Evolutionary analysis of hepatitis C virus gene sequences from 1953

Reconstructing the transmission history of infectious diseases in the absence of medical or epidemiological records often relies on the evolutionary analysis of pathogen genetic sequences. The precision of evolutionary estimates of epidemic history can be increased by the inclusion of sequences derived from ‘archived’ samples that are genetically distinct from contemporary strains. Historical sequences are especially valuable for viral pathogens that circulated for many years before being formally identified, including HIV and the hepatitis C virus (HCV). However, surprisingly few HCV isolates sampled before discovery of the virus in 1989 are currently available. Here, we report and analyse two HCV subgenomic sequences obtained from infected individuals in 1953, which represent the oldest genetic evidence of HCV infection. The pairwise genetic diversity between the two sequences indicates a substantial period of HCV transmission prior to the 1950s, and their inclusion in evolutionary analyses provides new estimates of the common ancestor of HCV in the USA. To explore and validate the evolutionary information provided by these sequences, we used a new phylogenetic molecular clock method to estimate the date of sampling of the archived strains, plus the dates of four more contemporary reference genomes. Despite the short fragments available, we conclude that the archived sequences are consistent with a proposed sampling date of 1953, although statistical uncertainty is large. Our cross-validation analyses suggest that the bias and low statistical power observed here likely arise from a combination of high evolutionary rate heterogeneity and an unstructured, star-like phylogeny. We expect that attempts to date other historical viruses under similar circumstances will meet similar problems.     

Characterization of long-term cultures of hepatitis C virus.

The human T- and B-cell lines HPBMa10-2 and Daudi produced infectious hepatitis C virus (HCV) for more than 1 year after infection. The infectivity titer of the cell culture-grown HCV and its genome titer were comparable. The virion density in sucrose was around 1.12 g/ml. Among the 13 variants detected in the inoculum, 7 were adsorbed by the cells and one particular HCV sequence which was present in minor quantities in the inoculum persisted.     

Detection of divergent hepatitis C virus envelope sequences

The nucleotide sequences of the putative envelope region (E1) and the junction between the E1 and envelope 2/nonstructural 1 (E2/NS1) region of the hepatitis C virus (HCV) genome are divergent among different genotypes. To characterize them, we introduced a set of nested primers that are conserved among four different genotypes (types I–IV) of HCV for polymerase chain reaction (PCR) amplification. The amplified products include the variable full-length E1 region, and the 5 end of the E2/NS1 region, the so-called hypervariable region-1 (HVR-1). Of 53 patients with histologically confirmed chronic liver disease and HCV viremia, type II virus was the most dominant strain as detected by the PCR genotyping method and the envelope region could be amplified in more than half of them irrespective of their genotypes. The specificity was confirmed by subsequent nucleotide sequence analysis. The positivity of envelope region PCR was not correlated with histologic diagnosis and hepatitis activities in these patients. Our results suggest that the nested primers can amplify the variable E1 and hypervariable 5 end of E2/NS1 of the HCV genome with moderate efficiency, and thus will be useful in future studies of HCV infections.     

Characterization of infectious hepatitis C virus from liver-derived cell lines

The efficient production of infectious HCV from the JFH-1 strain is restricted to the Huh7 cell line and its derivatives. However, the factors involved in this restriction are unknown. In this study, we examined the production of infectious HCV from other liver-derived cell lines, and characterized the produced viruses. Clones of the Huh7, HepG2, and IMY-N9, harboring the JFH-1 full-genomic replicon, were obtained. The supernatant of each cell clone exhibited infectivity for naïve Huh7. Each infectious supernatant was then characterized by sucrose density gradient. For all of the cell lines, the main peak of the HCV-core protein and RNA exhibited at approximately 1.15 g/mL of buoyant density. However, the supernatant from the IMY-N9 differed from that of Huh7 in the ratio of core:RNA at 1.15 g/mL and significant peaks were also observed at lower density. The virus particles produced from the different cell lines may have different characteristics.     

Alterations in hepatitis B virus nucleotide sequences in a chronic virus carrier from immunotolerant to immunoactive phase

Factors involved in transition from the immunotolerant to immunoactive phase in chronic hepatitis B virus (HBV) infection remain unclear. We investigated viral mutations occurring during transition and elucidated their virological and immunological significance. Full-length HBV DNA sequences were serially determined in a chronic HBV carrier from the immunotolerant to immunoactive phase. Viral replicative competence was examined by transfection analysis. HBV-specific CD8⁺ T cell response was evaluated by coculture of CD8⁺ T cells with autologous dendritic cells followed by interferon-γ Elispot assay. Eleven point mutations and two deletions appeared around the onset of the immunoactive phase. Viral replicative competence declined significantly after the onset of active hepatitis. Examination of the CD8⁺ T cell response against two putative T-cell epitopes, which contained substituted amino acids from the immunotolerant to immunoactive phase, showed that mutant HBV epitopes gave a lesser T cell response than wild-type HBV ones. In summary, point mutations and deletions may occur prior to or concurrent with the onset of the immunoactive phase during chronic HBV infection. These mutations may result in a significant decrease in both viral replicative competence and HBV-specific CD8⁺ T cell response, suggesting a possible adaptation for the maintenance of viral persistence.     

Comparison of the nucleotide sequences of wheat dwarf virus (WDV) isolates from Hungary and Ukraine

Wheat dwarf virus (WDV) is the most ubiquitous virus in cereals causing huge losses in both Hungary and Ukraine. The presence of barley-and wheat-adapted strains has been confirmed, suggesting that the barley strain is restricted to barley, while the wheat strain is present in both wheat and barley plants. Five WDV isolates from wheat plants sampled in Hungary and Ukraine were sequenced and compared with known WDV isolates from GenBank. Four WDV isolates belonged to the wheat strain. Our results indicate that WDV-Odessa is an isolate of special interest since it has originated from wheat, but belongs to the barley-adapted strain, providing novel data on WDV biology and raising issues of pathogen epidemiology.     

Characterization of Whitney's Clethrionomy gapperi virus isolates from Massachusetts.

Six strains of virus were recovered from the blood and/or liver of five Clethrionomys gapperi ochraceus trapped in southeastern Massachusetts during 1969. Biological, antigenic and physiochemical properties of these isolates are reported. USA M-2268a was selected as the reference strain. This strain was identical by complement-fixation and neutralization tests to Whitneys C. gapperie virus (USA 64-7855) from New York State and was related to, but distinct from, an unpublished agent (Johnson's Microtus montanus enterovirus USA M-1146) isolated in June, 1962 from voles trapped in Klamath County, Oregon. USA M-2268a was resistant to lipid solvents and acid pH and was stable at temperatures of 4 C, 22 C, and 37 C. Virus was detected over a 10-day observation period in four species of mosquitoes inoculated with USA M-2268a, although there was no evidence of infection or replication, and transmission attempts by bite failed. Neutralizing antibody was detected in C.g. gapperi and C. g. ochraceus in various habitats throughout the state.     

Characterization of functional hepatitis C virus envelope glycoproteins

Hepatitis C virus (HCV) encodes two envelope glycoproteins, E1 and E2, that assemble as a noncovalent heterodimer which is mainly retained in the endoplasmic reticulum. Because assembly into particles and secretion from the cell lead to structural changes in viral envelope proteins, characterization of the proteins associated with the virion is necessary in order to better understand how they mature to be functional in virus entry. There is currently no efficient and reliable cell culture system to amplify HCV, and the envelope glycoproteins associated with the virion have therefore not been characterized yet. Recently, infectious pseudotype particles that are assembled by displaying unmodified HCV envelope glycoproteins on retroviral core particles have been successfully generated. Because HCV pseudotype particles contain fully functional envelope glycoproteins, these envelope proteins, or at least a fraction of them, should be in a mature conformation similar to that on the native HCV particles. In this study, we used conformation-dependent monoclonal antibodies to characterize the envelope glycoproteins associated with HCV pseudotype particles. We showed that the functional unit is a noncovalent E1E2 heterodimer containing complex or hybrid type glycans. We did not observe any evidence of maturation by a cellular endoprotease during the transport of these envelope glycoproteins through the secretory pathway. These envelope glycoproteins were recognized by a panel of conformation-dependent monoclonal antibodies as well as by CD81, a molecule involved in HCV entry. The functional envelope glycoproteins associated with HCV pseudotype particles were also shown to be sensitive to low-pH treatment. Such conformational changes are likely necessary to initiate fusion.     

Detection and analysis of hepatitis C virus sequences in cerebrospinal fluid

Hepatitis C virus (HCV) sequences were detected in cerebrospinal fluid (CSF) in 8 of 13 HCV-positive patients. In four patients harboring different virus strains in serum and peripheral blood mononuclear cells (PBMC), CSF-derived virus was similar to that found in PBMC, which suggests that PBMC could carry HCV into the brain.     

Nature and distribution of feline sarcoma virus nucleotide sequences.

The genomes of three independent isolates of feline sarcoma virus (FeSV) were compared by molecular hybridization techniques. Using complementary DNAs prepared from two strains, SM- and ST-FeSV, common complementary DNA'S were selected by sequential hybridization to FeSV and feline leukemia virus RNAs. These DNAs were shown to be highly related among the three independent sarcoma virus isolates. FeSV-specific complementary DNAs were prepared by selection for hybridization by the homologous FeSV RNA and against hybridization by fline leukemia virus RNA. Sarcoma virus-specific sequences of SM-FeSV were shown to differ from those of either ST- or GA-FeSV strains, whereas ST-FeSV-specific DNA shared extensive sequence homology with GA-FeSV. By molecular hybridization, each set of FeSV-specific sequences was demonstrated to be present in normal cat cellular DNA in approximately one copy per haploid genome and was conserved throughout Felidae. In contrast, FeSV-common sequences were present in multiple DNA copies and were found only in Mediterranean cats. The present results are consistent with the concept that each FeSV strain has arisen by a mechanism involving recombination between feline leukemia virus and cat cellular DNA sequences, the latter represented within the cat genome in a manner analogous to that of a cellular gene.     

Characterization of cell lines carrying self-replicating hepatitis C virus RNAs

Subgenomic selectable RNAs of the hepatitis C virus (HCV) have recently been shown to self-replicate to high levels in the human hepatoma cell line Huh-7 (V. Lohmann, F. K?rner, J. O. Koch, U. Herian, L. Theilmann, and R. Bartenschlager, Science 285:110-113, 1999). Taking advantage of this cell culture system that allows analyses of the interplay between HCV replication and the host cell, in this study we characterized two replicon-harboring cell lines that have been cultivated for more than 1 year. During this time, we observed no signs of cytopathogenicity such as reduction of growth rates or ultrastructural changes. High levels of HCV RNAs were preserved in cells passaged under continuous selection. When selective pressure was omitted replicon levels dropped, but depending on culture conditions the RNAs persisted for more than 10 months. A tight coupling of the amounts of HCV RNA and proteins to host cell growth was observed. Highest levels were found in exponentially growing cells, followed by a sharp decline in resting cells, suggesting that cellular factors required for RNA replication and/or translation vary in abundance and become limiting in resting cells. Studies of polyprotein processing revealed rapid cleavages at the NS3/4A and NS5A/B sites resulting in a rather stable NS4AB5A precursor that was processed slowly into individual products. Half-lives (t(1/2)s) of mature proteins ranged from 10 to 16 h, with the exception of the hyperphosphorylated form of NS5A, which was less stable (t(1/2), approximately 7 h). Results of immunoelectron microscopy revealed an association of the majority of viral proteins with membranes of the endoplasmic reticulum, suggesting that this is the site of RNA replication. In summary, replicon-bearing cells are a good model for viral persistence, and they allow the study of various aspects of the HCV life cycle.     

Characterization of low- and very-low-density hepatitis C virus RNA-containing particles

The presence of hepatitis C virus (HCV) RNA-containing particles in the low-density fractions of plasma has been associated with high infectivity. However, the nature of circulating HCV particles and their association with immunoglobulins or lipoproteins as well as the characterization of cell entry have all been subject to conflicting reports. For a better analysis of HCV RNA-containing particles, we quantified HCV RNA in the low-density fractions of plasma corresponding to the very-low-density lipoprotein (VLDL), intermediate-density lipoprotein, and low-density lipoprotein (LDL) fractions from untreated chronically HCV-infected patients. HCV RNA was always found in at least one of these fractions and represented 8 to 95% of the total plasma HCV RNA. Surprisingly, immunoglobulins G and M were also found in the low-density fractions and could be used to purify the HCV RNA-containing particles (lipo-viro-particles [LVP]). Purified LVP were rich in triglycerides; contained at least apolipoprotein B, HCV RNA, and core protein; and appeared as large spherical particles with a diameter of more than 100 nm and with internal structures. Delipidation of these particles resulted in capsid-like structures recognized by anti-HCV core protein antibody. Purified LVP efficiently bind and enter hepatocyte cell lines, while serum or whole-density fractions do not. Binding of these particles was competed out by VLDL and LDL from noninfected donors and was blocked by anti-apolipoprotein B and E antibodies, whereas upregulation of the LDL receptor increased their internalization. These results suggest that the infectivity of LVP is mediated by endogenous proteins rather than by viral components providing a mechanism of escape from the humoral immune response.     

Secondary structure and hybridization accessibility of hepatitis C virus 3'-terminal sequences

The 3'-terminal sequences of hepatitis C virus (HCV) positive- and negative-strand RNAs contribute cis-acting functions essential for viral replication. The secondary structure and protein-binding properties of these highly conserved regions are of interest not only for the further elucidation of HCV molecular biology, but also for the design of antisense therapeutic constructs. The RNA structure of the positive-strand 3' untranslated region has been shown previously to influence binding by various host and viral proteins and is thus thought to promote HCV RNA synthesis and genome stability. Recent studies have attributed analogous functions to the negative-strand 3' terminus. We evaluated the HCV negative-strand secondary structure by enzymatic probing with single-strand-specific RNases and thermodynamic modeling of RNA folding. The accessibility of both 3'-terminal sequences to hybridization by antisense constructs was evaluated by RNase H cleavage mapping in the presence of combinatorial oligodeoxynucleotide libraries. The mapping results facilitated identification of antisense oligodeoxynucleotides and a 10-23 deoxyribozyme active against the positive-strand 3'-X region RNA in vitro.     

The 30S Moloney sarcoma virus RNA contains leukemia virus nucleotide sequences.

The 50S-70S RNA of a Moloney sarcoma-leukemia virus [Mo-MSV(MLV)] complex produced by a particular mouse cell line was shown by gel electrophoresis to contain a major (97%) 30S sarcoma-specific subunit species and a minor (3%) 38S leukemia virus-specific subunit. On the basis of its sedimentation coefficient and known complexity, the 30S Mo-MSV RNA was estimated to be a unique RNA molecule of about 6000 nucleotides. Hybridization experiments using viral RNA and DNA complementary to viral RNA (cDNA) made by viral DNA polymerase indicated that the 30S Mo-MSV RNA shared 70% of its sequences with Mo-MLV, 30% with another MLV derived from Mo-MLV, and 30% with Kirsten sarcoma-xenotropic leukemia virus. The 30S Mo-MSV RNA sequences shared with these viruses were not additive. The Tm of a Mo-MSV RNA-MLV cDNA hybrid was 83 degrees C, indicating that large contiguous nucleotide sequences were shared between the two nucleic acids. Mo-MSV RNA and Mo-MLV RNA shared possibly seven of 20-30 RNAase T1-resistant oligonucleotides, while Mo-MSV RNA contained three, and Mo-MLV RNA contained at least five specific oligonucleotides. We conclude that the 30S Mo-MSV RNA molecule consists of approximately 70% (about 4200 nucleotides) Mo-MLV-specific sequences and of 30% (1800 nucleotides) Mo-MSV-specific sequences covalently linked. Our results favor the hypothesis that 30S Mo-MSV RNA was generated by recombination between Mo-MLV and other genetic elements. We discuss whether all or only the MSV-specific sequences of the 30S Mo-MSV RNA function as sarcoma genes. Mo-MLV cDNA was hybridized about 45% by unfractionated Mo-MSV (MLV) RNA at RNA/DNA ratios of up to 10, about 50% by electrophoretically purified 30S Mo-MSV RNA at RNA/DNA ratios up to 500, but close to 100% by unfractionated Mo-MSV(MLV) RNA at RNA/DNA ratios over 900. This indicated that unfractionated RNA of our Mo-MSV(MLV) contained a complete complement of Mo-MLV, albeit at a low ratio.