zyxw zyxwvutsr zyxwv zy TRANSFU SI 0N COMPLICATIONS Exposure to GB virus type C or hepatitis G virus in selected Australian adult and children populations C.A. Hyland, L. Mison, N. Solomon, J. Cockerill,L. Wang,J. Hunt, L.A. Selvey,J. Faoagali, W.G.E.Cooksley, I.F. Young, R. Trowbridge, I. Borthwick, and E.J. Gowans zyxwvutsrqp zy z N zyxwvu BACKGROUND: The epidemiology and disease association for the GB virus type C (GBV-C) or hepatitis G virus (HGV) are poorly understood. STUDY DESIGN AND METHODS: This study describes the exposure rates to GBV-C/HGV in diverse Australian population groups by testing for current infection and evidence of past infection with a reverse transcriptase polymerase chain reaction and an anti-E2 enzymelinked immunosorbent assay, respectively. Subjects included volunteer blood donors, hepatitis C antibody (anti-HCV)-positivedonors, children, hemodialysis patients, pregnant women attending a prenatal clinic, injecting drug users (IVDUs), and adult hemophiliacs. RESULTS: Combined GBV-C RNA and E2 antibody prevalence was 6.5 percent (6/93) in children, 13.3 percent (75/565) in blood donors, 14 percent (14/99) in pregnant women, 22.5 percent (18/80) in hemodialysis patients, 80 percent (56/70) in anti-HCV-positive donors, 88.6 percent (31/35) in IVDUs, and 85.7 percent (54163) in adult hemophiliacs. Children had the lowest antibody rate, 1.1 percent, whereas the rate was 10.8 percent for blood donors and rose to 45.7 percent for IVDUs, 57.1 percent for anti-HCV-positive donors, and 74.6 percent for hemophiliacs. In contrast, current infection rates were comparable for children, blood donors, and pregnant women (5.4, 2.6, and 6%, respectively), rising to 11.1 percent for hemophiliacs, 24.3 percent for antiHCV-positive donors, and 48.6 percent for IVDUs. Ten of 12 blood donors had persistent viremia, while 2 had recent infections, 1 with apparent resolution. CONCLUSION: Exposure to GBV-C can commence at an early age, although ongoing exposure may also occur among adults with no apparent risk factors. GBV-C RNA positivity was not associated with abnormal plasma alanine aminotransferase levels among blood donors. ovel viruses, designated GB virus type C (GBVC) or hepatitis Gvirus (HGV),have been isolated from patients with non-A-to-E hepatitis by independent laboratories.'P2 The nucleotide sequences o f the genomes from these isolates are highly homologous (85%), with a predicted 95-percent homology at the amino acid level. These isolates may represent a new The nucleotide segenus within the family Fl~viuiridue.~~~ quences exhibit about 26-percent homologywith the hepat i t i s C virus (HCV) g e n ~ m e In . ~ contrast to HCV, it i s not clear if GBV-C/HGV i s hepatotropic or indeed if infection with the virus has any disease a s s ~ c i a t i o n . ~ - ~ The virus i s present in 1 to 4 percent o f blood donor populations, a figure that i s approximately 10 times the HCV infection rate.2s8-13 In contrast to HCV GBV-C/HGV does not appear to be associated with abnormal liver function as measured by alanine aminotransferase (ALT) levels, although some reports are conflicting? GBV-C i s transmissible by blood and blood products, and persistent viremia may develop in a proportion of case^.^^-^^ The virus is detected indirectly by amplification of the genome by the reverse transcriptase polymerase chain reaction (RT-PCR). ~ ~ _______~ ABBREVIATIONS: ALT = alanine arninotransferase;EIA(s) = enzyme irnrnunoassay(s);GBV-C = GB virus type C; HCV = hepatitis C virus; HGV = hepatitis G virus; IVDU(s) = intravenous drug user(s);RT-PCR = reverse transcriptase polymerase chain reaction; UTR = untranslated region. From the Australian Red Cross Blood Service, Queensland,Australia; Abbott Laboratories, Abbott Park, Illinois;the Cornrnunicable Diseases Unit, Queensland Health; the Queensland Health Pathology Services, Royal Brisbane Hospital Campus; and the Royal Brisbane Hospital Foundation, Brisbane, Australia; and the Sir Albert Sakzewski Virus Research Centre, Royal Children's Hospital, Herston, Australia. Supported by the National Health and Medical Research Council of Australia. Received for publication November 1 1 , 1997; revision received February 18,1998, and accepted February 26,1998. TRANSFUSION 1998;38:821-827. Volume 38, September 1998 TRANSFUSION 821 HYLAND ET AL. zyxwvutsrqpon zy zyxwv zyxwvutsrq zyxwvut Antigen and antibody assays, which usually correlate with viremia and hence could signal the presence of virus, are not available.The disappearance of the viral RNAis accompanied by the appearance of antibody to a GBV-CIHGV envelope protein (anti-E2),and assays have recently been developed to identify this marker.13J4J9*20 Thus, anti-E2 is considered to be a marker of convalescence. The aim of this study was to compare the rates of exposure to GBV-C in population groups by examining the prevalence rates of viral RNA and anti-E2. The groups examined included children, pregnant women, blood donors, and persons potentially at risk for parental exposure to blood-borne viruses, such as hemophiliacsand intravenous drug users (IVDUs). MATERIALS AND METHODS Subjects Subjects included volunteer blood donors (n = 391) with normal plasma AlT levels and donors with a history of abnormal ALT levels (n = 174).The former group was made up of consecutive donors who presented at two sites over 4 days. The latter group comprised donors with a history of elevated ALT who underwent phlebotomy consecutively over a period of 6 months at one of the sites. Although the two groups were composed in different time frames, it was impossible to perform the study in any other way. It was considered that this difference was unlikely to bias the study and that the samples collected were representative of the designated population. Both groups were negative for hepatitis B surface antigen, anti-HIV-l/2, anti-HC\S and antibody to humanT-lymphotropic virus in a current-generation enzyme immunoassay (EM) (AbbottLaboratories, Abbott Park, IL). Other subjects included children who presented at the Accident and Emergency Department of the Royal Children’s Hospital (n = 951, pregnant women (n = 991, IVDUs (n = 35), anti-HCV-positive blood donors (n = 70), adult hemophiliacs (n = 63), and hemodialysis patients (n = 80). Ethical clearance was obtained from the Australian Red Cross Blood Service-Queensland and from the Royal Children’sHospital Ethics Committees. Samples from respective groups were consecutive,with the exception of the hemophiliacs and hemodialysis patients, in whom sampling was random. Sample collection and storage Whole blood was collected from all population groups into EDTA, and the plasma was separated in a manner compatible with PCR testing and frozen as soon as possible. Plasma samples from children, blood donors, and IVDUs were frozen within 2 hours of collection. Plasma samples from other groups were frozen within 12 hours of collection. All samples were stored at -70°C. Samples from donors, chil- 822 TRANSFUSION Volume 38, September 1998 dren, pregnant women, and IVDUs were stored for between 2 to 6 months before testing, while samples from hemophiliacs and hernodialysispatients were stored for up to 12 months before testing. GBV-C RNAdetection. GBV-C RNA was detected by RTPCR (LCx,Abbott Laboratories,Abbott Park, IL).All reagents and equipment for GBV-C RNA extraction,RT-PCR, and the E x assay were supplied by Abbott.21Briefly, RNA was extracted from plasma by using an HCV kit (QIAamp, Qiagen, Hildern, Germany)accordingto the manufacturer‘sinstructions but with minor modifications: 25 pL of plasma was added to 280 pL of lysis buffer containing carrier RNA, the mixture was incubated at room temperature for 30 minutes, 280 pL of ethanol was added, and the solution was applied to a Qiagen column.The column was washed twice with 500 pL of wash buffer containing 70-percent ethanol, and the RNA was eluted with 100 pL of RNase-free water. Eluted RNA (20 pL) was added to a PCR mix (Abbott)and reversetranscribed into cDNA for 1 cycle (60OCfor 30 min); this was followed by PCR amplification for 35 cycles (denaturation at 94°C for 40 sec and annealing and extension at 63°C for 1 min) and probe hybridization (denaturation at 97°C for 5 min and hybridization at 15°C for 5 min). Primers used in the PCR targeted the 5’-untranslated region (UTR) of the GBV-C genome. Specific sequences amplified from GBV-C were detected by using the LCx automated probe-based assay,which generates a fluorescencerate-detection signal. Combined studies have established that the sensitivity of the assay is 96.04 percent and the specificity is 99.76 percent.21These studies also determined that the cutoff value for the assay was 75 counts per second per second. For the purpose of this study, samples with fluorescence rates repeatedly greater than 100 counts per second per second were classified as reactive. Sampleswere tested in groups of 18with an additional six controls including duplicates of a GBV-C RNA-negative plasma control, a GBV-C RNA-positiveplasma sample that was a borderline control, and an additional GBV-C RNA calibrator control including extracted RNA. The mean values for these three respective controls were 16 (*SD 12.5), 362 (1751, and 289 (131)counts per second per second. A run was considered invalid if any one control failed. Confirmation of specific GBV-C RNA sequences in Ex-reactive samples. Freshly extracted RNA (5 pL) was reverse-transcribed using RNase H (Superscript 11, Gibco BRL, Gaithersburg, MD) as described previously.22Ten pL of cDNA products were subjected to PCR in a 50-pL reaction mixture containing l x Taq buffer, 1.5 mMMgCl,, 0.2 mMof the four dNTPs, 10pmol of each primer, and 2.5 units of Tuq polymerase (Perkin Elmer Cetus, Norwalk, CT). Primer sets specific to the 5’-UTR and NS3 regions of the GBV-C genome were used. PCR products of 366 and 597 nt, respectively,were generated from all samples that reacted in the LCx assay, but not from control samples. zyxwvu EXPOSURE TO GBV-C IN AUSTRALIA GBV-C antibody detection. Antibody to the GBV-C envelope protein (anti-E2) was detected by using EIA as described previously.1gSamples that were reactive in both immunoassays (screeningand confirmatory)were defined as antibody positive. Other assays. A third-generation anti-HCV recombinant immunoblot assay (Chiron, Raritan, NJ) was used to confirm the specificity of anti-HCV EM-reactive samples. HCV RNA testing was performed with another RT-PCR (Amplicor, Roche Diagnostic, Branchburg, NJ). HCV genotyping was performed using a line probe assay (Innolipa HCV 11, Innogenetics, Zwijndrecht, Belgium). zy 68 were found to be positive by the in-house PCR using both primer sets and were thus defined as genuinely GBV-CI HGV RNA-positive. Therefore, all LCx-positive samples were positive by the in-house assays. Similarly, 10 samples that were LCx negative were also tested by the in-house assay, and all remained negative. These 10 samples were from four IVDUs, three anti-HCV-positivedonors, and three hemophiliacs; these subjects were selected because they were considered most at risk for infection and were all antiE2 negative. The rates of exposure to GBV-C were defined as the number of RNA-positive samples combined with the number of anti-E2 positives and were expressed as a percentage of the total samples tested within each group. Children. Five of the 95 childrenwere GBV-C RNA positive (aged from 10 months to 3 years) and another 3 were anti-E2positive (aged 2.7,4.0, and 12.3years).All 8 children were anti-HCVnegative and 7 of the 8 were under 5 years old (Tables 1 and 2). Tho of the anti-E2-positive children were on monthly intravenous gamma globulin injections zyxwv zyx zy zyxwvuts zyx RESULTS GBV-C exposure rates. All samples were tested initially for GBV-C RNA by the LCx system. The fluorescence distribution between negative and reactive samples is shown in Fig. 1. Of 1007 samples tested, 68 were repeatedly reactive. All 1000 800 -rn -a 0 a 5 E z 600 400 200 0 11-20 0.10 m o a14 I140 7140 11-100 XOlJOQ 4001400 Ml-lM 801400 1001-1100 1¶01-1~00 1401-11M 1M1.1700 101.200 a o i a MI-W 101401 wi-iow iioi-im i u i - 1 4 ~i s o i m a zyxwvutsrqpon 41.10 w o 8140 LCx fluorescence rate (countlseclsec) Pig. 1. Detection of GBV-C RNk distribution of the fluorescence rate (countslseclsec).GBV-C RNA was detected by using an automated probe-basedassay (LCx, Abbott Laboratories) that generates a fluorescence rate-detection signal. The distribution of the fluorescence is shown on the horizontal axis and the number of samples on the vertical axis on two scales, according to whether samples were GBV-C RNA negative (400countslseclsec,). or positive (>lo0countslseclsec, [ 1). The negative samples in the range of 80 to 100 were repeatedly negative. AU positive samples generated PCR fragments of the correct size by in-house PCR Msap using GBV-C-speciflcprimers. Volume 38, September 1998 TRANSFUSION 823 HYLAND ET AL. TABLE 1. ProDortion of individuals eXDOSed to GBV-C/HGV in various DoDulations RNA Number Percentage 5 5.4 Anti-E2 Number Percentage It 1.1 zyxw Exposure' Population Number Number Percentage Children 93 6 6.5 Blood donors Total 565 15 2.6 61 10.8 75* 13.3 Random 391 13 3.3 41 10.5 53 13.6 1.1 20 11.5 22 12.6 Elevated ALT 174 2 Pregnant women 6 8 8 14 14 99 6 1.2 17 21.2 18 22.5 ao 1 Hemodialysis patients 11.1 47 74.6 54 85.7 Hemophiliacs 63 7 80 Anti-HCV-positive donors 70 17 24.3 40 57.1 56$ HCV RNA-positive 50 14 28 25 50 38$ 76 15 15 75 18 90 HCV RNA-negative 20 3 IVDUs 35 17 48.6 16 45.7 31 88.6 Total 1005 68 190 254 The exposure rate was determined by combining the number of GBV-RNA- and antkE2-positive individuals. t An additional two children were anti-E2 positive; however, they were both receiving monthly intravenous gamma globulin injections that may have resulted in passive antibody transfer. They were not included in the calculations. Both GBV-C RNA and anti-E2 were present in one blood donor, two IVDUs, and one anti-HCV-positive donor. * * TABLE 2. Age distribution of children tested for GBV-C RNA and anti-E2 0-5 29 24 53 Age range (years) 6-10 > l l Unknown Total 7 11 6 53 8 5 5 42 15 16 11 95 Male Female Total Number exposed to GBV-C RNA-positive 5 0 0 0 5 Anti-E2-positive 2 ' 0 1 0 3 Proportion exposedt 5/51t 0/15 1/16 0111 6/93 Two children received monthly intravenous gamma globulin injections that may have resulted in passive antibody transfer. t Excluding the two antkE2-positive children receiving gamma globulin injections. for gamma globulin subclass deficiency, and, as this may have resulted in passive transfer of GBV-C anti-E2, they were not included in estimations of the overall exposure to GBV-C. The overall exposure rate was therefore 6 (6.5%)of 93, with 5 (5.4%)of 93 children currently GBV-C RNA positive and 1 (1.1%)of 93 having antibody, which is indicative of past exposure. The five GBV-C RNA-positive children presented to the emergency department with complaints ranging from suspected red-back spider bite, swallowing household detergent, gastroenteritis while on a family holiday, headache and fever, and tonsillitis. Plasma A U levels were measured in three children and found to be normal. The remaining anti-E2-positivechild (aged 12.3years) presented with an epileptic seizure. Blood donors with normal and abnormal ALT levels. GBV-C RNA was detected in 13 (3.3%)of 391 and 2 (1.1%) of 174 donors with normal and a b n o r m a l u levels, respectively. The anti-E2 detection rate among these two groups was 41 (10.5%)of 391 and 20 (11.5%)of 174, respectively. One donor had both RNAand anti-E2 markers, although the LCx fluorescence signal was weak (456 compared with av- erage signals above 1200 for other LCx-positive samples). Overall, 13.3 percent (75l565) of the donors had been exposed to GBV-CIHGV (Table 1). Pregnant women. Six (6%) of 99 sequential subjects attending a public antenatal clinic were GBV-C RNA positive. The age of these six ranged from 16 to 38 years. One was anti-HCVpositive with a history of injecting drug use. The other five subjects had no apparent risk factors. Another eight (8%)were anti-E2 positive, for an overall exposure rate of 14 percent (Table 1). Hemodialysis patients and hemophiliacs. Among hemodialysis patients, 1 (1.2%)of 80 and 17 (21.2%)of 80 were RNA and anti-E2 positive, respectively, for a total exposure rate of 22.5 percent (Table 1). This was the oldest group tested, with a mean age of 65 years. Three were antiHCV positive. Seven (11.1%) and 47 (74.6%) of 63 hemophiliacs (mean age, 34.6 f 11.8)were GBV-C RNA and anti-E2 positive, respectively, for a total exposure rate of 85.7 percent (Table 1).The high antibody rate relative to RNA positivity in this group may reflect passive antibody from blood products or a convalescent state. IVDUs. GBV-C RNA and anti-E2 rates among IVDUs were 17 (48.6%)and 16 (45.7%)of 35, respectively; 2 individuals were positive for both markers, albeit with low LCx signals (countslseclsec, 185and 754).Thus,the overall proportion exposed was 88.6 percent (31135,Table 1).Among the 17 GBV-C RNA-positive individuals, coinfection with HCVRNAwas observed in 13 (76%).TheHCVgenotypes for these were l a (n = 31, l b (n = 2), 2b (n = l), and 3a (n = 71, which is consistent with reports that coinfection occurs across all HCV genotypes.23,24 Anti-HCV-positive donors. Seventeen (24.3%)of 70 anti-HCV-positiveblood donors were GBV-C RNA positive. Coinfection in this group was also observed across HCV genotypes (la, lb, 2b, 3a). Anti-E2 was detected in 40 zyxw zyxwvu zyxwvutsr 824 TRANSFUSION Volume 38, September 1998 EXPOSURE TO GBV-C IN AUSTRALIA (57.1%)of the 70 anti-HCV-positivedonors, including one who was also GBV-C RNApositive.The proportion exposed to GBV-CIHGVin this group was 80 percent (56170). The 70 anti-HCV-positivedonors included 50 with detectable HCV RNA (Table 1). It was noted that the relative proportion of anti-E2-positivesamples and GBV-C-positive samples varied with the HCV RNA positivity state. For the HCV RNA-positive samples, anti-E2 positivity was 50 percent (251501,with a GBV-CRNApositivityof28 percent (141 50),while, in the HCVRNA-negativesamples, anti-E2positivitywas 75 percent (15120)and GBV-C RNApositivitywas 15 percent (3120) (Table 1). Relationship between GBV-Cpersistence and ALT history in blood donors. The ALT history was reviewed for 12 GBV-C-positive donors who were anti-HCV negative. Retrospective testing of stored plasma samples from these donors determined that 10showed persistent viremia since first enrolling as blood donors and had given a combined total of 84 donations over a 4-year period. Plasma ALT levels were normal for all 84 donations (mean, 18 IUIL; SD, 6 IU/L). The remaining two donors were infected with GBVC from 3 to 6 months before the initial positive test (Table 3). In both instances, the plasma ALT values were normal before and after the appearance of viremia. One of the donors subsequently became GBV-C RNA negative. It was noted that one of the donors who became positive between May 1996and July 1996had undergone acupuncture early in 1996 (Table 3). With that exception, neither of the two donors had any other identifiable risk factor in the 6 months before infection. It is interesting that both experienced flulike illness within the 3 months before becoming viremic. Coinfection with GBV-C and HCV RNA was not associated with higher p l a s m a m levels. Mean values were 100 IU per L (SD 48; n = 9) and 95 IU per L (SD 51; n = 16) for HCV RNA-positiveindividuals with elevated AIT with and without GBV-C infection, respectively. Similarly, among HCV RNA-positive individuals with normal ALT levels, mean ALT values were 36 IU per L (SD 15; n = 11)and 33 IU zy per L (SD 14; n = 34) with and without GBV-C infection, respectively. Overall, for the HCV RNA-infected donors, the mean ALT values were 63 f 46 and 55 f 44 IU per L with and without GBV-C RNA coinfection, respectively (p = NS). DISCUSSION This study describes the GBV-C exposure rates for a variety of population groups by detectingviral RNA as a marker for current infection and anti-E2 protein as a marker for past exposure to the virus. The presence of these markers is generally, but not always, mutually e x c l u ~ i v e , ~ and, ~ J ~in this study, only 4 of 254 exposed individuals had both markers. These 4 persons, including 1blood donor, 2 IVDUs, and 1 anti-HCV-positivedonor, all had low RNA levels, which is indicative of decreasing viremia prior to clearance. The GBV-C RNA and antibody prevalence rates reported here for blood donors and IVDUs are comparable with those in other s t u d i e ~ ? . ~ J ~ For blood donors, the anti-E2 positivity rate was about three to four times greater than that for RNA and was similar to ratios reported e1se~here.l~ After infection, GBV-C may establish a persistent viremia, or the virus may be cleared with the concomitant development of anti-E2.’J3J4 However, it is not clear whether infected individualswill go through an extended viremia and then, years later, clear the virus with the appearance of anti-E2,or whether some subset of the population remains viremic and another subset clears the virus relatively quickly. One donor in this study fit the latter category, as he seroconverted within 8 to 12 months. To our knowledge, the prevalence of GBV-C RNA among children has not been described before. This study shows that virus RNA infection rates for children (5%)are comparable to those in adult populations, such as blood donors and women attending antenatal clinics, who may be considered at low risk for blood-borne viral agents. It was beyond the scope of this study to determine whether the children had persistent or transient viremia or whether other family members were infected, and hence to ascertain the degree of familialtransmission. Individual cases of mother-to-infant and intrafamilialtransmission have been rep0rted.2~)~~ Additional studies are required to determine if mother-to-child transmission plays an important role in the incidence of this virus and, if so, whether a majority of such cases establish a chronic-carrier state in a manner analogous to hepatitis B. Alternatively, other factors may contribute to a constant exposure throughout childhood with or without viral clearance. In contrast to the RNA prevalence, the anti-E2 prevalence rate for children was lower than for any other group, even if the two children receiving gamma globulin treatment had not been excluded.The results for RNA and antibody prevalence indicate that infection and clearance may zyxwvuts zyxwvu zyxwvutsrq zyxwvutsrq TABLE 3. Detection of GBV-C RNA In two blood donors who were exposed to the virus 3 to 6 months before the studv Dates tested Donor No. 426 05111/95 02/07/96 07130196 10125196 02111197 Donor No. 484 10127193 05/07/96 GBV-C RNA’ 23 21 1297 159 23 Plasma ALT IIUIU 11 13 19 13 18 17 12 11 22 07130196 1213 20 10125196 1285 22 In countslseclsec. Rates less than 100 are negative. Volume 38, September 1998 TRANSFUSION 825 HYLAND ET AL. zyxwvutsrqpon occur at a very early age. New infections occur in the community, even in groups without apparent risk factors such as parental exposure to blood products. This is exemplified by a comparison of the antibody prevalence in blood donors and children (11%vs. 1%)and by the observation that two donors were exposed to the virus during the course of the study. The study shows that there was no apparent association between GBV-C persistence and the development of liver disease, as measured by ALT levels.This was examined from three perspectives.First, blood donors with a continuing history of idiopathic abnormal ALT levels had GBV-C RNA and antibody prevalence similar to that of those donors with normal ALT levels. Second, among the consecutively selected donors, the GBV-C RNA carriers had maintained normal plasma ALT levels throughout at least 4 years of viremia.This contrasts with the cyclicalALT pattern that can occur with HCV carriers.zBThird, donors coinfected with HCVand GBV-Cdid not have exacerbated liver abnormalities, as assessed by ALT levels. We noted that anti-HCV-positive donors without detectable HCV RNA demonstrated GBV-C exposure rates equivalent to or slightly higher than those in HCV RNApositive, anti-HCV-positivedonors (90%vs. 76%).Furthermore, within the anti-HCV-positive, HCV RNA-negative group, most donors (75%)were GBV-C anti-E2positive.This latter group may comprise blood donors who have been exposed to and have recovered from both HCVand GBV-C. Tache et al.I3 reported that the GBV-C anti-E2 prevalence increased with duration of exposure to intravenous drug use, while RNA prevalence decreased. Similarly,the HCV/ GBV-C RNA-negative antibody-positive group may represent those for whom there has been a longer time since exposure.29 Exposure to HGV/GBV-C is prevalent among populations with low risk for parental exposure to blood-borne agents, and it increases along with the risk of that exposure. Our results indicate that exposure commences at an early age and continues to rise. GBV-CIHGVinfectionhas yet to be linked to any disease. Nevertheless, the effect of longterm persistence of this virus is unknown, and long-term follow-up studies on cohorts of carriers, perhaps extending for decades, may be required to elucidate any associated pathogenic role. 3. Zuckerman AJ. Alphabet of hepatitis viruses. Lancet 1996;347:558-9. 4. Erker JC, Simons JN, Muerhoff AS, et al. Molecular cloning and characterization of a GB virus C isolate from a patient with non-A-E hepatitis. J Gen Virol 1996;77:2713-20. 5. Alter HJ. The cloning and clinical implications of HGV and HGBV-C (editorial).N Engl J Med 1996;334:1536-7. 6. Alter MJ, Gallagher M, Morris 'IT,et al. Acute non-A-E 7. hepatitis in the United States and the role of hepatitis G virus infection. Sentinel Counties Viral Hepatitis Study Team. N Engl J Med 1997;336:741-6. Alter HJ, Nakatsuji Y, Melpolder J, et al. The incidence of transfusion-associatedhepatitis G virus infection and its relation to liver disease. N Engl J Med 1996;336:747-54. Dawson GJ, Schlauder GG, Pilot-Matias TJ, et al. Prevalence studies of GB virus-C infection using reverse transcriptasepolymerase chain reaction. J Med Virol 1996;50:97-103. Nakatsuji Y, Shih JW, Tanaka E, et al. Prevalence and disease association of hepatitis G virus infection in Japan. J Viral Hepat 1996;3:307-16. Roth WK, Waschk D, Marx S, et al. Prevalence of hepatitis G virus and its strain variant, the GB agent, in blood donations and their transmission to recipients. Transfusion zyxw zyxwvu zyxw zyxwvutsrqp 8. 9. 10. 1997;37:651-6. 11. Loiseau P, Mariotti M, Corbi C, et al. Prevalence of hepatitis G virus RNA in French blood donors and recipients. Transfusion 1997;37:645-50. 12. Yoshikawa A, Fukuda S,Itoh K, et al. Infection with hepatitis G virus and its strain variant, the GB agent (GBV-C), among blood donors in Japan. Transfusion 1997;37:657-63. 13. Tacke M, Kiyosawa K, Stark K, et al. Detection of antibodies to a putative hepatitis G virus envelope protein. Lancet 1997;349:318-20;erratum appears Lancet 1997;349:736. 14. Pilot-MatiasTJ, Carrick RJ, Coleman PF, et al. Expression of the GB virus C E2 glycoprotein using the Semlii Forest virus vector system and its utility as a serologic marker. VirolO ~ Y 1996225~282-92. 15. Feucht HH, Fischer L, Sterneck M, et al. GB virus C transmission by blood products (letter).Lancet 1997;349:435. 16. JarvisLM,Davidson F, Hanley JP, et al. Infection with hepatitis G virus among recipients of plasma products. Lancet 19963481352-5. 17. Schmidt B, Korn K, Fleckenstein B. Molecular evidence for transmission of hepatitis G virus by blood transfusion (letter). Lancet 1996;347:909. 18. Schreier E, Hohne M, Kunkel U, et al. Hepatitis GBV-C sequences in patients infected with HCV contaminated antiD immunoglobulin and among i.v. drug users in Germany. J Hepatoll99625385-9. 19. Lou S, Qiu X,Tegtmeier G, et al. Immunoassays to study prevalence of antibody against GB virus C in blood donors. J Virol Methods 1997;68:45-55. zyxwv zyxwvutsr REFERENCES 1. Simons JN, Leary TP, Dawson GJ, et al. Isolation of novel virus-like sequences associated with human hepatitis. Nat Med 1995;1:564-9. 2. Linnen J, Wages J Jr, Zhang-Keck ZY, et al. Molecular cloning and disease association of hepatitis G virus: a transfusion-transmissible agent. Science 1996;271:505-8. 826 TRANSFUSION Volume 38, September 1998 EXPOSURE TO GBV-C IN AUSTRALIA 20. D u e BJ, Surowy TK, Gutierrez RA, et al. An ELISA for the detection of antibodies to the E2 protein of GB virus C. J Infect Dis 1997;175:458-61. 21. Marshall RL, Cockerill J, Friedman P, et al. Detection of GB virus C by the RT-PCR LCx@System. J Virol Methods (in press). 22. Beardsley AM, LaBrooy JT, Rozen L, Gowans EJ. A comparb son of hepatitis C virus (HCV)-RNAand -antibody as markers of infection and predictors of infectivity. Aust N Z J Med 1994;24:182-7. 23. Schleicher S, Chaves RL,Dehmer T, et al. Identification of GBV-C hepatitis G RNA in chronic hepatitis C patients. J Med Virol 1996;50:71-4. 24. Bralet MP, Roudot-Thoraval F, Pawlotsky JM, et al. Histopathologic impact of GB virus C infection o n chronic hepatitis C. Gastroenterology 1997;112:188-92. 25. Feucht HH, Zoller B, Polywka S, Laufs R. Vertical transmission of hepatitis G (letter). Lancet 1996;347:615-6. 26. Lin HH, Kao JH, Chen PJ, Chen DS. Mechanism of vertical transmission of hepatitis G (letter). Lancet 1996;347:1116. 27. Kew CM, Kassianides C. HGV hepatitis G virus or harmless G virus? Lancet 1996;348(Supp12):sIIlO. 28. Morgan C, Hyland C, Young IF. Hepatitis C antibody and transaminase activities in blood donors. Lancet 1990;335:921. 29. Kaldor JM, Archer GT, Buring ML, et al. Risk factors for hepatitis C virus infection in blood donors: a case-control study. Med J Aust 1992;157:227-30. zy AUTHORS Catherine A. Hyland, PhD, Scientist in Charge, Viral Serology, Australian Red Cross Blood Service, 480 Queen Street, Brisbane, Queensland, Australia 4000. [Reprint requests] Leigh Mison, BSc, Scientist, DNA Laboratory, Australian Red Cross Blood Service. Natalie Solomon, PhD, Research Scientist, Probe Discovery, Abbott Laboratories, Abbott Park, IL. James Cockerill, PhD, Research Scientist, Probe Discovery, Abbott Laboratories. Lucy Wang, PhD, Research Scientist, Probe Discovery, Abbott Laboratories. Jeffrey Hunt, PhD, Research Scientist, Probe Discovery, Abbott Laboratories. LA. Selvey, MD, PhD, Manager, Communicable Diseases, Queensland Health, Brisbane. Joan Faoagali, FRCPA, Director, Microbiology, Royal Brisbane Hospital, Brisbane. W.G.E. Cooksley, MD, Director, Clinical Research Centre, Royal Brisbane Hospital. Ian F. Young, FRCPA, FRCPA, Director, Australian Red Cross Blood Service-Queensland; current address: International Federation of Red Cross and Red Crescent Societies, Geneva, Switzerland. Rachel Trowbridge, PhD, Research Scientist, Sir Albert SakzewskiVirus Research Centre, Herston, Brisbane. Ian Borthwick, PhD, Research Scientist, Sir Albert SakzewskiVirus Research Centre. E.J. Gowans, PhD, Director, Sir Albert SakewskiVirus Research Centre. zyxwvutsr zyxwv Volume 38, September 1998 TRANSFUSION 827