- Email: [email protected]
HGV: a new human hepatitis-related virus
Res. Virol.
0 INSTITUT PASTEUFJELSEVIER
Paris 1997
1997, 148, 119-122
GBVUHGV: a new human hepatitis-related A.R. Zanetti
(l) (*), E. Tanzi (I), L. RomanB
virus
(l) and C. Galli
(2)
(I’ institute of Virology, University of Milan (Italy) and (2’ Abbott Diagnostic Division, Rome
SUMMARY
Two novel potentially hepatotropic flavi-like viruses were recently identified in patients with acute or chronic hepatitis and were provisionally called GBV-C and hepatitis G virus (HGW. The sequence identity analysis of these two viruses clearly indicated that GBV-C and HGV are two isolates of the same virus. In addition, the phylogenic analysis of the aligned viral polyprotein sequences showed that the GBV-C and HGV isolates are closely related to two Fhiviruses (GBV-A and GBV-B) that cause hepatitis in tamarins, and are distantly related to hepatitis C virus (HCV). Taken together, these results demonstrate that GBV-C/HGV belongs to the Fhviviridae family. GBV-C/HGV genomic RNA is detectable in both acute and chronic non-A, nonE hepatitis as well as in a minor proportion of patients with fulminant hepatic failure, hepatocellular carcinoma and in blood donors with or without abnormal alanine aminotransferase. However, the majority of patients with prospectively followed HGV infections have no evidence of liver damage. The high frequency of GBV-C/HGV infections in patients who are coinfected with HBV and/or HCV suggests that these viruses can share a common mode of transmission.
Key-words: Viral hepatitis, GBV-C/HGV, New hepatitis viruses.
Introduction
The existence of other viral hepatitis agents besides hepatitis A and B viruses (HAV, HBV) was postulated more than twenty years ago (Prince et al., 1974). The identification and characterization of hepatitis C virus (HCV) in 1989, and more recently, of hepatitis E virus (HEV) did not completely resolve the situation, since not all non-A, non-B hepatitis cases can be attributed to those viral agents. In Italy, a national surveillance of acute viral hepatitis (SEIEVA) has been established since 1985, and the latest report, updated to 1994 (Mele et al., 1996),
Received November
indicates that at least 8 % of all reported acute hepatitis cases could not be ascribed to any of the known viruses. A national survey of acute non-A, non-C conducted in 1995 (Roman6 et al., 1996) demonstrated that only about 10% of such cases are due to HEV infection. Thus, since the hepatitis “F’ virus (HFV) does not circulate in Europe (Deka et al., 1994), it can be estimated that approximately 7 % of all acute hepatitis with possible viral aetiology is related to unknown agents. For chronic viral hepatitis, while almost all non-B post-transfusion chronic hepatitis cases may be ascribed to HCV (Alter, 1994), a proportion estimated to be between 10 and
5, 1996.
(*) Correspondence: Prof. Alessandro Zanetti. Institute of Virology, University of Milan, Via C. Pascal 38, 20133 Milan, Italy.
A.R. ZANETTI
120
15 % of the so-called “sporadic”, or “communityacquired” chronic hepatitis, is still classified as nonB, non-C and therefore can be diagnosed only by exclusion of other known hepatotropic agents.
Discovery
of the GB viruses
Studies of the so-called “GB agent” started in the late sixties (Deinhardt et al., 1967). After almost three decades, in April 1995, the Virus Discovery Group at Abbott Diagnostics Division announced the identification and partial characterization of a group of novel viral agents, provisionally called “GB viruses” (from the initials of a surgeon with non-A, non-C hepatitis) (Simons et al., 1995a). The sera from this patient had been previously demonstrated to be able to transmit a hepatic disease to tamarin monkeys (Almeida et al., 1976; Karaiannis et al., 1989). Serial passages of the transmissible agents from animal to animal confiied that serum obtained during the periods of elevation of aminotransferases was infectious, and that the infection, along with the possible ensuing liver disease, was due to viral agents that could be filtered through O.l-nm filters. The genomic characteristics and organization of those viruses were eventually described (Simons et al., 1995a) and included the GB agents in the Flaviviridae family, to which HCV also belongs. The first two GB agents (GBV-A and GBV-B) could be found, together or separately, in the sera of infected monkeys. A study of the pattern of cytonecrosis markers after inoculation of one or both of the GB agents (Schlauder et al., 1995) established that alanine aminotransferase (ALT) increases always followed the inoculation of GBV-B, either alone or together with GBV-A, while GBV-A transmitted alone did not cause hepatic disease. Periodic monitoring of GB viraemia together with ALT and the antibody response towards epitopes of the putative NS3/4, core and NS5 regions of GBV-A and -B, carried out by enzyme immunoassays employing recombinant antigens expressed in Escherichia coli as fusion proteins, revealed that one or more ALT flares followed a period of detectable and often intermittent viraemia for GBV-B (Schlauder et al., 1995). The first ALT elevation was followed by an antibody response mainly directed against epitopes from the non-structural regions of the GBV-B genome. Conversely, transient GBV-A viraemia did not elicit cytonecrosis
ALT HAV HBV HCV
= = = =
alanine aminotransferase. hepatitis A virus. hepatitis B virus. hepatitis C virus.
ET AL. flares, nor raise antibody titres, confirming of pathogenicity of the latter agent.
the lack
During the studies aimed at the genomic characterization of GBV-A and GBV-B, the use of the polymerase chain reaction (PCR) with degenerated oligonucleotide primers from the NS3 region enabled identification of specific sequences from a gene coding for helicase activity of a third infectious agent in serum specimens obtained from several patients for whom the diagnosis of chronic cryptogenetic non-A, non-E hepatitis had been posed (Simons et al., 1995b). Since this putative agent revealed a high nucleotide (59%) and amino acid (64%) homology with GBV-A, it was provisionally named GB virus C (GBV-C). GBV-C
and HGV:
two isolates of the same virus
The nucleotide sequence of the GBV-C agent was later identified to cover almost the length of the entire genome (Leary et al., 1996), which has about 9,200 residues and encodes a single polyprotein of more than 3,000 amino acids. The genome of GBVC is organized like the genomes of GBV-A and GBV-B, with genes predicted to encode for the structural proteins at the 5’ end and the non-structural proteins at the 3’ end. Sequences consistent with coding for a serine protease, a helicase and an RNAdependent-RNA-polymerase are situated as in GBVA and GBV-B. The phylogenetic analysis of the aligned polyprotein demonstrated that this agent is also a member of the Flaviviridae family, with a homology with HCV of less than 25 % (fig. 1). This agent is transmissible by parenteraf routes (Schmidt et aZ., 1996), and unlike GBV-A and B, is found only in humans and appears to establish longterm infection. Several recent PCR studies have demonstrated the presence of specific GBV-C sequences (often associated with HCV) in subjects at high risk for parenteral infections (Aikawa et aZ., 1996; Egawa et al., 1996; Masuko et al., 1996; Tsuda et al., 1996), in some patients with hepatocellular carcinoma, in those with community-acquired acute and chronic hepatitis and in some patients with fulminant hepatitis of unknown origin (Yoshiba et al., 1995). A few months after the identification and characterization of the GB agents another research group reported the identification of a novel agent, which they named hepatitis G virus or HGV, whose trans-
HEV HGV PCR
= = =
hepatitis E virus. hepatitis G virus. polymerase chain reaction.
GBV-UHGV:
A NEW HUMAN
GBV-B
I
Hcf!~Q-<:8:’ ‘G
HCV-J8 Identity
-”
HLV--
BV-A
16
between
: HCV isolates 2 70% GBV-C and HGV > 95% GBV-A and GBV-C 48% GBV-B and GBV-A or C c 30%
Fig. 1. Phylogenetic tree based on analysis of viral polyproteins.
mission by transfusion or other parenteral routes and presence in high-risk groups were also documented (Linnen et al., 1996). The similarity between HGV and GBV-C appears to be very strong, since, based on the Gen-Bank sequences, the nucleotide homology should be higher than 85.5%. It is, then, very likely that the two agents represent different isolates of the same virus, although a complete analysis of the two genome sequences is still needed for definitive classification. Diagnosiq
of GBV-UHGV
infection
The identification of specific genomic sequences of GBV-C/I-IGV has made it possible to utilize PCR for evaluation of the viraemia. The procedures employed so far require a retrotranscription from viral RNA to cDNA (RT-PCR) and the use of a specific set of primers, either from the NS3 or from the 5’ non-coding (5’-UTR) regions of the GBVC/HGV genome. Studies are directed at optimizing serological tests for the detection of specific antibodies to GBV-C/HGV. Genomic characterization of the GB viruses makes it possible to recognize several clones encompassing the open reading frames of those agents. The clones were expressed in E. coli, as previously published (Pilot-Matias et al., 1996), and the bacterial lysates were analysed by Western blot against sera either from infected tamarins or from patients with (or at risk of) non-A, non-E hepatitis. Antigenic regions were thus found in the sequences of putative NS3, NS4 and NS5 proteins of all three GB viruses, and also in the putative “core” region of GBV-B. At least three immunogenic epitopes were identified for GBV-A, five for
HEPATITIS-RELATED
VIRUS
121
GBV-B and four for GBV-C. Preliminary studies in patients with liver disease of possible viral aetiology and in subjects at high risk, including cases for whom a positive PCR result had been obtained (Pilot-Matias et al., 1996), detected an antibody response mainly directed against epitopes of the NS5 and NS2/3 regions, with high cross-reactivity between GBV-C and GBV-A. Future availability of sensitive and specific serological assays will enable us to conduct extensive population surveys to understand the epidemiology of GBV-C/HGV infection. Clinical importance GBV-CYHGV
and modes of transmission
of
The high frequency of GBV-C/HGV infections in patients coinfected with HBV or HCV (Egawa et al., 1996; Mast&o et al., 1996; Tsuda et al., 1996) suggests that these new viruses share common routes of transmission with the major agents of chronic viral liver disease. The parenteral transmission of GBVC/HGV has been documented by prospective studies in transfused patients (Egawa et al., 1996 ; Linnen et al., 1996) who became positive for viral RNA during the course of infection. No data are yet available for sexual or vertical/perinataI transmission of the virus. The causal relationship between GBV-C/HGV infection and hepatic disease is suggested by the finding of viral RNA in patients with both acute and chronic non-A, non-E hepatitis, as well as in patients with fulminant hepatitis (Yoshiba et al., 1995). However, the real pathogenetic potential of this virus has not yet been clearly established. In fact, most of the GBV-CMGV-infected transfusion recipients have no biochemical evidence of liver damage (Egawa et al., 1996; Alter, 1994), and Masuko et al. (1996) recently reported that GBVC/HGV can establish a long-term asymptomatic viraemia in haemodialysis patients. Finally, it has been reported that in the few cases in which GBVC/HGV is the only agent associated with the hepatitis, the rises in ALT are usually modest and asynchronous with the appearance of viraemia (Alter, 1994). Prospective studies are therefore needed to document a possible progression from acute to chronic hepatitis and eventual development of cirrhosis and hepatocellular carcinoma in patients infected with GBV-C/HGV alone. Since studies reported from both the USA and Europe indicate that GBV-C/I-IGV is detectable in only a minor share of acute (less than 5 %) and chronic (around 15%) non-A, non-E hepatitis, it is still reasonable to hypothesize the presence of other as yet undiscovered viruses that may cause hepatitis (non-A, non-GBV-C/HGV viruses).
122
A.R. ZANETTI ET AL.
References Aikawa, T., Sugai, Y. & Okamoto, H. (1996), Hepatitis G infection in drug abusers with chronic hepatitis C. N. Engl. J. Med., 334, 195-196. Almeida, J.D., Deinhardt, F., Holmes, A.W., Peterson, D.A., Wolfe, L. & Zuckerman, A.J. (1976), Morphology of the GB hepatitis agent. Nature (Land.), 261, 608-609. Alter, H.J. (1994), Transfusion-transmitted hepatitis C and non-A, non-B, non-C. VOX Sang., 67, 19-24. Deinhardt, F., Holmes, A.W., Capps, R.B. & Popper, H. (1967), Studies on the transmission of disease of human viral hepatitis in marmoset monkeys. Transmission of disease, serial passage and description of liver lesions. J. Exp. Med., 125,673-678. Deka, N., Sharma, M.D. 8z Mukerjee, R. (1994), Isolation of the novel agent from human stool samples that is associated with sporadic non-A, non-B hepatitis. J. ViroZ., 68, 7810-7815. Egawa, K., Yukawa, T., Arakawa, S., Nakao, H., Inoue, T., Tanaka, T., Tsuda, F., Okamoto, H., Miyakawa, Y. & Mayumi, M. (1996), Infection with GB virus C in leprous patients in Japan. J. Med. Virol., 49, 110-114. Karaiannis, P., Petrovic, L.M., Fry, M., Moore, D., Enticott, M., McGarvey, M.J., Scheuer, P.J. & Thomas, H.C. (1989), Studies of GB hepatitis agent in tamarim. Hepatology, 9, 186-192. Leary, T.P., Scott Muerhoff, A., Simons, J.N., Pilot-Mat&, T.J., Erker, J.C., Chalmers, M.L., Schlauder, G.G., Dawson, G.J., Desai, S.M. & Mushahwar, I.K. (1996), Sequence and genomic organization of GBVC : a novel member of the Flaviviridae associated with human non-A-E hepatitis. J. Med. Virol., 48,60-67. Linnen, J., Wages, J., Jr, Zhang-Keck, Z.Y., Fry, K.E., Krawczynski, K.Z., Alter, H., Koonin, E., Gallagher, M., Alter, M., Hadziyarmis, S., Karayannis, P., Fung, K., Nakatsuji, Y., Shih, J.W., Young, L., Paiatk, M., Jr, Hoover, C., Femandez, J., Chen, S., Zou, J.-C., Morris, T., Hyams, K.C., Ismay, S., Lifson, J.D., Hess, G., Foung, S.K.H., Thomas, H., Bradley, D., Margolis, H. & Kim, J.P. (1996), Molecular cloning and disease association of hepatitis G virus: a transfusion-transmitted agent. Science, 271, 505-508. Masuko, K., Mitsui, T., Iwano, K., Yamazaki, C., Okuda, K., Meguro, T., Murayama, N., Inoue, T., Tsuda, F., Okamoto, H., Miyakawa, Y. & Maynmi, M. (1996), Infection with hepatitis GB virus C in patients on maintenance hemodialysis. N. Engl. J. Med., 334, 1485-1490.
Mele, A., Stroffolini, T. & Pasquini, P. (1996), SEIEVA integrated epidemiological system for acute viral hepatitis, Report 1985-1994, Rapp. ISTISAN 9613 (PP. 4-32). Pilot-Matias, T.J., Scott Muerhoff, A., Simons, J.N., Leary, T.P., Buijk, S.L., Chalmers, M.L., Erker, J.C., Dawson, G.J., Desai, S.M. & Mushahwar, I.K. (1996), Identification of antigenic regions in the GB hepatitis viruses GBV-A, GBV-B, and GBV-C. J. Med. Viral., 48, 329-338. Prince, A.M., Brotman, B., Grady, G.F., Kuhns, W.J., Hazzi, C., Levine, R.W. 8z Millian, S.W. (1974), Long-incubation post-transfusion hepatitis without serological evidence of exposure to hepatitis B virus. Lmzcet, 2, 241-246. Roman& L., Tanzi, E., Galli, C., Zanetti, A.R. & the Study Group of Hepatitis E (1996), Hepatitis E virus infection in acute non-Anon-B,non-C (nA-nC) hepatitis in Italy, in “IX Triennial Symposium on Viral Hepatitis and Liver Disease”, Rome, Italy (A134). S&lauder, G.G., Dawson, G.J., Simons, J.N., Pilot-Matias, T.J., Gutierrez, R.A., Heynen, CA., Knigge, M.F., Kurpiewski, G.S., Buijk, S.L., Leary, T.P., Scott Muerhoff, A., Desai, S.M. & Mushahwar, I.K. (1995), Molecular and serologic analysis in the transmission of the GB hepatitis agent. J. Med. Viral., 46, 81-90. Schmidt, B., Kom, K. & Fleckenstein, B. (1996), Molecular evidence for transmission of hepatitis G virus by blood transfusion. Lancet, 347, 909. Simons, J.N., Pilot-Matias, T.J., Leary, T.P., Dawson, G.J., Desai, S.M., Schlauder, G.G., Scott Muerhoff, A., Erker, J.C., Bujk, S.L., Chalmers, M.L., VanSant, C.L. & Mushahwar, I.K. (1995a). Identification of two flavivirus-like genomes in the GB hepatitis agent. Proc. Natl. Acad. Sci. USA, 92, 3401-3405. Simons, J.N., Leary, T.P., Dawson, G.J., Pilot-Matias, T.J., Scott Muerhoff, A., S&lauder, G.G., Desai, S.M. & Mushahwar, I.K. (1995b), Isolation of novel viruslike sequences associated with human hepatitis. Nat. Medicine, 1, 564-569. Tsuda, F., Hadiwandowo, H., Sawada, N., Fukuda, M., Tanaka, T., Okamoto, H., Miyakawa, Y. & Mayumi, M. (1996), Infection with GB virus C (GBVC) in patients with chronic liver disease or on maintenance hemodialysis in Indonesia. J. Med. Virol., 49, 248-252. Yoshiba, M., Okamoto, H. & Mishiro, S. (1995), Detection of the GBV-C hepatitis virus genome in serum from patients with fuhninant hepatitis of unknown aetiology. Luncet, 346, 1131-1132.
0 INSTITUT PASTEUFJELSEVIER
Paris 1997
1997, 148, 119-122
GBVUHGV: a new human hepatitis-related A.R. Zanetti
(l) (*), E. Tanzi (I), L. RomanB
virus
(l) and C. Galli
(2)
(I’ institute of Virology, University of Milan (Italy) and (2’ Abbott Diagnostic Division, Rome
SUMMARY
Two novel potentially hepatotropic flavi-like viruses were recently identified in patients with acute or chronic hepatitis and were provisionally called GBV-C and hepatitis G virus (HGW. The sequence identity analysis of these two viruses clearly indicated that GBV-C and HGV are two isolates of the same virus. In addition, the phylogenic analysis of the aligned viral polyprotein sequences showed that the GBV-C and HGV isolates are closely related to two Fhiviruses (GBV-A and GBV-B) that cause hepatitis in tamarins, and are distantly related to hepatitis C virus (HCV). Taken together, these results demonstrate that GBV-C/HGV belongs to the Fhviviridae family. GBV-C/HGV genomic RNA is detectable in both acute and chronic non-A, nonE hepatitis as well as in a minor proportion of patients with fulminant hepatic failure, hepatocellular carcinoma and in blood donors with or without abnormal alanine aminotransferase. However, the majority of patients with prospectively followed HGV infections have no evidence of liver damage. The high frequency of GBV-C/HGV infections in patients who are coinfected with HBV and/or HCV suggests that these viruses can share a common mode of transmission.
Key-words: Viral hepatitis, GBV-C/HGV, New hepatitis viruses.
Introduction
The existence of other viral hepatitis agents besides hepatitis A and B viruses (HAV, HBV) was postulated more than twenty years ago (Prince et al., 1974). The identification and characterization of hepatitis C virus (HCV) in 1989, and more recently, of hepatitis E virus (HEV) did not completely resolve the situation, since not all non-A, non-B hepatitis cases can be attributed to those viral agents. In Italy, a national surveillance of acute viral hepatitis (SEIEVA) has been established since 1985, and the latest report, updated to 1994 (Mele et al., 1996),
Received November
indicates that at least 8 % of all reported acute hepatitis cases could not be ascribed to any of the known viruses. A national survey of acute non-A, non-C conducted in 1995 (Roman6 et al., 1996) demonstrated that only about 10% of such cases are due to HEV infection. Thus, since the hepatitis “F’ virus (HFV) does not circulate in Europe (Deka et al., 1994), it can be estimated that approximately 7 % of all acute hepatitis with possible viral aetiology is related to unknown agents. For chronic viral hepatitis, while almost all non-B post-transfusion chronic hepatitis cases may be ascribed to HCV (Alter, 1994), a proportion estimated to be between 10 and
5, 1996.
(*) Correspondence: Prof. Alessandro Zanetti. Institute of Virology, University of Milan, Via C. Pascal 38, 20133 Milan, Italy.
A.R. ZANETTI
120
15 % of the so-called “sporadic”, or “communityacquired” chronic hepatitis, is still classified as nonB, non-C and therefore can be diagnosed only by exclusion of other known hepatotropic agents.
Discovery
of the GB viruses
Studies of the so-called “GB agent” started in the late sixties (Deinhardt et al., 1967). After almost three decades, in April 1995, the Virus Discovery Group at Abbott Diagnostics Division announced the identification and partial characterization of a group of novel viral agents, provisionally called “GB viruses” (from the initials of a surgeon with non-A, non-C hepatitis) (Simons et al., 1995a). The sera from this patient had been previously demonstrated to be able to transmit a hepatic disease to tamarin monkeys (Almeida et al., 1976; Karaiannis et al., 1989). Serial passages of the transmissible agents from animal to animal confiied that serum obtained during the periods of elevation of aminotransferases was infectious, and that the infection, along with the possible ensuing liver disease, was due to viral agents that could be filtered through O.l-nm filters. The genomic characteristics and organization of those viruses were eventually described (Simons et al., 1995a) and included the GB agents in the Flaviviridae family, to which HCV also belongs. The first two GB agents (GBV-A and GBV-B) could be found, together or separately, in the sera of infected monkeys. A study of the pattern of cytonecrosis markers after inoculation of one or both of the GB agents (Schlauder et al., 1995) established that alanine aminotransferase (ALT) increases always followed the inoculation of GBV-B, either alone or together with GBV-A, while GBV-A transmitted alone did not cause hepatic disease. Periodic monitoring of GB viraemia together with ALT and the antibody response towards epitopes of the putative NS3/4, core and NS5 regions of GBV-A and -B, carried out by enzyme immunoassays employing recombinant antigens expressed in Escherichia coli as fusion proteins, revealed that one or more ALT flares followed a period of detectable and often intermittent viraemia for GBV-B (Schlauder et al., 1995). The first ALT elevation was followed by an antibody response mainly directed against epitopes from the non-structural regions of the GBV-B genome. Conversely, transient GBV-A viraemia did not elicit cytonecrosis
ALT HAV HBV HCV
= = = =
alanine aminotransferase. hepatitis A virus. hepatitis B virus. hepatitis C virus.
ET AL. flares, nor raise antibody titres, confirming of pathogenicity of the latter agent.
the lack
During the studies aimed at the genomic characterization of GBV-A and GBV-B, the use of the polymerase chain reaction (PCR) with degenerated oligonucleotide primers from the NS3 region enabled identification of specific sequences from a gene coding for helicase activity of a third infectious agent in serum specimens obtained from several patients for whom the diagnosis of chronic cryptogenetic non-A, non-E hepatitis had been posed (Simons et al., 1995b). Since this putative agent revealed a high nucleotide (59%) and amino acid (64%) homology with GBV-A, it was provisionally named GB virus C (GBV-C). GBV-C
and HGV:
two isolates of the same virus
The nucleotide sequence of the GBV-C agent was later identified to cover almost the length of the entire genome (Leary et al., 1996), which has about 9,200 residues and encodes a single polyprotein of more than 3,000 amino acids. The genome of GBVC is organized like the genomes of GBV-A and GBV-B, with genes predicted to encode for the structural proteins at the 5’ end and the non-structural proteins at the 3’ end. Sequences consistent with coding for a serine protease, a helicase and an RNAdependent-RNA-polymerase are situated as in GBVA and GBV-B. The phylogenetic analysis of the aligned polyprotein demonstrated that this agent is also a member of the Flaviviridae family, with a homology with HCV of less than 25 % (fig. 1). This agent is transmissible by parenteraf routes (Schmidt et aZ., 1996), and unlike GBV-A and B, is found only in humans and appears to establish longterm infection. Several recent PCR studies have demonstrated the presence of specific GBV-C sequences (often associated with HCV) in subjects at high risk for parenteral infections (Aikawa et aZ., 1996; Egawa et al., 1996; Masuko et al., 1996; Tsuda et al., 1996), in some patients with hepatocellular carcinoma, in those with community-acquired acute and chronic hepatitis and in some patients with fulminant hepatitis of unknown origin (Yoshiba et al., 1995). A few months after the identification and characterization of the GB agents another research group reported the identification of a novel agent, which they named hepatitis G virus or HGV, whose trans-
HEV HGV PCR
= = =
hepatitis E virus. hepatitis G virus. polymerase chain reaction.
GBV-UHGV:
A NEW HUMAN
GBV-B
I
Hcf!~Q-<:8:’ ‘G
HCV-J8 Identity
-”
HLV--
BV-A
16
between
: HCV isolates 2 70% GBV-C and HGV > 95% GBV-A and GBV-C 48% GBV-B and GBV-A or C c 30%
Fig. 1. Phylogenetic tree based on analysis of viral polyproteins.
mission by transfusion or other parenteral routes and presence in high-risk groups were also documented (Linnen et al., 1996). The similarity between HGV and GBV-C appears to be very strong, since, based on the Gen-Bank sequences, the nucleotide homology should be higher than 85.5%. It is, then, very likely that the two agents represent different isolates of the same virus, although a complete analysis of the two genome sequences is still needed for definitive classification. Diagnosiq
of GBV-UHGV
infection
The identification of specific genomic sequences of GBV-C/I-IGV has made it possible to utilize PCR for evaluation of the viraemia. The procedures employed so far require a retrotranscription from viral RNA to cDNA (RT-PCR) and the use of a specific set of primers, either from the NS3 or from the 5’ non-coding (5’-UTR) regions of the GBVC/HGV genome. Studies are directed at optimizing serological tests for the detection of specific antibodies to GBV-C/HGV. Genomic characterization of the GB viruses makes it possible to recognize several clones encompassing the open reading frames of those agents. The clones were expressed in E. coli, as previously published (Pilot-Matias et al., 1996), and the bacterial lysates were analysed by Western blot against sera either from infected tamarins or from patients with (or at risk of) non-A, non-E hepatitis. Antigenic regions were thus found in the sequences of putative NS3, NS4 and NS5 proteins of all three GB viruses, and also in the putative “core” region of GBV-B. At least three immunogenic epitopes were identified for GBV-A, five for
HEPATITIS-RELATED
VIRUS
121
GBV-B and four for GBV-C. Preliminary studies in patients with liver disease of possible viral aetiology and in subjects at high risk, including cases for whom a positive PCR result had been obtained (Pilot-Matias et al., 1996), detected an antibody response mainly directed against epitopes of the NS5 and NS2/3 regions, with high cross-reactivity between GBV-C and GBV-A. Future availability of sensitive and specific serological assays will enable us to conduct extensive population surveys to understand the epidemiology of GBV-C/HGV infection. Clinical importance GBV-CYHGV
and modes of transmission
of
The high frequency of GBV-C/HGV infections in patients coinfected with HBV or HCV (Egawa et al., 1996; Mast&o et al., 1996; Tsuda et al., 1996) suggests that these new viruses share common routes of transmission with the major agents of chronic viral liver disease. The parenteral transmission of GBVC/HGV has been documented by prospective studies in transfused patients (Egawa et al., 1996 ; Linnen et al., 1996) who became positive for viral RNA during the course of infection. No data are yet available for sexual or vertical/perinataI transmission of the virus. The causal relationship between GBV-C/HGV infection and hepatic disease is suggested by the finding of viral RNA in patients with both acute and chronic non-A, non-E hepatitis, as well as in patients with fulminant hepatitis (Yoshiba et al., 1995). However, the real pathogenetic potential of this virus has not yet been clearly established. In fact, most of the GBV-CMGV-infected transfusion recipients have no biochemical evidence of liver damage (Egawa et al., 1996; Alter, 1994), and Masuko et al. (1996) recently reported that GBVC/HGV can establish a long-term asymptomatic viraemia in haemodialysis patients. Finally, it has been reported that in the few cases in which GBVC/HGV is the only agent associated with the hepatitis, the rises in ALT are usually modest and asynchronous with the appearance of viraemia (Alter, 1994). Prospective studies are therefore needed to document a possible progression from acute to chronic hepatitis and eventual development of cirrhosis and hepatocellular carcinoma in patients infected with GBV-C/HGV alone. Since studies reported from both the USA and Europe indicate that GBV-C/I-IGV is detectable in only a minor share of acute (less than 5 %) and chronic (around 15%) non-A, non-E hepatitis, it is still reasonable to hypothesize the presence of other as yet undiscovered viruses that may cause hepatitis (non-A, non-GBV-C/HGV viruses).
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