Prevalence of TT virus infection in US blood donors and populations at risk for acquiring parenterally transmitted viruses.

Two overlapping sets of TT virus (TTV)-specific polymerase chain reaction primers were used to test for presence of TTV, which was found in approximately 10% of US volunteer blood donors, 13% of commercial blood donors, and 17% of intravenous drug abusers. The rate of TTV infection among US non-A, non-B, non-C, non-D, non-E hepatitis patients was only 2%. Among commercial blood donors and intravenous drug abusers, only 1%-3% of the TTV-positive individuals were coinfected with GB virus C (GBV-C), a parenterally transmitted virus. This suggests that GBV-C and TTV may have different routes of transmission. Comparison of the sensitivities of 2 TTV polymerase chain reaction (PCR) primer sets showed that the majority of samples were detected with only 1 of the 2 sets. Therefore, previous studies in which only a single PCR primer pair was used may have significantly underestimated the true prevalence of TTV.

Molecular and biophysical characterization of TT virus: evidence for a new virus family infecting humans.

The recent isolation of a novel DNA virus from the serum of a Japanese patient (T.T.) has provided the latest possible candidate virus associated with cryptogenic hepatitis. In the present study, we report the complete nucleotide sequence of this virus (TTV) isolated from the serum of a West African. Based on PCR studies designed to amplify overlapping regions of the viral genome and sensitivity to digestion with mung bean nuclease, the viral genome is circular and negative stranded, and comprises 3,852 nt, which is 113 nt longer than the prototype isolate from Japan. Cesium chloride density gradient centrifugation demonstrated banding of the virus at 1.31-1.34 g/ml; filtration studies indicated that TTV had a particle size of 30-50 nm. These results suggest that the virus is similar to the Circoviridae, viruses known to infect plants and vertebrates (e. g., birds and swine); however, sequence similarity searches of available databases did not reveal identity between TTV and other viruses. Phylogenetic analyses of a 260-nt region from 151 globally distributed isolates demonstrated the existence of three major TTV genotypes. Several individuals at high risk for infection with parenterally transmitted viruses were infected with more than one genotype. There was no correlation between genotype and geographic origin. Finally, intravenous inoculation of TTV-positive human serum into chimpanzees demonstrated that TTV can be transmitted to primates; no biochemical or histological evidence for hepatitis was obtained. The distinct biophysical and molecular characteristics of TTV suggest that it is a member of a new family of viruses, which we have tentatively named the Circinoviridae.

Isolation of a GB virus-related genome from a chimpanzee.

Recently, two new flaviviruses, GB virus A (GBV-A) and GB virus B (GBV-B), were identified in the plasma of a tamarin infected with the hepatitis GB agent. A third virus, GB virus C (GBV-C), was subsequently identified in humans. In the current study, representational difference analysis (RDA) was used to search for a new virus in the serum of a chimpanzee that developed acute resolving hepatitis following inoculation with a pool of chimpanzee plasma. The plasma pool originated from serial passages of a human sample containing virus-like particles. Numerous cDNA clones were obtained that exhibited 62-80% identity with GBV-C. With the exception of the extreme 5' and 3' ends, the complete viral genome was sequenced, revealing a single large open reading frame encoding a 2833 amino acid polyprotein that contains two envelope proteins, two proteases, a helicase, and an RNA-dependent RNA polymerase. Phylogenetic analysis of the new virus indicates that it is closely related to GBV-C, yet still sufficiently divergent as to be placed in a separate group, tentatively labeled GB virus Ctroglodytes (GBV-Ctro). Numerous human samples were screened by reverse transcriptase-polymerase chain reaction (RT-PCR), but GBV-Ctro sequence was not detected. However, a second chimpanzee inoculated with the same plasma pool was shown to develop a GBV-Ctro infection. Although isolated from an Old World primate with hepatitis, the primary host of GBV-Ctro and any association with disease remains to be determined.

Expression of the GB virus C E2 glycoprotein using the Semliki Forest virus vector system and its utility as a serologic marker.

A 336-amino-acid segment of the GB virus C second envelope protein (E2) has been produced in BHK-21 cells using the Semliki Forest virus vector system. Secretion of this protein was facilitated by deletion of a hydrophobic region at the C-terminus that may represent the membrane anchoring domain. The E2 protein recovered from the culture supernatant exhibited a molecular mass of approximately 52 kDa, with the increase in size relative to the polyprotein backbone being contributed by N-linked glycosylation. A radioimmunoprecipitation assay using GBV-C E2 was developed to test for the presence of antibodies against this protein in human sera. The prevalence of antibodies to E2 was high among injection drug users and other individuals at risk for acquiring parenterally transmitted agents. There was a much higher percentage of anti-E2 seropositivity in GBV-C RT-PCR negative compared to GBV-C RT-PCR positive samples from these populations. In addition, serial samples from patients transfused with blood containing GBV-C showed seroconversion to anti-E2 positivity and loss of GBV-C viremia as measured by RT-PCR within 11 months of transfusion in five of seven individuals. Thus, this system provided a rapid means to identify GBV-C E2 as a useful antigen for the study of GBV-C exposure.

Molecular cloning and characterization of a GB virus C isolate from a patient with non-A-E hepatitis.

Recently, the isolation of a novel virus, GB virus C (GBV-C), associated with cryptogenic hepatitis has been reported. Following the molecular cloning of this virus genome, it became apparent that the genomic sequence did not encode a protein resembling a nucleocapsid or core-like protein similar to those observed in other flaviviruses, pestiviruses, hepatitis C virus (HCV) and GB virus B. Similar findings were subsequently observed in the cloning of two viral genomes representing isolates of GBV-C, namely hepatitis G virus (HGV). To verify the presence or absence of a viral nucleocapsid protein, identify conserved protein motifs and determine the overall genomic variability, an additional virus isolate has been characterized. Here we report the full-length genomic sequence of GBV-C(EA), isolated from an East African suffering from acute non-A-E hepatitis. GBV-C(EA) was compared with the prototype West African isolate (GBV-C) and the two HGV isolates from the United States. The analyses demonstrate several characteristics of these novel viruses. (1) The degree of variability within the 5' nontranslated region (NTR) approximates that observed between HCV isolates. (2) The nucleotide sequence of the coding region and the 3' NTR is highly conserved between these isolates, in contrast to the extensive variability observed between HCV isolates from distinct geographical locations. (3) There is a high degree of amino acid conservation across the precursor polyproteins of these isolates; most striking is the lack of 'hypervariable' regions within the envelope proteins. (4) There appears to be no nucleocapsid protein near the amino terminus of the GBV-C/HGV polyproteins.

Identification of conserved nucleotide sequences within the GB virus C 5'-untranslated region: design of PCR primers for detection of viral RNA.

Recently, the discovery of a new human RNA virus, GB virus C (GBV-C), was reported. GBV-C was isolated from the serum of a West African individual using degenerate oligonucleotide PCR primers designed from a consensus sequence of the NS3 helicase genes of hepatitis C virus (HCV), GBV-A, and GBV-B. Seven other individuals were shown to be infected with GBV-C via RT-PCR using these primers. Subsequently, degenerate PCR primers based upon a consensus sequence of the eight original isolates were designed. These primers were shown to be superior to the original set. However, since they were derived from a region of the viral genome exhibiting up to 17% nucleotide sequence divergence, mismatch between the primers and template may result in an underestimation of the true GBV-C prevalence. To overcome this potential problem, we obtained the sequences at the 5'-untranslated region (UTR) of the GBV-C genome from 35 infected individuals and identified regions of high sequence conservation among the isolates. We describe the design and testing of PCR primers derived from conserved sequences within the 5'-UTR of the GBV-C genome. These primers were shown to be as effective as the helicase-derived primers in detecting GBV-C RNA in human sera.

Translation initiation in GB viruses A and C: evidence for internal ribosome entry and implications for genome organization.

GB viruses A and C (GBV-A and GBV-C) are two recently described RNA viruses which appear to be members of the Flaviviridae. Although both viruses appear to contain long 5' nontranslated regions, the sites of polyprotein initiation and the presence of core-like proteins remain to be determined. Translation studies were undertaken to determine the mechanism and sites of polyprotein initiation in GBV-A and GBV-C. Rabbit reticulocyte lysates programmed with monocistronic RNAs containing 5' ends of GBV-A or GBV-C fused in-frame with the chloramphenicol acetyltransferase (CAT) open reading frame generated GBV-CAT fusion proteins in vitro. Site-specific mutagenesis and N-terminal sequencing located the sites of translation initiation immediately upstream of the putative signal sequence for the GBV E1 envelope glycoproteins. Efficient translation of the monocistronic GBV-CAT RNAs required the inclusion of GBV coding sequences. This, coupled with the presence of at least 523 nucleotides of 5' nontranslated RNA containing multiple AUG codons, suggests that translation initiation of these RNAs did not utilize a ribosome scanning mechanism. Translation of bicistronic RNAs containing 5' nontranslated sequences within the intercistronic space was consistent with the presence of a weakly active internal ribosome entry site in both GBV-A and GBV-C. Secondary structure predictions indicate that the 5' ends of these viruses assume similar complex structures distinct from those identified in the internal ribosome entry site-containing picornaviruses, pestiviruses, and hepatitis C viruses. The data indicate that GBV-A and GBV-C are unique members of the Flaviviridae that do not contain core-like proteins at the N termini of their putative polyproteins.

Prevalence studies of GB virus-C infection using reverse transcriptase-polymerase chain reaction.

Among the three recently described GB viruses (GBV-A, GBV-B, and GBV-C), only GBV-C has been linked to cryptogenic hepatitis in man. Because of the limited utility of currently available research tests to determine antibody response to GBV-C proteins, the prevalence of GBV-C RNA in human sera was studied using reverse transcription-polymerase chain reaction (RT-PCR). The prevalence of GBV-C is higher among volunteer blood donors with elevated serum alanine aminotransferase (ALT) levels (3.9%) than among volunteer blood donors with normal ALT levels (0.8%). Higher rates were also noted among commercial blood donors (12.9%) and intravenous drug users (16.0%). GBV-C was frequently detected in residents of West Africa, where the prevalence was > 10% in most age groups. Approximately 20% of patients diagnosed with either acute or chronic hepatitis C virus (HCV) were found to be positive for GBV-C RNA. In addition, GBV-C RNA sequences were detected in individuals diagnosed with non-A-E hepatitis, with clinical courses ranging from mild disease to fulminant hepatitis. Fourteen of sixteen subjects with or without clinically apparent hepatitis were positive for GBV-C RNA more than 1 year after the initial positive result.

Sequence heterogeneity within the 5'-terminal region of the hepatitis GB virus C genome and evidence for genotypes.

BACKGROUND: GB virus C is a positive-strand RNA virus that is associated with hepatitis in humans. GB virus C bears some resemblance to hepatitis C virus in its genomic sequence and organization. However, unlike hepatitis C virus, an open reading frame possessing a complete core protein was not identified in the original isolate. METHODS: To verify the sequence at the 5'-end of the GB virus C genome, we amplified approximately 600 nucleotides from this region from 35 globally distributed individuals. The nucleotide sequences were translated in all possible reading frames and then examined for conserved motifs indicative of nucleocapsid or core-like peptides. RESULTS: Forty-two unique GB virus C sequences were obtained from the 35 individuals. The deduced amino acid sequences upstream of the putative E1 gene from each isolate varied in length and composition, such that a conserved core-like sequence was not apparent. No core-like sequences were evident in the other reading frames. There was, however, a single methionine codon held in common among all isolates, although it was located very near the presumed amino-terminus of the putative E1 protein. Further analysis of the sequences for their evolutionary relatedness demonstrated the existence of five GB virus C subtypes that demonstrated a significant correlation with geographic distribution. CONCLUSIONS: GB virus C differs from hepatitis C virus and GB virus B in that it does not encode a nucleocapsid or core protein. The existence of GB virus C subtypes emphasizes the importance of investigating the correlation between infecting subtype and the severity of liver disease and/or responsiveness to treatment of GB virus C-associated hepatitis.

Identification of antigenic regions in the GB hepatitis viruses GBV-A, GBV-B, and GBV-C.

The genomes of two novel members of the Flaviviridae associated with GB agent hepatitis (GB viruses A and B) were cloned and sequenced recently. The genome of a third novel virus (GB virus C), related to but distinct from GB viruses A and B, has also been identified and characterized. Overlapping clones encompassing the large open reading frames of these three viruses have been expressed in E. coli as CTP:CMP-3-deoxy-D-manno-octulosonate cytidylyltransferase (CKS) fusion proteins. Bacterial lysates were subjected to Western blot analyses using sera from GB agent-infected tamarins and human sera from various individuals with or "at risk" for non-A, non-B, non-C, non-D, non-E hepatitis. Antigenic regions were identified in the putative NS3, NS4, and NS5 proteins from all three viruses. An antigenic region was also identified in the putative core protein of GB virus B. Many of the clones identified originally as encoding antigenic proteins were quite large. To map these regions more narrowly, smaller overlapping clones were generated by polymerase chain reaction (PCR), expressed as recombinant CKS fusion proteins and tested by Western blot. Additionally, a lambda gt11 expression library was generated from infectious tamarin sera and immunoscreened. These studies have identified at least three epitopes in GB virus A, five epitopes in GB virus B and four epitopes in GB virus C.

Sequence and genomic organization of GBV-C: a novel member of the flaviviridae associated with human non-A-E hepatitis.

Recently, sequences from a novel virus, termed GB virus C (GBV-C), were identified in serum from several patients with cryptogenic hepatitis. In the present study, the nucleotide sequence of this virus has been extended to near-genome length. GBV-C encodes a putative single large polyprotein in which the structural proteins are positioned at the N-terminal end, with the non-structural proteins located at the C-terminal end. Amino acid sequence analysis of this large polyprotein reveals the presence of protease, helicase, and replicase motifs. Sequence alignments of the polyprotein followed by phylogenetic analyses suggest that GBV-C is a member of the Flaviviridae, most closely related to the recently described GB virus A.

Consensus oligonucleotide primers for the detection of GB virus C in human cryptogenic hepatitis.

Recently, sequences from a putative member of the Flaviviridae, GB virus C (GBV-C), were isolated from the serum of patients with cryptogenic hepatitis. These sequences were 83-99% identical at the nucleotide level. Because of the divergence between these GBV-C isolates, it is likely that the PCR-based detection assay yields false negatives, underestimating dramatically the true prevalence of GBV-C in human hepatitis. We report the design of a GBV-C consensus oligonucleotide primer pair that is superior to those originally described. These primers identify GBV-C sequences in cases of cryptogenic hepatitis, allowing a better estimation of the prevalence of this virus in human populations.

Genomic organization of GB viruses A and B: two new members of the Flaviviridae associated with GB agent hepatitis.

The genomes of two positive-strand RNA viruses have recently been cloned from the serum of a GB agent-infected tamarin by using representational difference analysis. The two agent, GB viruses A and B (GBV-A and GBV-B, respectively), have genomes of 9,493 and 9,143 nucleotides, respectively, and single large open reading frames that encode potential polyprotein precursors of 2,972 and 2,864 amino acids, respectively. The genomes of these agents are organized much like those of other pestiviruses and flaviviruses, with genes predicted to encode structural and nonstructural proteins located at the 5' and 3' ends, respectively. Amino acid sequence alignments and subsequent phylogenetic analysis of the RNA-dependent RNA polymerases (RdRps) of GBV-A and GBV-B show that they possess conserved sequence motifs associated with supergroup II RNA polymerases of positive-strand RNA viruses. On the basis of similar analyses, the GBV-A- and GBV-B-encoded helicases show significant identity with the supergroup II helicases of positive-strand RNA viruses. Within the supergroup II RNA polymerases and helicases, GBV-A and GBV-B are most closely related to the hepatitis C virus group. Across their entire open reading frames, the GB agents exhibit 27% amino sequence identity to each other, approximately 28% identity to hepatitis C virus type 1, and approximately 20% identity to either bovine viral diarrhea virus or yellow fever virus. The degree of sequence divergence between GBV-A and GBV-B and other Flaviviridae members demonstrates that the GB agents are representatives of two new genera within the Flaviviridae family.

Isolation of novel virus-like sequences associated with human hepatitis.

Two viruses, GB virus A (GBV-A) and GB virus B (GBV-B), were recently identified in the GB hepatitis agent. Human sera containing antibodies that recognize GBV-A and/or GBV-B recombinant proteins were subjected to polymerase chain reaction studies with degenerate oligonucleotides capable of amplifying a segment of the putative helicase genes from GBV-A, GBV-B or hepatitis C virus. Novel sequences related to members of the Flaviviridae were identified in sera from 12 individuals including 4 individuals with hepatitis. The limited nucleotide sequence identity between GBV-A, GBV-B and HCV sequences suggests that a novel virus, tentatively named GB virus C, may be responsible for some cases of non-A, non-B, non-C, non-D, non-E hepatitis.

Molecular and serologic analysis in the transmission of the GB hepatitis agents.

Two flavivirus-like genomes have recently been cloned from infectious tamarin (Saguinus labiatus) serum, derived from the human viral hepatitis GB strain, which is known to induce hepatitis in tamarins. In order to study the natural history of GB infections, further transmission studies were carried out in tamarins. Reverse-transcription-polymerase chain reaction and enzyme-linked immunosorbant assays were developed for the detection of RNA and antibodies associated with the two agents, GB virus-A and GB virus-B. The infectivity of both of these agents was demonstrated in tamarins to be filterable through a 0.1 micron filter. Two distinct genomes were identified in the serum of eight of the infected tamarins, while in four tamarins, the genomes were detected independently of each other. Although specific antibodies to the GB virus-B epitopes were detected in the serum of animals inoculated with both agents or GB virus-B alone, antibodies to putative epitopes specific to GB virus-A were not detected in any of the animals. All tamarins inoculated with serum containing GB virus-B exhibited an elevation in liver enzyme levels after inoculation. Elevations of serum liver enzyme levels did not occur when GB virus-A was the only agent detected in the serum. Infection with the original infectious tamarin inoculum conferred protection from reinfection with GB virus-B but not with GB virus-A.

Identification of two flavivirus-like genomes in the GB hepatitis agent.

A subtractive PCR methodology known as representational difference analysis was used to clone specific nucleotide sequences present in the infectious plasma from a tamarin infected with the GB hepatitis agent. Eleven unique clones were identified, seven of which were examined extensively. All seven clones appeared to be derived from sequences exogenous to the genomes of humans, tamarins, Saccharomyces cerevisiae, and Escherichia coli. In addition, sequences from these clones were not detected in plasma or liver tissue of tamarins prior to their inoculation with the GB agent. These sequences were detected by reverse transcription-PCR in acute-phase plasma of tamarins inoculated with the GB agent. Probes derived from two of the seven clones detected an RNA species of > or = 8.3 kb in the liver of a GB-agent-infected tamarin by Northern blot hybridization. Sequence analysis indicated that five of the seven clones encode polypeptides that possess limited amino acid identity with the nonstructural proteins of hepatitis C virus. Extension of the sequences found in the seven clones revealed that plasma from an infected tamarin contained two RNA molecules > 9 kb long. Limited sequence identity with various isolates of hepatitis C virus and the relative positions of putative RNA helicases and RNA-dependent RNA polymerases in the predicted protein products of these molecules suggested that the GB agent contains two unique flavivirus-like genomes.