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Hepatitis E virus (HEV), the sole member of the genus Hepevirus in the family of Hepeviridae, is the major cause of several outbreaks of waterborne hepatitis in tropical and subtropical countries and of sporadic cases of viral hepatitis in endemic and industrialized countries. Transmission of HEV occurs predominantly by the fecal-oral route although parenteral and perinatal routes have been implicated. The overall death rate among young adults and pregnant women is 0.5-3% and 15-20%, respectively. HEV is a small non-enveloped particle that consists of a polyadenylated single-strand RNA molecule containing three discontinuous and partially overlapping open reading frames. There are four major genotypes of HEV and a single serotype. At present, there are approximately 1,600 sequences of HEV that are already available at INSDC of both human and animal isolates. Diagnostic and molecular assays have been described for the accurate differentiation of ongoing from remote infection of HEV. Identification and characterization of swine HEV in the United States, Japan, and many other countries and their close relationship to locally characterized human HEV found in the same geographic areas prove that HEV is indeed a zoonotic virus and that domestic swine, wild deer, and boars are reservoirs of HEV in nature. A cell culture system for the propagation of the virus has been described, and a very successful phase 2 vaccine trial has been completed. This review summarizes the current knowledge on the molecular biology, clinical features, transmission, diagnosis, epidemiology, and prevention of HEV.

In the last 30 years, tremendous progress in identifying transfusion-transmitted viruses such as HBV, HCV, and HIV in industrialized countries has been achieved. Currently, the residual risk of transmitting these viruses through transfusion is very low especially after the introduction of "minipool" nucleic acid-amplification tests. Despite these major technical advances, there remains a legitimate concern as to the transmission of other blood-borne infectious agents through blood transfusion. Among these agents are HBV mutants, occult HBV, and HCV infections, malaria, Chagas, West Nile, dengue, and vesiviruses, bacterial infections such as Yersinia enterocolitica, and tick borne diseases such as human monocytic ehrlichiosis, human granulocytic ehrlichiosis, Rocky Mountain spotted fever, and Lyme and prion diseases such as Creutzfeldt and variant Creutzfeldt. Most of these agents are very rarely transmitted by transfusion in industrialized countries. However, an awareness of their possible transmission is essential for the control of spread of these diseases among the public by human-to-human transmission via blood transfusion. This review summarizes the current status of prevalence and diagnosis of these emerging diseases and also updates our knowledge on recently discovered non-pathogenic blood-borne viruses such as GB virus C and Torque Tenoviruses.

A fully automated chemiluminescent microparticle immunoassay (Architect HBsAg QT) was used for the detection and quantitation of hepatitis B surface antigen (HBsAg). The assay is capable of processing up to 800 HBsAg tests per hour. The concentration of HBsAg is determined by utilizing a previously generated Architect HBsAg calibration curve. Architect HBsAg QT sensitivity was found to be around 0.2ng/ml which is equivalent or superior to other known and commercially available enzyme immunoassays and/or chemiluminescent immunoassays. We performed a quantitative study of HBsAg, HBeAg, HBV-DNA and HBV-DNA polymerase in over 733 sera obtained from 43 chronic hepatitis B carriers. Serum HBsAg levels detected by Architect HBsAg QT were found to be higher in HBeAg-positive than in anti-HBe-positive HBV chronic carriers and correlated with the level of serum HBV-DNA and HBV-DNA polymerase.

Sequences of 234 complete genomes and 631 hepatitis B surface antigen genes were used to assess the worldwide diversity of hepatitis B virus (HBV). Apart from the described two subgenotypes each for A and F, also B, C, and D divided into four subgenotypes each in the analysis of complete genomes supported by significant bootstrap values. The subgenotypes of B and C differed in their geographical distribution, with B1 dominating in Japan, B2 in China and Vietnam, B3 confined to Indonesia, and B4 confined to Vietnam, all strains specifying subtype ayw1. Subgenotype C1 was common in Japan, Korea, and China; C2 in China, South-East Asia, and Bangladesh, and C3 in the Oceania comprising strains specifying adrq-, and C4 specifying ayw3 is encountered in Aborigines from Australia. This pattern of defined geographical distribution was less evident for D1-D4, where the subgenotypes were widely spread in Europe, Africa, and Asia, possibly due to their divergence having occurred a longer time ago than for genotypes B and C, with D4 being the first split and still the dominating subgenotype of D in the Oceania. The genetic diversity of HBV and the geographical distribution of its subgenotypes provide a tool to reconstruct the evolutionary history of HBV and may help to complement genetic data in the understanding of the evolution and past migrations of man., (2004 S. Karger AG, Basel.)

A modification of the Representational Difference Analysis (RDA) method for subtractive hybridization, termed Selectively Primed Adaptive Driver (SPAD) RDA, is described. It differs from conventional RDA primarily in the manner by which initial driver (D) and tester (T) amplicon complexities are determined, and by optimizing the composition of D with respect to T for each round of subtraction. Total nucleic acid is extracted from serum or plasma and converted to double-stranded DNA/cDNA. A polymerase chain reaction (PCR) primer containing a selective nucleotide(s) at its 3'-end is used to generate amplicons of reduced complexity. Parallel subtractions are carried out, D vs. T (DT) for enrichment of tester-unique sequences and D vs. D (Driver Control or DC) to generate an optimized driver for use in the subsequent round. Following each round, agarose gel electrophoresis is used to visually identify any DT-unique bands through a side-by-side comparison of DT and DC subtraction products. In comparison to conventional RDA, SPAD-RDA achieved greater enrichment of viral sequences from an HCV infected chimpanzee, resulting in isolation of 13.7% of the viral genome, and an overall enrichment for HCV sequences of 239-fold. Virus fragments were also obtained from an HCV-infected human sample subtracted against non-paired human driver sequences. J. Med. Virol. 71:150-159, 2003., (Copyright 2003 Wiley-Liss, Inc.)

DNA-based immunization may be of prophylactic and therapeutic value for hepatitis C virus (HCV) infection. In efforts to improve the immunogenicity of a plasmid expressing the second envelope protein (E2) of HCV, we evaluated in mice the role of the antigen localization and demonstrated that membrane-bound and secreted forms induced higher titers of E2-specific antibodies, as well as earlier and higher seroconversion rates, than the intracellular form, but all three forms induced strong CTL. We also investigated whether E2-specific antibody responses could be enhanced by CpG optimization of the plasmid backbone and showed that removal of neutralizing CpG dinucleotides did not have a significant effect but addition of 64 immunostimulatory CpG motifs significantly enhanced anti-E2 titers. These results may have implications for the design and development of HCV DNA vaccines.

A rapid, reverse transcription-polymerase chain reaction (RT-PCR) procedure for the detection of reovirus RNA in cell culture is described. Total nucleic acids are extracted from a small volume of cell culture supernatant and reverse transcribed using random hexamers. An aliquot of cDNA is then utilized in nested PCR. The PCR primers correspond to sequences conserved between prototype reovirus strains type 1 Lang, type 2 Jones, and type 3 Dearing, as well as those of several reovirus field-isolate strains. Reactions are analyzed by agarose gel electrophoresis, and samples showing a band of the appropriate size in the first and second amplification, or in the second amplification alone, are designated as positive. This protocol allows for the rapid and sensitive detection of reovirus in cell culture. The RT-PCR methods described below can easily be adapted to the amplification of reovirus from other media, including preserved tissues, clinical specimens, and water.

Reoviruses infect virtually all mammalian species, and infection of humans is associated with mild gastrointestinal or upper respiratory illnesses. To improve reovirus detection strategies, we developed a reverse transcription-PCR technique to amplify a fragment of the reovirus L1 gene segment. This assay was capable of detecting 44 of 44 reovirus field isolate strains and was sufficiently sensitive to detect nearly a single viral particle (1.16 +/- 0.13) per PCR of prototype strain type 3 Dearing. Pairwise comparisons of the 44 partial L1 gene sequences revealed that nucleotide variability ranged from 0 to 24.7%, with most of the nucleotide polymorphism occurring at synonymous positions. Phylogenetic trees generated from amplified L1 gene sequences suggest that multiple alleles of the L1 gene cocirculate in nature and that genetic diversity of the L1 gene is largely independent of the host species, geographic locale, or date of isolation. Phylogenetic trees constructed from the L1 gene sequences are distinct from those constructed from the four reovirus S-class gene segments, which supports the hypothesis that reovirus gene segments reassort in nature. This study establishes a new sensitive and specific technique for the identification of mammalian reoviruses and enhances our understanding of reovirus evolution.

Hepatitis E virus (HEV) infection has been considered a disease associated with developing regions and attributed to oral-fecal transmission due to inadequate sanitation. Several recent findings, however, have led to a new understanding of this virus. A number of novel isolates have been identified in patients with acute hepatitis from regions not considered endemic for HEV, and these individuals reported no recent travel to HEV endemic areas. In addition, a number of HEV-like sequences have also been isolated from swine worldwide, suggesting the potential of an animal reservoir. Although full-length sequence is available for some strains, the majority of HEV isolates have only been sequenced partially. Sequence comparisons and phylogenetic analyses were performed to determine the genotypic distribution of HEV isolates, based on the partial sequence data available. It has been suggested that HEV isolates segregate into four major genotypes based on full-length comparisons. These analyses, however, indicate that HEV may be distributed into at least nine different groups., (Copyright 2001 Wiley-Liss, Inc.)

In recent years, molecular biology advances have enabled many investigators to discover a number of viruses that have been difficult to characterise by cell culture techniques. Two blood-borne viruses have been identified. These are GB virus C (GBV-C) and TT virus (TTV). GBV-C was discovered in 1995. It is a flavivirus-like enveloped particle measuring 50-100 nm in diameter with a density of 1.08-1.13 g/cm3. The genome of GBV-C is a single-stranded, positive strand ribonucleic acid of approximately 8600 nucleotides. The TTV was discovered in 1997. It is a circular single-stranded deoxyribonucleic acid virus, non-enveloped of approximately 3900 nucleotides. It has a density of 1.31-1.34 g/cm3 and a particle size of 30-50 nm. Both viruses are distributed widely throughout the world. Most GBV-C infections are asymptomatic, transient and self-limiting. To date, solid evidence for any association of TTV with disease has not been demonstrated.

We isolated a novel hepatitis E virus (HEV-Au1) variant from a patient in Austria suffering from acute viral hepatitis, who had no known risk factors for acquiring hepatitis E. The clinical presentation and initial serological findings have been reported previously. In this paper we report the results of sequence and phylogenetic analysis of HEV products from viral RNA isolated from acute phase serum. The results show that HEV-Au1 is significantly divergent from other HEV isolates. The nucleotide identity of analysed fragments from the novel isolate ranges from 76.6 to 78.4% when compared to isolates from endemic regions and 84.6 to 87.9% when compared to isolates from non-endemic regions. Divergent results were obtained when serum samples taken from the convalescent phase of disease were tested with three different immunoassays (EIAs). An EIA based on United States isolate-specific peptides showed enhanced reactivity whereas EIAs based on recombinant proteins derived from prototype HEV strains from Burma and Mexico were unable to detect antibodies to HEV (anti-HEV) in late phase serum. The findings verify the presence of an additional HEV variant in an industrialized country and provide information about possible problems in detecting anti-HEV.

Immune responses to two recombinant envelope 2 (E2) proteins, representing genotypes 1 and 2 of the GB virus C, or the hepatitis G virus (GBV-C/HGV), were studied in mice and in 48 individuals with, or without, chronic, or past GBV-C/HGV infection. Immunised mice developed E2-specific antibodies (mean titres, 1:1,167 to 1:9,360), recognising linear antigenic regions and proliferative and IL-2, IL-6 and gammaIFN cytokine responses regardless of the viral genotype. Individuals with past GBV-C infection had E2 antibody titres from 1:1,500 to 1:7,500 that did not recognise the E. coli derived E2 protein or linear antigenic regions. Proliferative E2-specific responses were detected in peripheral blood mononuclear cells from 6/22 (27%) persons with, and in none without GBV-C markers (P<0.05). Thus, E2-specific immune responses are mainly crossreactive between different variants of GBV-C/HGV, although proliferative responses appear to be rare.

Hepatitis C virus (HCV) is an important cause of chronic liver disease worldwide. Development of vaccines to prevent HCV infection, or at least prevent progression to chronicity, is a major goal. In mice and rhesus macaques, a DNA vaccine encoding cell-surface HCV-envelope 2 (E2) glycoprotein stimulated stronger immune responses than a vaccine encoding intracellular E2. Therefore, we used DNA encoding surface-expressed E2 to immunize chimpanzees 2768 and 3001. Chimpanzee 3001 developed anti-E2 after the second immunization and antibodies to hypervariable region 1 (HVR1) after the third immunization. Although chimpanzee 2768 had only low levels of anti-E2 after the third immunization, an anamnestic response occurred after HCV challenge. CTL responses to E2 were not detected before challenge, but a strong response was detected after HCV challenge in chimpanzee 2768. An E2-specific CD4+ response was detected in chimpanzee 2768 before challenge and in both chimpanzees postchallenge. Three weeks after the last immunization, animals were challenged with 100 50% chimpanzee-infectious doses (CID(50)) of homologous monoclonal HCV. As a control, a naive chimpanzee was inoculated with 3 CID(50) of the challenge virus. The vaccine did not generate sterilizing immunity because both vaccinated chimpanzees were infected. However, both vaccinated chimpanzees resolved the infection early whereas the control animal became chronically infected. Compared with the control animal, hepatitis appeared earlier in the course of the infection in both vaccinated chimpanzees. Therefore, DNA vaccine encoding cell surface-expressed E2 did not elicit sterilizing immunity in chimpanzees against challenge with a monoclonal homologous virus, but did appear to modify the infection and might have prevented progression to chronicity.

Hepatitis E virus (HEV) has been identified in 2 Argentine patients with acute hepatitis who reported no history of travel to regions in which HEV is considered endemic. These isolates are the first to be identified in South America. By use of degenerate primers from open reading frames 1 and 2, HEV sequences were obtained from these patients' serum and compared with published HEV sequences. The Argentine isolates are different from all previously identified HEV isolates and are most closely related to each other. The Argentine isolates are distinct from the most geographically related isolate from Mexico as well as isolates from other endemic (China, Southeast Asia, and India) and nonendemic (the United States and Europe) regions. Phylogenetic analysis indicate that the Argentine isolates represent a new genotype of HEV, genotype 8, distinct from the Burmese-like genotype 1, Mexican genotype 2, US genotype 3, Chinese/Taiwan genotype 4, and European genotypes 5-7.

A polymerase chain reaction (PCR)-based procedure for the detection of TT virus DNA is described. In this method. total nucleic acid extracted from a small volume of serum or plasma is utilized as a template in PCR employing TT virus specific primers designed to highly conserved regions of the virus genome. Additional sensitivity is obtained by carrying out a second round of amplification. Reactions are analyzed by agarose gel electrophoresis, and samples having an ethidium bromide stainable fragment of the appropriate size in the first and/or second amplification are designated as positive. This protocol allows for the rapid and sensitive detection of TT virus in human plasma or serum.

The increasing use of hepatitis B vaccination has had an overwhelming positive impact on the prevention of hepatitis B viral infection. Mutations in the hepatitis B surface antigen (HBsAg) gene occur as a result of vaccine escape mutants, anti-hepatitis B surface antigen immunotherapy, or in chronic hepatitis B viral infection. These mutants may present a challenge to immunoassay detection. Evaluation of the immunodetection of various HBsAg mutants has been sporadic, as the occurrence of these mutants is not common, and sufficient volume of serum samples is difficult to obtain. To investigate mutant detection, recombinant antigens were constructed to reflect mutations described in the literature occurring throughout the S gene. A limited number of serum samples exhibiting discordant immunoassay reactivity were also used to construct recombinant antigens. The evaluation of 25 HBsAg mutants across nine commercial assays of differing formats is described. Mutations affecting immunoassay performance were characterized as occurring mainly in loop 2 of the "a" determinant of HBsAg. It was determined that reagent epitope recognition was more significant for mutant detection than assay format.

A rapid reverse transcription-polymerase chain reaction (RT-PCR) procedure for the detection of Hepatitis E virus (HEV) RNA in serum is described. Total nucleic acids are extracted from a small volume of human serum and reverse transcribed using random hexamers. An aliquot of cDNA is then utilized in nested PCR employing degenerate HEV consensus primers. These primers are designed to sequences conserved between the Burma, Mexico, and US HEV strains, generating amplicons within each of the three open reading frames. Reactions are analyzed by agarose gel electrophoresis and samples showing an ethidium bromide stained band of the appropriate size in the first and second amplification, or in the second amplification only, are designated as positive. This protocol allows for the rapid and sensitive detection of HEV infection in human serum.

TT virus is a newly described agent infecting humans. Initially isolated from a patient (initials T.T.) with unexplained hepatitis, the virus has since been found in both normal and diseased individuals. In the present study, we utilized genomic-length sequences from distinct genotypes of TT virus to design PCR-based assays using conserved oligonucleotide primers from three independent regions of the virus genome. Each of the three assays was found to be superior to the PCR-based assays previously published. The most sensitive of the new assays was utilized to demonstrate the prevalence of TT virus to be at least 34.1% in volunteer blood donors, 39.6% in commercial blood donors, 59.6% in non-A-GB hepatitis cases, 81.7% in injectable drug users and 95.9% in haemophiliacs. In an attempt to identify a possible source of human infection, we found TT virus sequences to be present in 19% of chickens, 20% of pigs, 25% of cows and 30% of sheep. Sequence determination and phylogenetic analyses demonstrated that isolates from farm animals were not genetically distinct from those found in humans. This study clearly demonstrates that previously reported PCR assays dramatically underestimate the true prevalence of TT virus within the human population. Due to the high rate of infection in both blood donors and those with non-A-GB hepatitis, these results question the causal role of TT virus in cases of unexplained hepatitis. Further, it is possible that domesticated farm animals serve as a source of human infection.

Since the identification of TT virus, only one full-length and two near full-length sequences representing a single subtype of the virus have been reported. In order to understand further the nature of the TT virus genome, nine of the most divergent TT virus sequences have been extended to full-length or near full-length. Phylogenetic analysis demonstrated that these sequences represent three distinct TT virus genotypes and two subtypes. A high degree of nucleotide sequence variability (approximately 30%) was observed across the genomes with several significantly more divergent regions. Three conserved ORFs were identified, none of which shared significant amino acid sequence identity to sequences present in public databases. Additionally, sequence motifs, such as those necessary for protein translation and for rolling circle replication, were found to be partially conserved between all TT virus isolates.

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.

We analyzed the humoral immune response elicited by hepatitis C virus (HCV) E2 protein expressed in vivo after injection of plasmid DNA into mice and rhesus macaques. Three plasmids were used for immunization: a plasmid containing the entire sequence of the E2 and p7 genes (pE2); a plasmid encoding a truncated form of the E2 protein targeted to the cell surface (pE2surf); a control plasmid (pDisplay) lacking an HCV insert. Each plasmid was injected intramuscularly into 5 mice and intraepidermally (via gene gun) into 5 mice. Immunization was repeated three times at three week intervals. Five macaques were injected intramuscularly (two with pE2, two with pE2surf and one with pDisplay) and immunization was repeated after 8 weeks. All mice immunized via gene gun with pE2 or pE2surf developed anti-E2. The animals immunized with pE2surf developed an earlier and stronger humoral immune response than those immunized with pE2. Only 2 of the mice injected by the intramuscular route, both immunized with pE2surf, developed detectable anti-E2. One of the two macaques immunized with pE2 and both macaques immunized with pE2surf developed anti-E2; the humoral immune response was much stronger in the animals immunized with pE2surf. Our results suggest that presentation of HCV E2 on the cell surface may increase its immunogenicity while preserving its ability to react with antibodies generated during a natural infection.

Hepatitis E infection is typically associated with areas in which hepatitis E virus (HEV) is endemic. Except for a few cases in Europe and in the United States, acute hepatitis E is usually associated with travel to endemic areas. We set out to determine the etiologic role of HEV in acute non-A-C hepatitis in Italy. The presence of HEV-RNA and antibody was determined in 218 patients diagnosed with acute viral non-A-C hepatitis. Acute hepatitis E infection was defined by the presence of HEV-RNA in sera and positivity for IgM anti-HEV and seroconversion to IgG anti-HEV. Acute hepatitis E was found in 10.1% of the patients with acute non-A-C, with 95.5% exhibiting a benign course. A more severe course was observed in a patient co-infected with HAV and HEV. Most cases were travelers to endemic areas, although 18.2% reported no travel. One patient was from a household with an infected patient. Sequence analyses of the polymerase chain reaction (PCR) product derived from a patient who never visited endemic areas, identified an isolate that is divergent significantly from all reported isolates of HEV (79.5-85.8% nucleotide identity). Evidence from this study suggests that HEV accounts for approximately 10% of acute non-A-C viral hepatitis in Italy, diagnosed generally in travelers returning from endemic areas. However, the identification of a new HEV variant in an individual who never indicated travel or contact with individuals associated with endemic areas, suggests that this virus may be native to Italy.

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.

Hepatitis E infection is associated with areas in which hepatitis E virus (HEV) infection is endemic. Acute infections in industrialized nations are usually linked to travel to endemic areas. Recently, an acute hepatitis infection in a patient from the United States (US), with no recent foreign travel history, was linked to a novel strain of HEV. Although a few additional cases have been reported from patients who have not traveled to endemic areas, the source of these infections has not been determined. The objective of this study was to identify additional HEV isolates from patients with acute infection who had no recent history of travel to areas where HEV is considered endemic, and to determine the genetic relationship between these and other HEV isolates. Viral RNA was isolated from serum and polymerase chain reaction (PCR) was performed using consensus primers based on a number of HEV isolates. HEV sequence in open reading frame (ORF) 1 and ORF2 was identified in three patients from nonendemic areas, one from Italy and two from Greece. Comparative and phylogenetic analyses were performed. The Greek and Italian isolates were significantly divergent from two isolates from the US and isolates identified previously from HEV-endemic regions. The Italian isolate was distinct from the two Greek isolates. In addition, the two Greek isolates were significantly divergent from each other. Phylogenetic analysis indicated that the Italian and two Greek isolates represent three new genotypes of HEV, distinct from the Burmese, Mexican, and US genotypes.

The partial sequence of a hepatitis E virus (HEV-US1) isolated from a patient in the United States (US), suffering from acute viral hepatitis with no known risk factors for acquiring HEV, has been reported. These sequences were significantly different from previously characterized HEV isolates, alluding to the existence of a distinct human variant. In this paper, we report the near full-length sequences of HEV-US1 and a second US isolate (HEV-US2). HEV-US2 was identified in a US patient suffering from acute viral hepatitis. These sequences verify the presence of a new HEV strain in North America and provide information as to the degree of variability between variants. The HEV-US nucleotide sequences are 92% identical to each other and only 74% identical to the Burmese and Mexican strains. Amino acid and phylogenetic analyses also demonstrate that the US isolates are genetically distinct, suggesting the presence of three genotypes of HEV. Serum from the second US patient induced hepatitis following inoculation into a cynomolgus macaque. Within 2-4 weeks, HEV-US2 RNA was detectable in both the serum and faecal material coinciding with elevated serum alanine transaminase levels. Infection resolved as antibody titres increased 8 weeks post-inoculation.

Recent studies have reported and provided nucleotide sequence data from divergent isolates of hepatitis E virus (HEV), including isolates from North America and Africa. Sera were investigated from 29 Chinese patients with a diagnosis of acute hepatitis and who were negative for hepatitis viruses A-E by serology (HEV was excluded by testing for IgG antibody only). To determine whether some patients were infected with HEV but had yet to seroconvert to antibody positivity, RT-PCR was carried out with primers designed within conserved sequences of the HEV open reading frame (ORF) 1 and ORF2 regions. Fifteen patients were found to harbour sequences related to HEV. Analysis of the HEV products revealed that nucleotide sequences from nine of the sera closely matched Burmese-like HEV sequences (more than 92% nucleotide identity across ORF1 and 88% in ORF2). The remaining six HEV isolates were similar to each other but divergent from all other known HEV sequences (74 to 83% nucleotide identity in ORF1 or ORF2). Phylogenetic analysis suggests that the six divergent isolates represent a fourth genotype of HEV, distinct from the previously described Burmese, Mexican and United States variants (genotypes 1, 2 and 3). This novel variant, referred to here as the Chinese genotype (genotype 4), may be responsible for a significant proportion of cases of acute hepatitis in China, as seen by the fact that 40% of the HEV-infected patients in this study were genotype 4 positive.

Prior to the recent discovery of the swine hepatitis E virus (swine HEV) in pigs from the midwestern United States, HEV was not considered endemic to this country. Since swine HEV is antigenically and genetically related to human strains of HEV, it was important to characterize this new virus further. The infectivity titer of a pool of swine HEV in pigs was determined in order to prepare a standardized reagent and to evaluate the dose response in pigs. Although the sequence of swine HEV varied extensively from those of most human strains of HEV, it was very closely related to the two strains of human HEV (US-1 and US-2) isolated in the United States. The U.S. strains which were recently recovered from two patients with clinical hepatitis E in the United States shared >/=97% amino acid identity with swine HEV in open reading frames 1 and 2. Phylogenetic analyses of different regions of the genome revealed that swine HEV and the U.S. strains grouped together and formed a distinct branch. These results suggested that swine HEV may infect humans. When we inoculated rhesus monkeys and a chimpanzee, experimental surrogates of humans, with swine HEV, the primates became infected. Furthermore, in a reciprocal experiment, specific-pathogen-free pigs were experimentally infected with the US-2 strain of human HEV that is genetically similar to swine HEV. These results provided experimental evidence for cross-species infection by the swine virus. Thus, humans appear to be at risk of infection with swine HEV or closely related viruses.

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.

The etiology of liver disease remains unknown in about 4 to 23% of dialysis patients and 10 to 16% of renal transplant recipients. A search for other causative agents of liver disease led to the discovery of the GB group of viruses. We studied the association between the presence of GB virus C (GBV-C) infection, known risk factors for parenterally-transmitted infections and history or laboratory evidence of liver disease among end-stage renal disease (ESRD) patients referred for renal transplantation to the New England Organ Bank, MA. Stored sera from patients on the renal transplantation waiting list between November 1986 and June 1990 were tested for antibody to hepatitis C virus (HCV). Sera were available in 1544 of 3243 (48%) patients, and anti-HCV was detected by ELISA3 in 287 (19%). All 287 anti-HCV positive patients formed the anti-HCV positive cohort and 286 randomly selected anti-HCV negative patients formed the anti-HCV negative cohort (573 patients overall). Additional sera were available for GBV-C RNA testing in 465 of 573 (81%) patients, and GBV-C RNA was detected by RT-PCR in 146. The overall extrapolated prevalence of serum GBV-C RNA was 29%. The prevalence of serum GBV-C RNa among anti-HCV positive patients (35%) was not significantly different from that among anti-HCV negative patients (29%; P = 0.22). In a univariate analysis, compared to patients without GBV-C RNA, patients with serum GBV-C RNA were younger [odds ratio (OR) 0.98 per year of age, P = 0.01], had a lower proportion of males (OR 0.64, P = 0.03), lower proportion of patients with diabetes mellitus (OR 0.44, P = 0.01), higher proportion of patients with a previous transplantation (OR 1.53, P = 0.04), longer duration of dialysis at the time of enrollment (OR 1.004 per month on dialysis, P = 0.03), and a higher proportion of patients with history of transfusions (OR 4.58, P = 0.01). Serum GBV-C RNA was not associated with a significantly increased OR for history of liver disease or non-A, non-B hepatitis, or elevated serum alanine aminotransferase levels. In a step-wise multivariate regression analysis, a younger age (OR 0.98 per year of age, P = 0.03), and history of blood transfusions (OR 3.89, P = 0.03) were associated with an increased OR for serum GBV-C RNA, while diabetes mellitus was associated with a decreased OR for GBV-C RNA (OR 0.47, P = 0.01). Anti-HCV was not a predictor of serum GBV-C RNA (OR 1.07, P = 0.77). The results of this study support the fact that GBV-C is a parenterally transmitted virus and shed light on the modes of transmission of GBV-C among ESRD patients. However, the association with liver disease remains to be established.

We investigated the possible role of hepatitis G virus (HGV or GBV-C) in the aetiology of acute non-A-E hepatitis in Argentina by detecting viral RNA in sera by reverse transcriptase-polymerase chain reaction (RT-PCR) using primers specific for the putative NS3 helicase region of HGV. Sixty two patients with acute hepatitis were included in this study. The absence of hepatitis A-E was confirmed by serological testing, and all patients were negative for HCV RNA and autoimmune markers. All patients denied alcohol intake and the use of hepatotoxic drugs. Their mean age was 35.3 years and 37 were males. HGV RNA was present in 19/62 (30.6%) of the patients with non-A-E acute hepatitis. Among HGV-positive patients, three had parenteral risk factors within 3 months of onset, one was a health care worker, one was sexually promiscuous, one had travelled to the Middle East and 13 (68.4%) had no history of parenteral exposure. Epidemiological, clinical and biochemical features between HGV-positive and negative patients did not achieve statistical significance. Hence, HGV appears to play a role in the pathogenesis of acute viral hepatitis; however, the etiology of a significant number of hepatitis cases remains unclear, suggesting the existence of an additional agent(s). The absence of parenteral exposure in most of the HGV RNA-positive patients in this study shows that routes of community-acquired HGV infection are not yet completely understood.

Viral characterization studies were carried out on GB virus C (GBV-C) RNA positive plasma from normal human donors and from donors co-infected with GBV-C and hepatitis C virus (HCV). GBV-C RNA was detected by reverse-transcriptase polymerase chain reaction (RT-PCR) and probe hybridization in a single tube assay. Sequential filtration of GBV-C positive plasma indicated that GBV-C RNA is associated with a particle 50-100 nm in diameter. The peak of GBV-C RNA in sucrose gradients was observed at a buoyant density of 1.05-1.13 g/ml. GBV-C RNA titer was reduced following treatment with chloroform or with five detergents indicating that GBV-C has a lipid-containing envelope. Sucrose density gradients and self-forming cesium chloride gradients of detergent-treated GBV-C showed a shift in the RNA peak to heavier buoyant density only when RNase inhibitor (RNasin) and high detergent concentrations were present. The treated material was non-filterable and the RNA had a density of > 1.5 gm/ml.

A variant of hepatitis E virus (HEV), designated HEV US-1, was identified in a hepatitis patient in the United States (US); the patient had no history of travel to areas where HEV is endemic. Nucleotide sequences were obtained from the 5' end of open reading frame (ORF) 1 (1418 nt), the 3' end of ORF1 (1359 nt), the entire ORF2 and ORF3 regions, and the 3'-untranslated region (2127 nt). The HEV US-1 strain is significantly divergent from other human HEV isolates with nucleotide identities ranging from 76.8 to 77.5%. Phylogenetic analyses indicate that HEV US-1 and a recently discovered HEV variant from swine may represent separate isolates of a new strain of HEV, significantly divergent from the Mexican and Burmese strains. Synthetic peptides derived from the carboxyl amino acids of ORF2 and ORF3 were shown to be useful for detecting exposure to HEV. In addition, IgM class antibodies directed against HEV US-1 synthetic peptides were detected in the US patient infected with HEV US-1, but were absent using synthetic peptides from the Burmese or Mexican strains of HEV. A preferential reactivity to HEV US-1 specific peptides has lead to the identification of a second isolate of this virus also from a patient with acute hepatitis from the US. The discovery of these HEV variants may be important in understanding the worldwide distribution of HEV infection.

The recent isolation of GB viruses A and B from GB agent infected tamarins and their lack of involvement in human hepatitis has sparked interest in the origin of these viruses. Several healthy non-human primate species have been shown to harbour sequences 52-79% identical to the GBV-A 5' nontranslated region. In this paper we report the near genome length sequence of GBV-Amx 70047 and GBV-Atri 1122. These sequences support previous observations about the genomic organization of GBV-A and provide insight into the genomic variability within this virus genus. Although the GBV-A variant polyproteins possess many motifs conserved between other members of the Flaviviridae, they do not encode a basic core-like protein. Amino acid sequence comparisons and phylogenetic analysis demonstrate variability within the GBV-A genus similar to that observed between hepatitis C virus (HCV) types. However, genomic organization and disease association demonstrate a closer evolutionary relationship to GBV-C than to HCV.

A simple reverse transcription-polymerase chain reaction (RT-PCR) procedure for the detection of GB virus C (GBV-C) RNA in serum or plasma is described. In this method, total nucleic acid, extracted from a small volume of human plasma, is reverse transcribed using random hexamers. An aliquot of cDNA is then utilized in PCR employing GBV-C specific primers designed to highly conserved regions of the 5'nontranslated region (NTR). For additional sensitivity, a second round of nested amplification is performed. Reactions are analyzed on an agarose gel and samples showing an ethidium bromide stained band of the appropriate size in the first and second amplification, or in the second amplification only, are designated to be positive. This protocol allows for the rapid and sensitive detection of GBV-C infection in human plasma or serum.

The aim of this study was to evaluate, in patients with chronic hepatitis C, 1) the prevalence and the epidemiological characteristics of GB virus C (GBV-C) infection, 2) the influence of GBV-C on hepatitis C virus (HCV) infection, 3) the pathogenicity of GBV-C in the absence of treatment and under interferon therapy, and 4) the effect of interferon alfa on GBV-C and HCV replications. One hundred fifteen patients with chronic hepatitis C were studied. Before treatment, they were tested for GBV-C RNA by PCR and GBV-C genotype was determined for positive samples. Pretreatment information was collected, including age, gender, source of HCV, estimated duration of HCV infection, alanine aminotransferase and gamma-glutamyl transpeptidase activities, cirrhosis and Knodell's score on liver biopsy, HCV genotype, HCV viral burden and anti-HCV core IgM antibodies. The genetic complexity of the hypervariable region 1 (HVR1) of HCV was studied by PCR-Single Strand Conformation Polymorphism. All patients were treated with 3 to 9 mega units of interferon alfa-2a three times per week for 3 to 6 months. The influence of GBV-C on the evolution of ALT and HCV replication during and after treatment was studied, and GBV-C and HCV RNA were monitored monthly by PCR during this period. Eighteen patients (16%) were GBV-C RNA-positive. Among 11 samples studied, GBV-C genotype 2a was present in 9 cases, 2b in one case and type 3 in one case. GBV-C RNA-positive patients were significantly younger than GBV-C RNA-negative ones (38.4 +/- 11.5 vs. 47.4 +/- 14.0, P = 0.012), a result independent of the route of transmission and the disease duration. No difference between GBV-C RNA-positive and -negative patients was found for other epidemiological parameters (e.g. gender, risk factor for parenteral viral infections, disease duration and HCV genotypes), or for the characteristics of HCV infection and related liver disease (e.g. HCV RNA level, genetic complexity of the HVR1, anti-HCV core IgM, alanine aminotransferase and gamma-glutamyl transpeptidase activities, cirrhosis and Knodell's score). GBV-C did not influence the rates of ALT normalization at months 3, 6 and 12 and of sustained hepatitis C virological response at month 12 of treatment follow-up. During treatment, GBV-C viremia became undetectable in 12 patients (67%) but relapse occurred after treatment withdrawal in all the nine patients with sufficient follow-up. In the remaining six patients (33%), GBV-C resisted interferon. Whatever the effect of interferon on GBV-C replication, the ALT levels correlated with the presence of HCV RNA. In conclusion, GBV-C infection is frequent in patients with chronic hepatitis C, who are mainly, but not exclusively, infected by GBV-C genotype 2a. GBV-C positive patients are significantly younger than GBV-C negative ones. GBV-C does not seem to affect HCV replication, liver disease and responses of HCV infection and liver disease to interferon therapy. GBV-C is sensitive to 3 mega units of interferon alfa administered three times per week in two-thirds of the patients, but relapse is constant with this dosage after treatment withdrawal.

An enzyme linked immunosorbent assay (ELISA) for detection of antibodies to mammalian cell-expressed E2 protein of GB virus C (GBV-C E2) is described. Antibodies to GBV-C E2 are captured on a solid phase coated with affinity purified E2 protein. Bound antibody is detected in an indirect assay format using horseradish peroxidase (HRPO) labeled goat anti-human IgG as the secondary antibody. Following a color development step, absorbance at 492 nm is measured. A population of 100 volunteer blood donors was tested to assess the specificity of this assay. Individuals reactive for antibody to GBV-C E2 can be considered to have been exposed to GB virus C.

Exposure to GB virus C (GBV-C) was determined in several U.S. populations by both reverse-transcription-polymerase chain reaction (RT-PCR) and by an enzyme linked immunosorbent assay (ELISA) for antibodies to mammalian cell-expressed GBV-C envelope protein, E2 (GBV-C E2). Most individuals exposed to GBV-C were either RNA positive/ELISA negative or ELISA positive/RNA negative. Exposure, therefore, was measured as the sum of GBV-C RNA positive and GBV-C E2 antibody positive specimens, and was higher in commercial plasmapheresis donors (40.5%) than in volunteer blood donors (5.5%). In intravenous drug users (IVDUs), GBV-C exposure was 89.2%. Serial bleed specimens tested for GBV-C RNA indicate that some patients remain viremic for at least 3 years and fail to produce detectable antibodies to GBV-C E2. In other exposed individuals who tested negative for GBV-C RNA, antibodies to E2 appear to be similarly long-lived (greater than 3 years) with a fairly constant titer (ranging in reciprocal endpoint dilution from 336 to 21,504). Since the detection of GBV-C RNA and GBV-C E2 antibody are mutually exclusive in most exposed individuals, studies pertaining to incidence and prevalence of GBV-C infection require both antibody and nucleic acid detection.

Recently, gene fragments of several novel variants of GB virus A were isolated from the serum of distinct monkey species that had not been experimentally inoculated with an infectious agent. These variants appeared to be species-specific in that sequences isolated within a species were virtually identical, though sequences were strikingly different when compared between each species. In the present study, the nucleotide sequence of one of these variants, GBV-Alab, was extended to near-genome length. Similar to the other GB viruses, GBV-Alab appears to encode a single large polyprotein of 2967 amino acids that is post-translationally cleaved by cellular and viral proteases into the individual viral proteins. The structural proteins are found at the N-terminal end of the polyprotein, while the nonstructural proteins are found at the C teminus. Amino acid sequence comparisons of the large polyprotein demonstrate that GBV-Alab is 74% identical to GBV-A and 48% identical to GBV-C, sharing only marginal identity with GBV-B and HCV-1 at 27%. Examination of the GBV-Alab polyprotein reveals that structural motifs are conserved for a protease, a helicase and a replicase. Phylogenetic analysis of the polyprotein confirms previous results that GBV-Alab is a member of the Flaviviridae, distinct from GBV-B and HCV, though more closely related to GBV-A and GBV-C.

Phylogenetic analysis of 44 GB virus C (GBV-C) 5'-untranslated region (5'-UTR) sequences from 37 individuals suggested the presence of GBV-C genotypes (A. S. Muerhoff, J. N. Simons, T. P. Leary, J. C. Erker, M. L. Chalmers, T. J. Pilot-Matias, G. J. Dawson, S. M. Desai, and I. K. Mushahwar, J. Hepatol. 25:379-384, 1996) that correlated with geographic origin: type 1, 2a and 2b, and 3 isolates are found predominantly in West Africa, the United States and Europe, and Japan, respectively. We have extended our analysis to include 5'-UTR sequences from 129 globally distributed GBV-C isolates and sequences from the second envelope protein (E2) gene and nonstructural (NS) regions 3 and 5b from a subset of these isolates. Bootstrap analysis of a 157-nucleotide segment of the 5'-UTR from 129 sequences provided weak support for the existence of the four major groups of GBV-C isolates previously described, although phylogenetic analysis of a 374-nucleotide segment of the 5'-UTR from 83 isolates provided stronger support. Thus, the groups of GBV-C variants previously identified upon analysis of the entire 5'-UTR can be distinguished by analysis of the shorter, 374-nucleotide region from the 5'-UTR. In contrast, independent analysis of the E2, NS3, or NS5b region sequences does not identify groups of GBV-C variants that correlate with geographic origin. However, bootstrap analysis of these coding sequences, when linked to form colinear sequences, demonstrates that longer coding regions can produce GBV-C groupings that are similar to that determined from 5'-UTR sequence analysis. The inability to distinguish between GBV-C variants by using small segments of coding sequence suggests that the GBV-C genome is constrained. As a result of these constraints, there is a high degree of nucleotide and amino acid sequence conservation between isolates from widely separated geographic areas. Hence, substitutions at many nucleotide positions are not tolerated, so that substitutions at the positions which can change are saturated, thereby obscuring the evolutionary relationships.

A 315 amino acid recombinant segment of the GB virus C (GBV-C) E2 envelope glycoprotein (E2-315) was expressed and secreted from CHO cells. E2-315 was purified by affinity chromatography using a monoclonal antibody directed to a FLAG sequence genetically engineered onto the C terminus of the recombinant protein. The secreted protein had a molecular mass of 48-56 kDa and was shown to be N-glycosylated. Amino acid sequencing confirmed the expected N-terminal sequence. Purified E2-315 was used to develop an ELISA for detection of E2 antibodies in human sera. Antibodies to GBV-C E2 appeared to be directed toward conformational epitopes since human sera reactivity was detected in ELISA using native E2-315, but it was extremely weak or non-existent with denatured E2 protein. The use of an ELISA which can detect human GBV-C E2 antibodies will be important in further understanding of the clinical significance and epidemiology of GBV-C.

Among renal transplant recipients with posttransplantation liver disease, the etiology remains unknown in 10 to 16% of patients. The discovery of yet another parenterally transmitted hepatitis virus, GB virus C (GBV-C), has opened avenues to study the prevalence and risk factors for GBV-C infection among patients undergoing renal transplantation and its impact on posttransplantation clinical outcomes. A cohort of 103 randomly selected recipients of kidneys were examined from anti-hepatitis C virus (HCV)-negative donors between 1986 and 1990. Pretransplantation sera were available in 99 of 103 (96%) recipients and were tested for anti-HCV, using a second-generation ELISA, and for GBV-C RNA by reverse transcription PCR. Pretransplantation GBV-C RNA was present in 18 of 99 (18%, 95% confidence interval [CI], 17.2 to 18.8%) recipients. GBV-C RNA was present in 5 of 22 (23%) anti-HCV-positive recipients compared with 13 of 77 (17%) anti-HCV-negative recipients (P = 0.53). The median number of pretransplantation blood transfusion among recipients with GBV-C RNA before transplantation was significantly higher than among recipients without GBV-C RNA (10 versus 7, P = 0.05). Posttransplantation liver disease and non-A, non-B hepatitis (NANBH) was observed in 35 and 18%, respectively, of GBV-C RNA-positive recipients compared with 28 and 10%, respectively, of GBV-C RNA-negative recipients. Using Cox regression analysis, the relative risk (RR) of posttransplantation liver disease among recipients with GBV-C RNA before transplantation was 1.37 (95% CI, 0.55 to 3.41), and posttransplantation NANBH was 2.09 (95% CI, 0.64 to 6.79). The RR of graft loss and death were not increased (0.88 and 0.92, respectively). When adjusted for pretransplantation anti-HCV, the RR of posttransplantation liver disease, NANBH, graft loss, and death did not change appreciably. In summary, although a higher risk of posttransplantation liver disease was observed among recipients with pretransplantation GBV-C infection, the analyses presented here do not allow for a precise estimate of this risk.

Background: The cloning of yet another hepatitis virus, GB virus-C (GBV-C), has provided the opportunity to study the prevalence, and clinical and laboratory characteristics, associated with GBV-C infection among cadaver organ donors and recipients of organs from infected donors., Methods: Stored sera from a cohort of cadaver organ donors from eight organ procurement organizations, representing different geographic regions of the United States previously screened for hepatitis C virus (HCV) infection, were tested for GBV-C RNA by polymerase chain reaction using degenerate primers derived from the NS3 helicase and 5'-untranslated regions of the GBV-C genome. Pre- and posttransplantation clinical data, and prevalence of GBV-C RNA among recipients of organs from GBV-C RNA-positive and -negative donors, were studied at one of the organ procurement organizations., Results: Twenty-one of 76 (27.6%) anti-HCV ELISA1-positive donors tested positive for GBV-C RNA compared with 6 of 82 (7.3%) ELISA1-negative donors (P=0.001). The prevalence of GBV-C RNA, extrapolated to all cadaver organ donors, was 8.3% (95% confidence interval [CI]: 5.6-11.1%) and was higher than the prevalence of HCV RNA (2.4%). Among ELISA1-positive donors, GBV-C RNA was present in 13 of 35 (37%) donors with HCV RNA, compared with 8 of 41 (20%) donors without HCV RNA (odds ratio [OR]=2.44, P=0.09). Blood alcohol level of more than 100 mg/dl (OR=9.43, P=0.05) and a positive anti-HCV ELISA2 (OR=4.58, P=0.001) were significantly associated with GBV-C infection. In addition, there was a trend toward an association between history of drug abuse (OR=5.23, P=0.06) and younger age (OR=0.97/year, P=0.06) with GBV-C infection. Organs from four GBVC-positive donors and 47 GBV-C-negative donors procured by the New England Organ Bank (Newton, MA) were transplanted into 6 and 79 recipients, respectively. Among recipients of organs from GBV-C RNA. positive donors, the posttransplantation prevalence of GBV-C RNA (25%) was not significantly higher than among recipients of organs from GBV-C RNA-negative donors (23%). Among recipients in whom both pre- and posttransplantation sera were available, one of three (33%) recipients of kidneys from GBV-C RNA-positive donors acquired GBV-C RNA after transplantation, compared with 4 of 40 (10%) recipients of kidneys from GBV-C RNA-negative donors. After a median follow up of 6 years, the posttransplantation prevalence of liver disease, and graft and patient survival, were not significantly different between recipients of organs from GBV-C RNA-positive and -negative donors., Conclusions: Although GBV-C could be transmitted by organ transplantation, the results of this study preclude definitive conclusions. Further studies are required to determine the risk of transmission of GBV-C by organ transplantation and its role in posttransplantation liver disease.