Jungsuh P. Kim

Molecular Cloning and Disease Association of Hepatitis G Virus: A Transfusion-Transmissible Agent

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 (9392 nucleotides) encoding a polyprotein of 2873 amino acids. The virus is closely related to GB virus C (GBV-C) and distantly related to hepatitis C virus, GBV-A, and GBV-B. HGV was associated with acute and chronic hepatitis. Persistent viremia was detected for up to 9 years in patients with hepatitis. The virus is transfusion-transmissible. It has a global distribution and is present within the volunteer blood donor population in the United States.

The Incidence of Transfusion-Associated Hepatitis G Virus Infection and Its Relation to Liver Disease

BACKGROUND The role of hepatitis G virus (HGV) in transfusion-associated infection and its relation to liver disease are not well understood. METHODS Serum samples collected between 1972 and 1995 from 357 transfusion recipients, 157 controls who did not receive transfusions, 500 randomly selected volunteer blood donors, and 230 donors of blood received by HGV-infected patients were tested for HGV RNA by qualitative and quantitative polymerase-chain-reaction assays. Samples obtained before transfusion and serially after transfusion from 79 of the 81 transfusion recipients who had transfusion-associated non-A, non-B hepatitis were available for testing. RESULTS Of the 79 patients with transfusion-associated hepatitis, 63 (80 percent) had infections related to the hepatitis C virus (HCV) and 3 had preexisting HCV and the cause of their acute hepatitis could not be determined; of the remaining 13 patients, 3 had acute HGV infection, and 10 were infected with unidentified agents. Six of the 63 patients with HCV infection who were tested (10 percent) were also infected with HGV. The three patients infected only with HGV had mild hepatitis (mean peak alanine aminotransferase level, 198 U per liter; none had jaundice); the levels of alanine aminotransferase and HGV RNA were not well correlated. The combined HCV and HGV infections were no more severe than HCV infections alone; the alanine aminotransferase values paralleled the levels of HCV RNA, but not those of HGV RNA. There were 35 HGV infections among the 357 transfusion recipients; only 3 had hepatitis with HGV as the sole viral marker. One of the 157 controls and 7 of the 500 randomly selected blood donors (1.4 percent) had detectable HGV RNA. In all eight instances in which a transfusion recipient had acute HGV infection after transfusion and samples from all donors could be tested, at least one HGV-positive donor was identified. CONCLUSIONS HGV was common in a group of volunteer blood donors, and it can be transmitted by transfusion. Most HGV infections were not associated with hepatitis. HGV did not worsen the course of concurrent HCV infection. No causal relation between HGV and hepatitis has been established.

Acute non-A-E hepatitis in the United States and the role of hepatitis G virus infection

BACKGROUND Little is known about the relation of the newly discovered hepatitis G virus (HGV) to the cause and clinical course of acute and chronic viral hepatitis. METHODS We selected patients from a surveillance study of acute viral hepatitis in four U.S. counties who had acute disease during 1985 to 1986 or 1991 to 1995. Serum samples were tested for HGV RNA by the polymerase chain reaction. RESULTS HGV RNA was detected in 4 of 45 patients with a diagnosis of non-A-E hepatitis (9 percent), 23 of 116 patients with hepatitis C (20 percent), 25 of 100 patients with hepatitis A (25 percent), and 32 of 100 patients with hepatitis B (32 percent) (P<0.05 for the comparison of hepatitis B with hepatitis non-A-E or C). The clinical characteristics of the acute illness were similar for patients with HGV alone and those with hepatitis A, B, or C with or without HGV infection. During a follow-up period of one to nine years, chronic hepatitis did not develop in any of the patients with HGV alone, but 75 percent were persistently positive for HGV RNA, as were 87 percent of those with both hepatitis C and HGV infection. The rates of chronic hepatitis were similar in patients with hepatitis C alone (60 percent) and those with both hepatitis C and HGV infection (61 percent). CONCLUSIONS The evidence from this surveillance study does not implicate HGV as an etiologic agent of non-A-E hepatitis. Persistent infection with HGV was common, but it did not lead to chronic disease and did not affect the clinical course in patients with hepatitis A, B, or C.

EFFECT OF HEPATITIS G VIRUS INFECTION ON CHRONIC HEPATITIS C

Hepatitis C virus (HCV) is a major cause of acute and chronic hepatitis throughout the world [1-3]. A new virus, tentatively called hepatitis G virus (HGV), was recently cloned and sequenced [4]. This virus is closely related to HCV in genomic structure; like HCV, HGV is transmitted through transfusion and may be associated with acute and chronic hepatitis [4-6]. We analyzed the role of HGV infection in patients with chronic hepatitis C, including patients treated with interferon-. Methods Patients We enrolled 189 patients with chronic hepatitis C who were seen at Shinshu University Hospital, Matsumoto, Japan (122 men and 67 women; mean age, 51.1 11.0 years). All patients were positive for antibody to HCV according to a second-generation assay and were negative for hepatitis B virus (HBV) surface antigen and antibody to human immunodeficiency virus. No patients had hepatocellular carcinoma or a history of alcohol intake exceeding 80 g/d. Of the 189 patients, 101 (66 men and 35 women; mean age, 50.0 11.2 years) had been retrospectively selected so that our sample would include all patients who had received a single course of interferon- therapy between October 1991 and December 1993. The remaining 88 (47%) patients (56 men and 32 women; mean age, 52.5 10.7 years) were consecutively selected from patients with chronic hepatitis C who had been followed for more than 1 year and had had liver biopsy within the same period but had not received antiviral therapy. Interferon- 2a had been administered at a dosage of 9 million U daily for 2 weeks, followed by 9 million U three times a week for 22 weeks (total dose, 720 million U). Treated patients had histologic examination within the 6 months before interferon- therapy was initiated and were followed for at least 6 months after therapy was completed. For all patients, serum samples were obtained at the time of liver biopsy and were stored at 70C until testing. For patients receiving interferon- therapy, serum samples were also collected just before therapy began, just after therapy was completed, and 6 months after therapy was completed. Serum alanine aminotransferase levels (normal range, 5 to 45 IU/L) were measured at liver biopsy and, in patients receiving interferon- therapy, at least once every 4 weeks during therapy and follow-up. The grade (extent of hepatic inflammation and hepatocellular destruction) and stage (degree of fibrosis) of liver histologic findings [7] were judged by three authors; the final diagnosis was established by consensus. Investigators involved in separate portions of the study were blinded to the results of other portions. Serologic Markers of and Molecular Assays for Hepatitis C Virus RNA Levels of HCV antibody, HBV surface antigen, and antibody to human immunodeficiency virus were measured by using commercially available second-generation enzyme-linked immunosorbent assays (Abbott Laboratories, North Chicago, Illinois). Serum levels of HCV RNA were measured by using nested reverse-transcription polymerase chain reaction (PCR) with primers in the 5 noncoding region [8] and were quantified by using the branched-DNA signal amplification assay [9, 10]. The detection limit of the branched-DNA assay was set at 105.7 equivalents/mL. Hepatitis C virus genotypes were tested by nested PCR using genotype-specific primers of core region [11] and were categorized according to the classification system of Simmonds and colleagues [12]. Serum Levels of Hepatitis G Virus RNA Levels of HGV RNA in serum were measured by using reverse-transcription PCR as described elsewhere [5]. Total nucleic acids were extracted from 50 mL of serum. After reverse transcription, 45 PCR cycles (for detection) or 35 PCR cycles (for quantitation) were done with primers in the putative NS5 region of the HGV RNA genome. The PCR products were analyzed by dot-blot hybridization with a 32P-labeled oligonucleotide probe. The sensitivity of this assay system was 20 RNA copies/mL of starting serum. Quantitative measurement of HGV RNA levels was done using standards of known HGV RNA levels. Statistical Analysis Statistical analyses were done using the Student t-test, the Mann-Whitney U test, the Wilcoxon rank-sum test, the chi-square test, the Fisher exact test, and the Somers D test. A P value of 0.05 or less indicated statistical significance. Results Clinical Features and Hepatitis C Virus Markers Twenty-one (11.1%) of the 189 enrolled patients were positive for HGV RNA. The rate of detection of HGV RNA was similar in the subgroup of 88 untreated patients (12.5%; 11 of 88) and the 101 patients who received interferon- therapy (9.9%; 10 of 101). Mean age and the number of men and women were also similar in the two subgroups. Thus, the clinical features of patients who had chronic hepatitis C could be compared with those of patients who had HCV and HGV infection without respect to interferon- therapy (Table 1). Patients with HGV infection were significantly younger than those without HGV infection. Other clinical features did not differ between HGV RNA-positive and HGV RNA-negative patients (Table 1). Table 1. Clinical Features in HGV RNA-Positive and HGV RNA-Negative Patients with Chronic Hepatitis C* Hepatitis C virus genotype and serum HCV RNA level were compared between the 21 patients with HGV RNA and the 52 patients without HGV RNA who were randomly selected from the 168 HGV-negative persons. The HCV genotypes and HCV RNA levels were distributed similarly in the two groups (Table 2). Table 2. Markers of HCV and Response of HCV to Interferon- in HGV RNA-Positive and HGV RNA-Negative Patients with Chronic Hepatitis C* Effect of Hepatitis G Virus Infection on Response of Hepatitis C Virus to Interferon- Therapy Of the 101 patients receiving interferon-, 36 had a sustained loss of HCV RNA and normalization of alanine aminotransferase levels; they were therefore considered to have responded to interferon-. The remaining 65 patients were positive for HCV RNA 6 months after completing therapy and thus were classified as nonresponders. The rate of HCV response to interferon- did not differ between patients with and those without HGV infection (Table 2). Response of Hepatitis G Virus to Interferon- Response of HGV to interferon- was analyzed in 9 of 10 patients with HGV infection. The serum HGV RNA level decreased during interferon- therapy in all 9 patients. The geometric mean titer of HGV RNA just after interferon- therapy (mean, 6.3 RNA copies/mL; range, 0.0 to 5000 RNA copies/mL) was significantly (P = 0.008; Wilcoxon rank-sum test) lower than the titer just before therapy (mean, 3200 RNA copies/mL; range, 20 to 1 000 000 RNA copies/mL). Two patients had a sustained loss of HGV RNA 6 months after discontinuation of therapy. In the remaining 7 patients, HGV RNA level increased after cessation of therapy to levels similar to those just before therapy. Thus, the sustained response rate of HGV (22%; 2 of 9 patients) was lower than but not significantly different from (P > 0.2; Fisher exact test) the sustained response rate of HCV (36%; 36 of 101 patients). However, a dichotomy was seen in the response to interferon-; two patients who had a sustained loss of HGV RNA did not clear HCV RNA, and three patients who had a sustained loss of HCV RNA did not clear HGV RNA. In the latter three HCV responders, alanine aminotransferase levels remained normal despite the reappearance of HGV viremia. Discussion Approximately 10% of patients with chronic hepatitis C are also infected with HGV [5, 6]. Although the precise routes of HGV transmission have not been established, this agent is parenterally transmitted through blood transfusion and exposure to shared needles during injection drug use [4]. In our study, the frequency of previous blood transfusion was similar in patients with HGV and HCV co-infection and patients with hepatitis C alone. The apparent link between HGV and HCV infections probably reflects common exposures and transmission patterns rather than a specific interdependence of the two agents. Patients with chronic hepatitis often harbor more than one hepatitis agent [4, 13-15], and important interactions between HBV and HCV have been documented [15]. The relation between HCV and HBV replication is reciprocal: Increasing replication of one agent can diminish the replication of the other. The key question underlying our study is whether coexistent HGV infection alters the level of viremia, clinical course, or treatment response of HCV infection. Although it is unclear whether our findings are applicable to non-Japanese persons, we found no evidence of such an effect on any of these variables. The following findings support this claim. First, the HCV RNA level in patients with HGV and HCV infection was the same as in patients with HCV infection alone. In addition, Nakatsuji and colleagues [5] reported that the serum level of HGV RNA did not differ between these two groups of patients. These two studies showed no evidence of unidirectional or bidirectional viral interference between HGV and HCV. Second, no evidence suggested that HGV infection increased the severity of hepatitis C. When patients with chronic hepatitis C were compared with those who had HCV and HGV infection, no differences were seen in the mean alanine aminotransferase level or liver histologic findings. Further, in five co-infected patients in whom HGV and HCV responses to interferon- therapy were dissociated, hepatic inflammation after discontinuation of therapy seemed to depend on HCV replication, not on HGV replication. These data suggest that HGV has limited pathogenicity compared with HCV, and they are consistent with previous observations that HGV-positive blood donors were no more likely to have elevated alanine aminotransferase than were HGV-negative donors [4]. Several viral and host factors have been reported to influence the response of HCV to interferon- [8, 16-18]. We found no effect of HGV on the HCV respo

Hepatitis C virus antigen in hepatocytes: immunomorphologic detection and identification.

Hepatitis C virus (HCV) antigen was detected immunohistochemically using fluorescein isothiocyanate-labeled immunoglobulin G fractions from chimpanzee and human sera strongly reactive with recombinant hepatitis C virus structural and non-structural proteins. The antigen was localized in the cytoplasm of hepatocytes in all 9 chimpanzees with acute hepatitis C, in 5 of 10 chimpanzees with chronic HCV infection, and in 11 of 12 patients with chronic hepatitis C. The specificity of the hepatocellular HCV and FITC-labeled probes for HCV was ascertained by blocking studies with paired serum samples obtained from 8 infected and uninfected chimpanzees or from 14 patients during the acute and chronic phases of HCV infection. Absorption experiments on FITC-labeled probes with selected host proteins (normal liver homogenate, plasma proteins, red blood cells) did not indicate cross reactivity of the probes with these antigens. Direct immunomorphologic evidence for the HCV specificity of hepatocellular HCV antigen deposits and the FITC-labeled polyclonal anti-HCVAg probe was established in absorption experiments using recombinant HCV nonstructural proteins. The putative HCV NS3 protein was the most prominent component of hepatocellular HCV antigen.

Sequence-independent, single-primer amplification (SISPA) of complex DNA populations

Sequence-Independent, Single-Primer Amplification (SISPA) is a primer initiated technique that requires target sequence modification to achieve the non-selective logarithmic amplification of heterogeneous DNA populations. The method contrasts with the polymerase chain reaction (PCR), and its modified approaches, that have as their objective the amplification of unique or homologous sequences. SISPA requires the directional ligation of an asymmetric linker/primer oligonucleotide onto the target population of blunt ended DNA molecules. The common end sequence allows one strand of the double-stranded linker/primer to be used in repeated rounds of annealing, extension and denaturation in the presence of thermostable Taq DNA polymerase. The linker/primers contain restriction endonuclease sites to facilitate the molecular cloning of as little as 1 pg of starting material after amplification. SISPA is especially useful when the nucleotide sequence of the desired molecule is both unknown and present in limited amounts making its recovery by standard cloning procedures technically difficult. These conditions are present in the initial isolation and cloning of previously uncharacterized viral genomes. The application and quantitation of SISPA is described, together with its utility in the cloning and recovery of low abundance genetic sequences, as illustrated here with the hepatitis C virus.

Prevalence and disease association of hepatitis G virus infection in Japan

SUMMARY. A reverse transcriptase‐polymerase chain reaction procedure (RT‐PCR) for the detection of hepatitis G virus (HGV) RNA was used to examine the prevalence of HGV infection and HGV‐related disease in Japan. Among 48 patients with acute non‐A, B, C, D, E (non‐A‐E) hepatitis (five transfusion‐associated cases and 43 sporadic cases), only one patient (2%), a transfusion recipient, was HGV RNA positive. Similarly, among 50 patients with established chronic non‐A‐E hepatitis, only two (4%) were positive for HGV RNA. These frequencies were not significantly different from those in 129 voluntary blood donors (0.8%). By contrast, HGV infection was relatively common among patients who were also infected with other hepatitis viruses. HGV co‐infection or superinfection was found in seven of 53 (13%) patients with acute hepatitis C, in 15 of 126 (12%) patients with chronic hepatitis C, in three of 21 (14%) patients with acute hepatitis B and in four of 81 (5%) patients with chronic hepatitis B. Among the 29 dually infected patients, 15 (52%) had a history of blood transfusion. HGV was also detected in seven (10%) of 69 haemodialysis patients, of whom only one had a dual infection with hepatitis C virus (HCV) and an elevated aminotransferase level. In conclusion: HGV RNA was found in only a low percentage of patients with either acute or chronic non‐A‐E hepatitis; HGV appears to co‐infect or superinfect in 10–15% of HCV infections and in 5–15% of HBV infections; the prevalence of HGV infection (0.8%) among voluntary blood donors in Japan is similar to that for HCV infection; a history of blood transfusion was obtained in 22 (55%) of the total 40 HGV‐positive subjects; and isolated HGV infection appears to have a low disease burden.

Experimental Infection of Chimpanzees with Hepatitis G Virus and Genetic Analysis of the Virus

Hepatitis G virus (HGV) was transmitted to 2 chimpanzees by inoculation with human plasma containing approximately 10(8) genome equivalents (GE) of HGV. The infection was characterized by the late appearance (weeks 10 and 11 after inoculation [pi]) of viremia that persisted throughout the 120-week follow-up. Serum HGV titer increased steadily until it plateaued at 10(6)-10(7) GE/mL. However, despite this relatively high titer, neither of the chimpanzees developed hepatitis. The sequence of the viral genome, recovered from each chimpanzee at week 77 pi, differed from that of the inoculum by 5 nt (2 aa) and 27 nt (2 aa). Two more chimpanzees were inoculated with a first-passage plasma pool. The chimpanzee inoculated with approximately 10(6.7) GE of HGV had viremia at week 1 pi. However, the viral titer increased with the same kinetics as observed in the first passage. The second chimpanzee inoculated with approximately 10(4.7) GE of HGV had late appearance (week 7 pi) of viremia.

Hepatitis G virus infection: clinical characteristics and response to interferon

A new member of the Flaviviridae family has recently been cloned and completely sequenced. The new virus, tentatively named hepatitis G virus (HGV) and known to be closely related to GB virus C (GBV‐C), is transmitted by blood and blood products, intravenous drug use and other behaviour associated with a high risk of parenteral exposure to blood. The association of the virus with hepatitis is demonstrated by the presence of raised liver transaminase (alanine aminotransferase, ALT) levels in patients infected with HGV in the absence of other identifiable causes of hepatitis. No patient sera from groups exposed to blood and blood products were found to be positive when tested for the presence of GBV‐A or GBV‐B sequences, two other recently described flaviviruses. Forty‐five per cent of the HGV‐infected patients investigated had normal ALT suggesting the existence of a normal carrier state. Persistent infection of up to 13 years duration was observed. Co‐infection with hepatitis B or hepatitis C viruses (HBV and HCV) was commonly seen presumably because of shared risk factors. None of five patients with fulminant hepatic failure was positive for HGV infection. The virus is sensitive to interferon‐α, but sustained responses were not seen with the treatment regimens used for HBV and HCV. Viral titres increased during immunosuppression following liver transplantation and the higher levels of viraemia were in one case accompanied by elavated ALT. Whether HGV (GBV‐C) replicates in the liver in some or all cases remains to be established. Preliminary data suggest that it is present within peripheral blood lymphocytes.

Hepatitis E virus (HEV): Strain variation in the nonstructural gene region encoding consensus motifs for an RNA-dependent RNA polymerase and an ATP/GTP binding site

Hepatitis is transmitted by a number of infectious agents. The epidemiological characterization of waterborne or enterically transmitted non-A, non-B hepatitis (ET-NANBH) is unique when compared with other known hepatitides. We have reported on the molecular cloning of a cDNA clone derived from the etiologic agent associated with ET-NANBH, the hepatitis E virus (HEV). The complete sequence of these first molecular clones, isolated from an HEV-infected human after passage inMacaca fascicularis (cynomolgus macaques), illustrates a distant relationship to other known positive-strand RNA viruses of plants and animals. The translated major open reading frame (ORF-1) from these clones indicates that this portion of the genome encodes a polyprotein with consensus sequences found in RNA-dependent RNA polymerase and ATP/GTP binding domains. The latter activity has been associated with putative helicases of positive-strand RNA viruses. These viral-encoded enzymatic activities identify this region and ORF-1 as containing at least two different nonstructural genes involved in HEV replication. Molecular clones obtained from two other geographically distinct HEV isolates demonstrated sequence heterogeneity in this nonstructural gene region. Further study will be required to elucidate the pathogenic significance (if any) of this observed divergence in the nonstructural region.