation did not fit the relationship between 0 molecules, defined by isotype staining controls, and the lowest expressing cell line. To calculate the phase distributions of molecules in the lowest cell line, a second linear equation from 0 to 110,000 molecules was used. CONCLUSIONS: This work describes a system where fixed cells expressing various levels of a target antigen quantified by Western blots can be used to standardize flow cytometric measurements of gene expression in absolute terms.

Effects of iron loading on pathogenicity in hepatitis C virus-infected chimpanzees.

Elevated iron levels have been associated with raised serum alanine transaminase (ALT) levels in hepatitis C virus (HCV)-infected humans. However, it is not clear if HCV infection causes increased iron accumulation by the liver or if the severity of HCV infection is actually worsened by higher iron levels in the host. To better understand the relationship between iron and persistent HCV infections, we examined the effect of excess dietary iron on disease severity in HCV-infected chimpanzees. Iron was supplemented in the diets of four HCV-infected and two uninfected chimpanzees for 29 weeks to achieve iron loading. Iron loading was confirmed by increases in serum iron levels, percentages of transferrin saturation, ferritin levels, elevations in hepatic iron concentration (HIC), and by histological examination. The majority of HCV-infected chimpanzees had higher iron levels before iron feeding than the uninfected animals. Although various degrees of iron loading occurred in all chimpanzees, HCV-infected animals exhibited increased loading in comparison with uninfected animals. The effects of iron loading on HCV disease expression was determined by comparing disease parameters during an extended baseline period before iron loading with the period during iron loading and immediately following iron loading. Iron loading did not influence the viral load, but did exacerbate liver injury in HCV-infected chimpanzees, as evidenced by elevated ALT and histological changes. Because all chimpanzees on high iron diets experienced iron loading, but pathological effects were only observed in HCV-infected chimpanzees, HCV infection appears to increase the susceptibility of the liver to injury following iron loading. These results confirm and extend previous observations made in human populations and serve to further validate the chimpanzee model of chronic hepatitis C.

Properties of monoclonal antibodies directed against hepatitis B virus polymerase protein.

Hepadnavirus polymerases are multifunctional enzymes that play critical roles during the viral life cycle but have been difficult to study due to a lack of a well-defined panel of monoclonal antibodies (MAbs). We have used recombinant human hepatitis B virus (HBV) polymerase (Pol) expressed in and purified from baculovirus-infected insect cells to generate a panel of six MAbs directed against HBV Pol protein. Such MAbs were subsequently characterized with respect to their isotypes and functions in analytical and preparative assays. Using these MAbs as probes together with various deletion mutants of Pol expressed in insect cells, we mapped the B-cell epitopes of Pol recognized by these MAbs to amino acids (aa) 8 to 20 and 20 to 30 in the terminal protein (TP) region of Pol, to aa 225 to 250 in the spacer region, and to aa 800 to 832 in the RNase H domain. Confocal microscopy and immunocytochemical studies using various Pol-specific MAbs revealed that the protein itself appears to be exclusively localized to the cytoplasm. Finally, MAbs specific for the TP domain, but not MAbs specific for the spacer or RNase H regions of Pol, appeared to inhibit Pol function in the in vitro priming assay, suggesting that antibody-mediated interference with TP may now be assessed in the context of HBV replication.

Generation of transmissible hepatitis C virions from a molecular clone in chimpanzees.

Multiple alignments of hepatitis C virus (HCV) polyproteins from six different genotypes identified a total of 22 nonconsensus mutations in a clone derived from the Hutchinson (H77) isolate. These mutations, collectively, may have contributed to the failure in generating a "functionally correct" or "infectious" clone in earlier attempts. A consensus clone was constructed after systematic repair of these mutations, which yielded infectious virions in a chimpanzee after direct intrahepatic inoculation of in vitro transcribed RNAs. This RNA-infected chimpanzee has developed hepatitis and remained HCV positive for more than 11 months. To further verify this RNA-derived infectivity, a second naive chimpanzee was injected intravenously with serum collected from the first chimpanzee. Infectivity analysis of the second chimpanzee demonstrated that the HCV infection was successfully transmitted, which validated unequivocally the infectivity of our repaired molecular clone. Amino acid sequence comparisons revealed that our repaired infectious clone had 4 mismatches with the isogenic clone reported by Kolykhalov et al. (1997, Science 277, 570-574) and 8 mismatches with that reported by Yanagi et al. (1997, Proc. Natl. Acad. Sci. USA 94, 8738-8743). At the RNA level, more mismatches (43 and 67, respectively) were identified; most of them were synonymous substitutions. Further comparisons with 16 isolates from different genotypes demonstrated that our repaired clone shares greater consensus than the reported isogenic clones. This approach of generating infectious HCV RNA validates the importance of amino acid sequence consensus in relation to the biology of HCV.

Mapping of the hepatitis B virus reverse transcriptase TP and RT domains by transcomplementation for nucleotide priming and by protein-protein interaction.

Hepadnavirus polymerases initiate reverse transcription in a protein-primed reaction. We previously described a complementation assay for analysis of the roles of the TP and RT domains of HBV reverse transcriptase (pol) in the priming reaction. Independently expressed TP and RT domains form a complex functional for in vitro priming reactions. To map the minimal functional TP and RT domains, we prepared baculoviruses expressing amino- and carboxyl-terminal deletions of both the TP and RT domains and analyzed the proteins for the ability to participate in transcomplementation for the priming reaction. The minimal TP domain spanned amino acids 20 to 175; however, very little activity was observed without a TP domain spanning amino acids 1 to 199. The minimal RT domain spanned amino acids 300 to 775; however, little activity was observed unless the carboxyl end of the RT domain extended to amino acid 800. Thus, most of the RNase H domain was required. In previous studies, we observed a TP inhibitory domain between amino acids 199 and 344. The current analysis narrowed this domain to residues 300 to 334, which is a portion of the minimal RT domain. In addition, the ability of TP and RT deletion mutants to form stable TP-RT complexes was examined in coimmunoprecipitation assays. The minimal TP and RT domains capable of protein-protein interaction were considerably smaller than the domains required for functional interaction in the transcomplementation assays, and unlike priming activity, TP-RT interaction did not require the epsilon RNA stem-loop. These studies help to further define the complex protein-protein interactions required in HBV genome replication.

Presecretory degradation of apolipoprotein [a] is mediated by the proteasome pathway.

Plasma levels of atherogenic lipoprotein [a] (Lp[a]) vary over a 1000-fold range and are largely determined by the gene for its unique glycoprotein, apolipoprotein [a] (apo[a]). The apo[a] locus comprises more than 100 alleles, encoding proteins from <300 to >800 kDa. Using primary baboon hepatocyte cultures, we previously demonstrated that differences in the secretion efficiency of apo[a] allelic variants contribute to the variation in plasma Lp[a] levels. In the current study, we investigated the mechanism of apo[a] presecretory degradation. The proteasome inhibitors, acetyl-leucyl-leucyl-norleucinal and lactacystin, prevented apo[a] degradation and increased apo[a] secretion. Transfection with an HA-tagged ubiquitin construct demonstrated the accumulation of ubiquitinated apo[a] in the presence of lactacystin. These results suggest a role for the cytoplasmic proteasome in apo[a] proteolysis. Apo[a] that accumulated intracellularly in the presence of lactacystin remained sensitive to endo-B-N-glucosaminidase H, and apo[a] degradation was reversibly inhibited by brefeldin A, suggesting that transport to a post-endoplasmic reticulum (ER) pre-medial Golgi compartment is required for apo[a] degradation. Newly synthesized apo[a] bound to the ER chaperone calnexin and conditions that enhanced this interaction prevented apo[a] degradation, suggesting that calnexin can protect apo[a] from proteolysis. These studies provide further support for the role of the proteasome in endoplasmic reticulum quality control, and expand this role to one that influences plasma levels of the atherogenic lipoprotein Lp[a].-White, A. L., B. Guerra, J. Wang, and R. E. Lanford. Presecretory degradation of apolipoprotein[a] is mediated by the proteasome pathway.

Viral persistence, antibody to E1 and E2, and hypervariable region 1 sequence stability in hepatitis C virus-inoculated chimpanzees.

The relationship of viral persistence, the immune response to hepatitis C virus (HCV) envelope proteins, and envelope sequence variability was examined in chimpanzees. Antibody reactivity to the HCV envelope proteins E1 or E2 was detected by enzyme-linked immunosorbent assay (ELISA) in more than 90% of a human serum panel. Although the ELISAs appeared to be sensitive indicators of HCV infection in human serum panels, the results of a cross-sectional study revealed that a low percentage of HCV-inoculated chimpanzees had detectable antibody to E1 (22%) and E2 (15%). Viral clearance, which was recognized in 28 (61%) of the chimpanzees, was not associated with an antibody response to E1 or E2. On the contrary, antibody to E2 was observed only in viremic chimpanzees. A longitudinal study of animals that cleared the viral infection or became chronically infected confirmed the low level of antibody to E1, E2, and the HVR-1. In 10 chronically infected animals, the sequence variation in the E2 hypervariable region (HVR-1) was minimal and did not coincide with antibody to E2 or to the HVR-1. In addition, low nucleotide and amino acid sequence variation was observed in the E1 and E2 regions from two chronically infected chimpanzees. These results suggest that mechanisms in addition to the emergence of HVR-1 antibody escape variants are involved in maintaining viral persistence. The significance of antibodies to E1 and E2 in the chimpanzee animal model is discussed.

Strand-Specific rTth RT-PCR for the Analysis of HCV Replication.

Because of the very low level of HCV present in the serum of infected individuals, as well as the low level of replication in the host, reverse transcription-polymerase chain reaction (RT-PCR) assays are the only method suitable for the routine detection of HCV RNA. The use of RT-PCR to monitor HCV replication in vivo as well as the in vitro inoculation of cultured cells presents unique problems. The extreme sensitivity of PCR permits the detection of HCV RNA in tissues not permissive for replication of the virus, and following in vitro infections, the residual inoculum can be detected for extended time periods depending on the sensitivity of the PCR procedure. Since HCV is a positive-stranded RNA virus, the detection of negative-strand RNA should be indicative of active viral RNA replication, assuming that the inoculum contains primarily positive-strand RNA. Early attempts to detect negative-strand RNA employed a strand-specific PCR technique that utilized only one primer during cDNA synthesis, followed by inactivation of the RT and amplification of the cDNA by PCR. During the course of our studies, we found this technique to lack significant strand specificity using synthetic RNA. A series of experiments suggested that the lack of specificity was probably owing to a combination of factors, including false priming of the incorrect strand (e.g., the positive strand in a negative-strand assay) by the cDNA primer, self-priming of the RNA, and random priming by extraneous nucleic acids (illustrated in Fig. 1 A). Fig. 1 Schematic diagram of conventional strand-specific RT-PCR and rTth RT-PCR procedures. (A) The amplification of HCV negative-strand RNA using conventional strand-specific RT-PCR and the false amplification of the HCV positive-strand RNA by the same procedure A cDNA copy of the RNA is made with primer complementary to negative-strand RNA. At the reduced temperatures used for cDNA synthesis, this primer can misanneal at sites on the positive-strand RNA with partial homology to the primer. cDNA synthesis can also be primed by contaminating nucleic acids and by terminal hairpin structures that can occur anywhere in genome if the RNA is partially degraded Following cDNA synthesis, the RT activity is inactivated and PCR amplification is performed with Taq. False-primed positive-strand RNA yields the same product as negative-strand RNA provided that the cDNA produce from false priming spans the sequence encompassed by the PCR primers. (B) The amplification of HCV negative-strand RNA by rTth RT-PCR is depicted A cDNA copy of the RNA is made with a primer complementary to the negative strand of RNA using the rTth thermostable reverse transcriptase at 70°C The RT activity of rTth is inactivated by chelation of Mn(2+) with EGTA. Following the addition of Mg(2+) and the reverse primer, PCR is conducted using the thermostable DNA polymerase activity of rTth.

A cultivation method for highly differentiated primary chimpanzee hepatocytes permissive for hepatitis C virus replication.

The liver performs a wide array of functions, a few of which include the synthesis and secretion of most of the plasma proteins, including the lipoproteins, cholesterol, and bile acid metabolism, and detoxification of the blood. In vitro analysis of most liver functions has been hampered by the difficulties encountered in isolating and maintaining functional cultures of primary hepatocytes. Although in vivo the liver has an amazing capacity for regeneration, hepatocytes in culture have limited proliferation capacity and are normally short-lived. We have developed methods for the isolation and cultivation of highly differentiated primate hepatocyte cultures that can be maintained for over 100 d without significant loss of differentiated function. This system has been used in our lab for the analysis of lipoprotein synthesis and hepatotropic virus replication. This chapter is designed to provide a detailed methodology of our approach. Numerous alternative hepatocyte cultivation systems have been described, but owing to space limitations, these systems will not be described here. A number of excellent reviews are dedicated to this subject, one of which is in a previous volume of this series (1), and another that is an entire book dedicated to the subject (2).

Amplification of simian retroviral sequences from human recipients of baboon liver transplants.

Investigations into the use of baboons as organ donors for human transplant recipients, a procedure called xenotransplantation, have raised the specter of transmitting baboon viruses to humans and possibly establishing new human infectious diseases. Retrospective analysis of tissues from two human transplant recipients with end-stage hepatic disease who died 70 and 27 days after the transplantation of baboon livers revealed the presence of two simian retroviruses of baboon origin, simian foamy virus (SFV) and baboon endogenous virus (BaEV), in multiple tissue compartments. The presence of baboon mitochondrial DNA was also detected in these same tissues, suggesting that xenogeneic "passenger leukocytes" harboring latent or active viral infections had migrated from the xenografts to distant sites within the human recipients. The persistence of SFV and BaEV in human recipients throughout the posttransplant period underscores the potential infectious risks associated with xenotransplantation.