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Interferon sensitivity determining sequence of the hepatitis C virus genome
HEPATOLOGY Elsewhere T. JAKE LIANG, EDITOR
ADVISORY COMMITTEE BRUCE R. BACON, St. Louis, MO HENRY C. BODENHEIMER, New York, NY JAMES M. CRAWFORD, Boston, MA NORMAN D. GRACE, Boston, MA SANJEEV GUPTA, Bronx, NY JOEL LAVINE, San Diego, CA RICHARD H. MOSELEY, Ann Arbor, MI
Gastrointestinal Unit GRJ-724 Massachusetts General Hospital 32 Fruit St Boston, MA 02114
INTERFERON SENSITIVITY DETERMINING SEQUENCE OF THE HEPATITIS C VIRUS GENOME
Enomoto N, Sakuma I, Asahina Y, Kurosaki M, Murakami T, Yamamoto C, Izumi N, et al. Comparison of full-length sequences of interferon-sensitive and -resistant hepatitis C virus 1b. J Clin Invest 1995;96:224-230. ABSTRACT
We have previously demonstrated that sensitivity to interferon is different among hepatitis C virus (HCV) quasispecies simultaneously detected in same individuals and that interferon-resistant HCV quasispecies are selected during the treatment. To determine the genetic basis of their resistance to interferon, HCV genotype 1b was obtained from serum of three patients before and during interferon therapy, and their full-length nucleotide and deduced amino acid sequences were determined. Comparison of the pairs of interferon-resistant and interferon-sensitive HCV isolates in respective individuals demonstrated clusters of amino acid differences in the COOH-terminal half of the NS5A region (codon 2154-2383), which contained a common unique amino acid difference at codon 2218. Additional sequence data of the COOH-terminal half of the NS5A region obtained from six interferon-resistant and nine interferon-sensitive HCV confirmed the exclusive existence of missense mutations in a 40–amino acid stretch of the NS5A region around codon 2218 (from codon 2209 to 2248) in interferon-sensitive HCV. On the other hand, this region of interferon-resistant HCV was identical to that of prototype HCV genotype 1b (HCV-J, HCV-JTa, or HC-J4). We designated this region as the interferon sensitivity–determining region. Thus, HCV genotype 1b, with the prototype interferon sensitivity determining region, appears to be an interferon-resistant strain. The specific nature of these mutations might make it possible to predict prognostic effects of interferon treatment. Enomoto N, Sakuma I, Asahina Y, Kurosaki M, Murakami T, Yamamoto C, Ogura Y, et al. Mutations in the nonstructural protein 5A gene and response to interferon in patients with chronic hepatitis C virus 1b infection. N Engl J Med 1996; 334:77-81. ABSTRACT
Background: A region associated with sensitivity to interferon has been identified in the nonstructural protein 5A (NS5A) of hepatitis C virus (HCV) genotype 1b. The region spans amino acid residues 2209 to 2248 (NS5A2209-2248) of HCV-J, a strain of HCV-1b whose complete genomic sequence has been identified. We examined whether the NS5A2209-2248 sequence present before therapy could be used as a predictor of the response to interferon (IFN) therapy in patients with
chronic HCV-1b infection. Methods: We retrospectively analyzed 84 patients with chronic HCV-1b infection who had received interferon alfa (total dose, 516-880 MU) for 6 months. Pretreatment serum samples were analyzed. The amino acid sequence of NS5A2209-2248 was determined by direct sequencing of the HCV genome amplified by polymerase chain reaction (PCR) and was compared with the established sequence for HCV-J. Results: A complete response, as evidenced by the absence of HCV RNA in serum on nested reverse-transcription PCR for 6 months after therapy, did not occur in any of the 30 patients whose NS5A2209-2248 sequences were identical to that of HCV-J (wild type). Five of 38 patients (13%) with 1 to 3 changes in NS5A2209-2248 (intermediate type) had complete responses as did all 16 patients with 4 to 11 amino acid substitutions (mutant type), indicating that the mutant type was significantly associated with a complete response (P õ .001). Although baseline serum HCV RNA levels, as measured by a branched-chain DNA assay, were lower in patients with the mutant type of NS5A2209-2248 than in those with the other types (P õ .001), multivariate analyses revealed that the number of amino acid substitutions in NS5A2209-2248 was the only variable associated with an independent effect on the outcome of interferon therapy (odds ratio, 5.3, 95% confidence interval, 1.6 to 18; P Å .007). Conclusions: In patients with chronic HCV-1b infection, there is a substantial correlation between responses to interferon and mutations in the NS5A gene. COMMENTS
The efficacy of interferon (IFN) treatment is a major concern of hepatologists who are treating patients with hepatitis C. IFN treatment is the only strategy at present to potentially eliminate hepatitis C virus (HCV) from infected cells and liver tissues and consequently to cure the disease. However, only one third of patients with HCV infection are sensitive to IFN. The determinants of IFN responsiveness may be complicated. Multiple host factors may interact with each other.1 It is believed that viral factor(s) may influence responsiveness as well. These include: (1) viral load (the more virus amount, the less responsive), (2) viral type (type 1b is less reactive than type 2), and (3) viral heterogeneity (the more heterogeneous the viral population, the less responsive). Enomoto et al.2 had previously reported that sensitivity to interferon treatment was different according to HCV quasispecies and suggested the presence of interferon-sensitive or -resistant HCVs. In the first paper, the same group examined HCV RNA sequences from three patients infected with genotype 1b HCV who did not respond to high doses of interferon alfa. By polymerase chain reaction (PCR) amplification and direct nucleotide sequencing, they compared the nucleotide and amino acid sequences of the coding and noncoding regions between samples collected before and after interferon treatment. The authors considered that among quasispecies
460
AID
Hepa 0035
/
5P11$$$681
07-20-96 11:52:03
hpta
WBS: Hepatology
HEPATOLOGY Vol. 24, No. 2, 1996
HEPATOLOGY Elsewhere
of HCV, the major population represented is interferon-sensitive, but during or following IFN treatment in nonresponders, an IFN-resistant clone(s) constitutes the major population. The difference was seen mainly in the E2, NS2, and NS5A regions in patient 1, in the E2 and NS5A region in patient 2, and in the NS5A region in patient 3. Although there were other scattered differences, the main differences were considered to be clustered in the C-terminal region of the NS5A, particularly codons 2154-2383, and in the E2-hypervariable region (HVR). After further pairwise comparison, these three pairs were found to have unique amino acid substitutions at codon 2218. They were glutamine, arginine, and cysteine in patients 1, 2, and 3, respectively, pretreatment. All changed to histidine posttreatment. The previously published HCV type 1b (HCV-J, HCV-J4, and HCV-JTa) possesses a histidine residue at 2218. This was the only commonly observed amino acid difference before and after IFN treatment in three nonresponders. Amino acid differences were detected in other regions, particularly the E2 hypervariable region. However, there were no common changes among the three HCV isolates obtained from patients 1, 2, and 3. The authors considered that the pretreatment HCV sequence represented IFN-sensitive HCV and the posttreatment sequence that of an IFNresistant clone. It should be cautioned that the nucleotide sequence was determined by the direct method, and it was thus not clear whether the IFN-resistant clone was originally present in the pretreatment samples as a minor population. The above results were clear but not conclusive. The authors extended the study by examining HCV sequences from six IFN nonresponders and nine complete responders. This time, they only examined sequences of the C-terminal half of the NS5A. The 9 IFN-resistant clones were from ‘‘after’’ samples of the previous patients 1 through 3 and new nonresponder patients 4 through 9. The 12 IFN-sensitive clones were ‘‘before’’ samples of patients 1 through 3 and responder patients 10 through 18. The amino acid sequences from 2209 to 2248 of the former group were identical to those of prototype HCV 1b. In contrast, clones obtained from the latter group had at least one amino acid change in this region. The clone from patient 18 even had an eight–amino acid insertion between codon 2221 and 2222. Particularly illuminating findings were again revealed at codon 2218. Clones from the former group and from previous isolates (HCV-J, HCV-J4, and HCV JTa) possessed histidine at 2218. However, the amino acid at this position of the clones obtained from complete responders prior to IFN treatment was different. Three of 12 were histidine, but four were arginine, three cysteine, one glutamine, and another proline. More interestingly, the clone from one nonresponder (patient 18) possessed arginine at 2218, but after unsuccessful IFN treatment was replaced by histidine. From this meticulously performed sequence analysis, together with individual clinical data, the authors concluded that there are IFN-sensitive and -resistant clones of HCV. The main difference between these two groups lies in the Cterminal half of the NS5A region, particularly in codons 2209 to 2248, which the authors designate as the IFN sensitivity determining region (ISDR). Within the ISDR, the authors consider codon 2218 most important. When the same nonresponder patients were examined, IFN-sensitive clones were obtained in the pretreatment phase and IFN-resistant clones in posttreatment. As the authors discuss, it may be possible to prospectively predict IFN responsiveness. It is very important to study whether the amino acid changes described were caused by ‘‘selection’’ or ‘‘mutation.’’ To analyze this, it would be more appropriate to sequence as many subclones as possible, instead of performing direct sequencing, which provides information only on major populations. It is known that the viral quasispecies population may change under selective pressure.3,4 In the second paper, the same group retrospectively examined samples obtained from 84 patients who had shown dif-
AID
Hepa 0035
/
5P11$$$682
07-20-96 11:52:03
461
ferent responses to IFN treatment. There were two groups: one consisted of nonresponders (n Å 63); the other consisted of complete responders (n Å 21). The amino acid sequence analysis of the NS5A-ISDR indicated that there were three groups: (1) wild-type with sequences identical to that of prototype HCV-J (n Å 30), (2) intermediate-type with only one to three amino acid substitutions (n Å 38), and (3) mutant-type with more than four amino acid changes (n Å 16). The results were clear. All of the 30 patients infected with wild-type HCV were nonresponders, and all of the 16 mutant HCV-infected patients were complete responders. Only 5 of the 38 patients infected with intermediate-type HCV were complete responders. This time, however, the striking difference was not observed between sensitive and resistant clones at codon 2218. Statistical analysis revealed that HCV RNA levels in sera before treatment were significantly lower in the patient group with mutant-type than those with wild or intermediate types. However, for each individual the amount of HCV RNA did not correlate with IFN responsiveness. Thus, it seems likely that the HCV clones that have identical or very similar amino acid sequences to that of wild-type in the ISDR are resistant to IFN therapy. Viruses of the Flaviviridae family have a similar region corresponding to HCV NS5A. However, the biological function of these NS5As is not yet known. It was recently shown that it is a hyperphosphorylated protein. The candidate hyperphosphorylation site is positioned at serine residues located near the ISDR described by these papers.5 These papers by Enomoto et al.2 provide a potentially important new predictor of IFN responsiveness. They also may provide clues regarding potential virus-cell interactions in HCV infection. When an efficient in vitro replication system is developed, it would be very interesting to compare the virological behavior between HCV clones with wild-type or mutant sequences in the ISDR, particularly with regard to their replication activity. TATSUO MIYAMURA, M.D. Department of Virology II National Institutes of Health Tokyo, Japan REFERENCES 1. Kimura T, Nakayama K, Penningner J, Kitagawa M, Harada H, Matsuyama T, Tanaka N, et al. Involvement of the IRF-1 transcription factor in antiviral responses to interferons. Science 1994;264:1921-1924. 2. Enomoto N, Kurosaki M, Tanaka Y, Marumo F, Sato C. Fluctuation of hepatitis C virus quasispecies in persistent infection and interferon treatment revealed by single-strand conformation polymorphism analysis. J Gen Virol 1994;75:1461-1369. 3. Aizaki H, Saito A, Kusakawa I, Ashiwara Y, Nagamori S, Toda G, Suzuki T, et al. Mother-to-child transmission of a hepatitis C virus variant with an insertional mutation in its hypervariable region. J Hepatol (in press). 4. Nakajima N, Hijikata M, Yoshikura H, Shimizu Y. Characterization of longterm culture of hepatitis C virus. J Virol (in press). 5. Tanji Y, Kaneko T, Satoh S, Shimotohno K. Phosphorylation of hepatitis C virus-encoded nonstructural protein NS5A. J Virol 1995;69:3980-3986.
HEPATITIS G: A VIRUS IN SEARCH OF A DISEASE
Linnen J, Wages J, Zhen-Yong Z-K, Fry KE, Krawczynski KZ, Alter H, Koonin E, et al. Molecular cloning and disease association of hepatitis G virus: a transfusion-transmissible agent. Science 1996;271:505-508. ABSTRACT
An RNA virus, designated hepatitis G virus (HGV), was identified from the plasma of a patient with chronic hepatitis. Extension from an immunoreactive complementary DNA clone yielded the entire genome (9,392 nucleotides) encoding a polyprotein of 2,873 amino acids. The
hpta
WBS: Hepatology
ADVISORY COMMITTEE BRUCE R. BACON, St. Louis, MO HENRY C. BODENHEIMER, New York, NY JAMES M. CRAWFORD, Boston, MA NORMAN D. GRACE, Boston, MA SANJEEV GUPTA, Bronx, NY JOEL LAVINE, San Diego, CA RICHARD H. MOSELEY, Ann Arbor, MI
Gastrointestinal Unit GRJ-724 Massachusetts General Hospital 32 Fruit St Boston, MA 02114
INTERFERON SENSITIVITY DETERMINING SEQUENCE OF THE HEPATITIS C VIRUS GENOME
Enomoto N, Sakuma I, Asahina Y, Kurosaki M, Murakami T, Yamamoto C, Izumi N, et al. Comparison of full-length sequences of interferon-sensitive and -resistant hepatitis C virus 1b. J Clin Invest 1995;96:224-230. ABSTRACT
We have previously demonstrated that sensitivity to interferon is different among hepatitis C virus (HCV) quasispecies simultaneously detected in same individuals and that interferon-resistant HCV quasispecies are selected during the treatment. To determine the genetic basis of their resistance to interferon, HCV genotype 1b was obtained from serum of three patients before and during interferon therapy, and their full-length nucleotide and deduced amino acid sequences were determined. Comparison of the pairs of interferon-resistant and interferon-sensitive HCV isolates in respective individuals demonstrated clusters of amino acid differences in the COOH-terminal half of the NS5A region (codon 2154-2383), which contained a common unique amino acid difference at codon 2218. Additional sequence data of the COOH-terminal half of the NS5A region obtained from six interferon-resistant and nine interferon-sensitive HCV confirmed the exclusive existence of missense mutations in a 40–amino acid stretch of the NS5A region around codon 2218 (from codon 2209 to 2248) in interferon-sensitive HCV. On the other hand, this region of interferon-resistant HCV was identical to that of prototype HCV genotype 1b (HCV-J, HCV-JTa, or HC-J4). We designated this region as the interferon sensitivity–determining region. Thus, HCV genotype 1b, with the prototype interferon sensitivity determining region, appears to be an interferon-resistant strain. The specific nature of these mutations might make it possible to predict prognostic effects of interferon treatment. Enomoto N, Sakuma I, Asahina Y, Kurosaki M, Murakami T, Yamamoto C, Ogura Y, et al. Mutations in the nonstructural protein 5A gene and response to interferon in patients with chronic hepatitis C virus 1b infection. N Engl J Med 1996; 334:77-81. ABSTRACT
Background: A region associated with sensitivity to interferon has been identified in the nonstructural protein 5A (NS5A) of hepatitis C virus (HCV) genotype 1b. The region spans amino acid residues 2209 to 2248 (NS5A2209-2248) of HCV-J, a strain of HCV-1b whose complete genomic sequence has been identified. We examined whether the NS5A2209-2248 sequence present before therapy could be used as a predictor of the response to interferon (IFN) therapy in patients with
chronic HCV-1b infection. Methods: We retrospectively analyzed 84 patients with chronic HCV-1b infection who had received interferon alfa (total dose, 516-880 MU) for 6 months. Pretreatment serum samples were analyzed. The amino acid sequence of NS5A2209-2248 was determined by direct sequencing of the HCV genome amplified by polymerase chain reaction (PCR) and was compared with the established sequence for HCV-J. Results: A complete response, as evidenced by the absence of HCV RNA in serum on nested reverse-transcription PCR for 6 months after therapy, did not occur in any of the 30 patients whose NS5A2209-2248 sequences were identical to that of HCV-J (wild type). Five of 38 patients (13%) with 1 to 3 changes in NS5A2209-2248 (intermediate type) had complete responses as did all 16 patients with 4 to 11 amino acid substitutions (mutant type), indicating that the mutant type was significantly associated with a complete response (P õ .001). Although baseline serum HCV RNA levels, as measured by a branched-chain DNA assay, were lower in patients with the mutant type of NS5A2209-2248 than in those with the other types (P õ .001), multivariate analyses revealed that the number of amino acid substitutions in NS5A2209-2248 was the only variable associated with an independent effect on the outcome of interferon therapy (odds ratio, 5.3, 95% confidence interval, 1.6 to 18; P Å .007). Conclusions: In patients with chronic HCV-1b infection, there is a substantial correlation between responses to interferon and mutations in the NS5A gene. COMMENTS
The efficacy of interferon (IFN) treatment is a major concern of hepatologists who are treating patients with hepatitis C. IFN treatment is the only strategy at present to potentially eliminate hepatitis C virus (HCV) from infected cells and liver tissues and consequently to cure the disease. However, only one third of patients with HCV infection are sensitive to IFN. The determinants of IFN responsiveness may be complicated. Multiple host factors may interact with each other.1 It is believed that viral factor(s) may influence responsiveness as well. These include: (1) viral load (the more virus amount, the less responsive), (2) viral type (type 1b is less reactive than type 2), and (3) viral heterogeneity (the more heterogeneous the viral population, the less responsive). Enomoto et al.2 had previously reported that sensitivity to interferon treatment was different according to HCV quasispecies and suggested the presence of interferon-sensitive or -resistant HCVs. In the first paper, the same group examined HCV RNA sequences from three patients infected with genotype 1b HCV who did not respond to high doses of interferon alfa. By polymerase chain reaction (PCR) amplification and direct nucleotide sequencing, they compared the nucleotide and amino acid sequences of the coding and noncoding regions between samples collected before and after interferon treatment. The authors considered that among quasispecies
460
AID
Hepa 0035
/
5P11$$$681
07-20-96 11:52:03
hpta
WBS: Hepatology
HEPATOLOGY Vol. 24, No. 2, 1996
HEPATOLOGY Elsewhere
of HCV, the major population represented is interferon-sensitive, but during or following IFN treatment in nonresponders, an IFN-resistant clone(s) constitutes the major population. The difference was seen mainly in the E2, NS2, and NS5A regions in patient 1, in the E2 and NS5A region in patient 2, and in the NS5A region in patient 3. Although there were other scattered differences, the main differences were considered to be clustered in the C-terminal region of the NS5A, particularly codons 2154-2383, and in the E2-hypervariable region (HVR). After further pairwise comparison, these three pairs were found to have unique amino acid substitutions at codon 2218. They were glutamine, arginine, and cysteine in patients 1, 2, and 3, respectively, pretreatment. All changed to histidine posttreatment. The previously published HCV type 1b (HCV-J, HCV-J4, and HCV-JTa) possesses a histidine residue at 2218. This was the only commonly observed amino acid difference before and after IFN treatment in three nonresponders. Amino acid differences were detected in other regions, particularly the E2 hypervariable region. However, there were no common changes among the three HCV isolates obtained from patients 1, 2, and 3. The authors considered that the pretreatment HCV sequence represented IFN-sensitive HCV and the posttreatment sequence that of an IFNresistant clone. It should be cautioned that the nucleotide sequence was determined by the direct method, and it was thus not clear whether the IFN-resistant clone was originally present in the pretreatment samples as a minor population. The above results were clear but not conclusive. The authors extended the study by examining HCV sequences from six IFN nonresponders and nine complete responders. This time, they only examined sequences of the C-terminal half of the NS5A. The 9 IFN-resistant clones were from ‘‘after’’ samples of the previous patients 1 through 3 and new nonresponder patients 4 through 9. The 12 IFN-sensitive clones were ‘‘before’’ samples of patients 1 through 3 and responder patients 10 through 18. The amino acid sequences from 2209 to 2248 of the former group were identical to those of prototype HCV 1b. In contrast, clones obtained from the latter group had at least one amino acid change in this region. The clone from patient 18 even had an eight–amino acid insertion between codon 2221 and 2222. Particularly illuminating findings were again revealed at codon 2218. Clones from the former group and from previous isolates (HCV-J, HCV-J4, and HCV JTa) possessed histidine at 2218. However, the amino acid at this position of the clones obtained from complete responders prior to IFN treatment was different. Three of 12 were histidine, but four were arginine, three cysteine, one glutamine, and another proline. More interestingly, the clone from one nonresponder (patient 18) possessed arginine at 2218, but after unsuccessful IFN treatment was replaced by histidine. From this meticulously performed sequence analysis, together with individual clinical data, the authors concluded that there are IFN-sensitive and -resistant clones of HCV. The main difference between these two groups lies in the Cterminal half of the NS5A region, particularly in codons 2209 to 2248, which the authors designate as the IFN sensitivity determining region (ISDR). Within the ISDR, the authors consider codon 2218 most important. When the same nonresponder patients were examined, IFN-sensitive clones were obtained in the pretreatment phase and IFN-resistant clones in posttreatment. As the authors discuss, it may be possible to prospectively predict IFN responsiveness. It is very important to study whether the amino acid changes described were caused by ‘‘selection’’ or ‘‘mutation.’’ To analyze this, it would be more appropriate to sequence as many subclones as possible, instead of performing direct sequencing, which provides information only on major populations. It is known that the viral quasispecies population may change under selective pressure.3,4 In the second paper, the same group retrospectively examined samples obtained from 84 patients who had shown dif-
AID
Hepa 0035
/
5P11$$$682
07-20-96 11:52:03
461
ferent responses to IFN treatment. There were two groups: one consisted of nonresponders (n Å 63); the other consisted of complete responders (n Å 21). The amino acid sequence analysis of the NS5A-ISDR indicated that there were three groups: (1) wild-type with sequences identical to that of prototype HCV-J (n Å 30), (2) intermediate-type with only one to three amino acid substitutions (n Å 38), and (3) mutant-type with more than four amino acid changes (n Å 16). The results were clear. All of the 30 patients infected with wild-type HCV were nonresponders, and all of the 16 mutant HCV-infected patients were complete responders. Only 5 of the 38 patients infected with intermediate-type HCV were complete responders. This time, however, the striking difference was not observed between sensitive and resistant clones at codon 2218. Statistical analysis revealed that HCV RNA levels in sera before treatment were significantly lower in the patient group with mutant-type than those with wild or intermediate types. However, for each individual the amount of HCV RNA did not correlate with IFN responsiveness. Thus, it seems likely that the HCV clones that have identical or very similar amino acid sequences to that of wild-type in the ISDR are resistant to IFN therapy. Viruses of the Flaviviridae family have a similar region corresponding to HCV NS5A. However, the biological function of these NS5As is not yet known. It was recently shown that it is a hyperphosphorylated protein. The candidate hyperphosphorylation site is positioned at serine residues located near the ISDR described by these papers.5 These papers by Enomoto et al.2 provide a potentially important new predictor of IFN responsiveness. They also may provide clues regarding potential virus-cell interactions in HCV infection. When an efficient in vitro replication system is developed, it would be very interesting to compare the virological behavior between HCV clones with wild-type or mutant sequences in the ISDR, particularly with regard to their replication activity. TATSUO MIYAMURA, M.D. Department of Virology II National Institutes of Health Tokyo, Japan REFERENCES 1. Kimura T, Nakayama K, Penningner J, Kitagawa M, Harada H, Matsuyama T, Tanaka N, et al. Involvement of the IRF-1 transcription factor in antiviral responses to interferons. Science 1994;264:1921-1924. 2. Enomoto N, Kurosaki M, Tanaka Y, Marumo F, Sato C. Fluctuation of hepatitis C virus quasispecies in persistent infection and interferon treatment revealed by single-strand conformation polymorphism analysis. J Gen Virol 1994;75:1461-1369. 3. Aizaki H, Saito A, Kusakawa I, Ashiwara Y, Nagamori S, Toda G, Suzuki T, et al. Mother-to-child transmission of a hepatitis C virus variant with an insertional mutation in its hypervariable region. J Hepatol (in press). 4. Nakajima N, Hijikata M, Yoshikura H, Shimizu Y. Characterization of longterm culture of hepatitis C virus. J Virol (in press). 5. Tanji Y, Kaneko T, Satoh S, Shimotohno K. Phosphorylation of hepatitis C virus-encoded nonstructural protein NS5A. J Virol 1995;69:3980-3986.
HEPATITIS G: A VIRUS IN SEARCH OF A DISEASE
Linnen J, Wages J, Zhen-Yong Z-K, Fry KE, Krawczynski KZ, Alter H, Koonin E, et al. Molecular cloning and disease association of hepatitis G virus: a transfusion-transmissible agent. Science 1996;271:505-508. ABSTRACT
An RNA virus, designated hepatitis G virus (HGV), was identified from the plasma of a patient with chronic hepatitis. Extension from an immunoreactive complementary DNA clone yielded the entire genome (9,392 nucleotides) encoding a polyprotein of 2,873 amino acids. The
hpta
WBS: Hepatology