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The recently discovered GB virus C (GBV-C) (13), or hepatitis G virus (HGV) (7), has a single-stranded positive-sense RNA genome and causes transient and persistent infections in humans. The viral genome has been detected in blood, liver tissue, semen, and saliva (1, 2, 5, 12). GBV-C is known to be transmitted by parenteral routes, such as contaminated blood products and intravenous drug use. However, several reports have described mother-to-infant transmission of GBV-C, which often seem to result in persistent infections (4, 6). Also, sexual transmission of GBV-C has been suggested (8-11, 14). Which of these routes that help to explain the high prevalence of both GBV-C RNA (1 to 3%) (7, 13) and antibodies (3 to 8%) (3) in the general population is not known. One hypothesis for the elevated prevalence of GBV-C markers in the blood donor population may be vertical transmission over several generations, similar to that of hepatitis B virus. Alternatively, GBV-C infections may be caused by sexual or even social contacts (8). The aim of the present study was to further characterize the transmission routes of GBV-C by analyzing for GBV-C markers in up to three generations of family members in three families with documented mother-to-infant transmission of GBV-C.

View larger version (55K): [in this window] [in a new window]   FIG. 1.   Results from analysis of GBV-C RNA by 5'-noncoding reverse transcription-PCR in serum and saliva samples from the two twins, the GBV-C-infected mother, and the father, from family A sampled in 1997 (a) and 1999 (d). Lanes M, size marker lanes. Ser, serum; Sal, saliva. A known GBV-C RNA-positive serum sample served as a positive control (Pos). Also shown are the analyses of family 1 for antibodies to the 42-kDa mtE2 protein (b) and the 56-kDa GBV-C E2 protein (c) by Western blotting. A known GBV-C-negative human serum sample served as a negative control (Neg), and a hyperimmune serum sample from a C57/BL6 mouse immunized with the GBV-C E2 protein served as a positive control (Pos).

Samples were collected from three previously identified families with known or suspected mother-to-infant transmission of GBV-C (4, 6). In family A, where a male twin was vertically infected in 1993 (4), saliva and/or serum samples were obtained from both parents and the two twins in 1996, 1997, and 1999. In family B, where the daughter was suspected to have been infected by the mother between 1990 and 1993, serum samples were obtained from the infected daughter (6), both parents, and the maternal grandparents in 1998. A stored sample from a younger brother obtained in 1993 was also analyzed. In family C, where vertical transmission occurred in 1991 (6), samples were obtained from the infected son, the infected mother, and the maternal grandparents in 1998.

All samples were analyzed for the presence of GBV-C RNA by a reverse transcription-PCR using primers from the 5'-noncoding region as described previously (2, 4). All serum samples were also tested at a dilution of 1:100 for antibodies to a recombinant genotype 1 GBV-C E2 protein (kindly provided by I. K. Mushahwar, Abbott Laboratories) by an enzyme immunoassay (EIA) as previously described (3). To confirm mother-to-infant transmission between the mother and the infected daughter in family B, the sequence of an 82-bp GBV-C nonstructural 3 fragment was determined and analyzed by phylogenetic analysis by standard protocols as previously described (6).

Cloning and expression of the E2 gene (mtE2) from the GBV-C genotype 2-infected male twin of family A (4) were performed in Escherichia coli by standard techniques and will be described in detail elsewhere. The mtE2 and the GBV-C E2 proteins were used in Western blot analysis for antibody detection after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PhastGelSystem; Pharmacia, Uppsala, Sweden) in accordance with the manufacturer's instructions. A hyperimmunized mouse serum (dilution 1:7,500) raised against the GBV-C E2 protein was used as a positive control in the Western blot. In family A, only the previously described mother and twin boy (4) had serum GBV-C RNA in the 1997 and 1999 samples. Saliva samples from these two contained GBV-C RNA in 1996 (4) and 1997 but not in 1999 (Fig. 1). Neither of them had antibodies to GBV-C E2 or to the mtE2 protein as determined by EIA (Table 1) or by Western blotting (Fig. 1). In contrast, both the father and the twin girl were negative for GBV-C RNA in serum but were positive for E2 antibodies in 1996 and 1997 as determined by EIA (Table 1) or by Western blotting (Fig. 1), respectively. The twin girl developed antibodies to the mtE2 protein in 1996 and became clearly seropositive in 1997 as determined by Western blotting (Fig. 1). However, since the antibodies did not recognize the GBV-C E2 protein by EIA (data not shown), we do not wish to overemphasize this observation. Her alanine aminotransferase and -glutamyl-transferase levels were persistently normal, whereas the asparagine aminotransferase levels were slightly elevated (0.92 µkat/liter; normal range, 0.50 to 0.90 µkat/liter) in the 1996 sample. She had neither undergone any type of surgical treatment nor received any blood products. Collectively, in the absence of other known exposures to GBV-C, these data suggest that the father, and possibly also the twin girl, may have acquired GBV-C through intrafamilial transmission.

In family B, where mother-to-infant transmission was presumed to have occurred between 1990 and 1993 (6), the daughter remained serum GBV-C RNA positive in 1998 (Table 1). Sequence analysis showed that the only other GBV-C NS3 sequence identical to that of the 1993 sample from the daughter (6) was that of the 1998 sample from the mother (Table 1), confirming the presumed mother-to-infant transmission. The father was negative for serum GBV-C RNA by PCR but was positive for GBV-C E2 antibodies by EIA in 1998 (Table 1). The maternal grandparents of the infected child were negative for all GBV-C markers (Table 1). Thus, an intrafamilial transmission of GBV-C may have occurred, although not over three generations. In family C, where vertical transmission occurred in 1991 (6), the mother and the boy remained positive for serum GBV-C RNA in 1998 (Table 1). No sample was available from the father. The maternal grandparents of the infected child were negative for both GBV-C RNA and GBV-C E2 antibodies (Table 1). No evidence for GBV-C transmission over three generations was found.

The transmission routes for GBV-C have not been completely defined. In these three cases of mother-to-infant transmission of GBV-C, the infants remained viremic and asymptomatic until 8 years of age. This may have implications for the prevalence of GBV-C RNA in adults. Transmission of GBV-C over three generations could not be confirmed since all maternal grandparents tested lacked GBV-C markers. They may have cleared their GBV-C infections many years ago, resulting in the levels of GBV-C E2 antibodies being below the detection limit at the time of analysis. This needs to be confirmed by others. GBV-C E2 antibodies were detected in samples from both tested fathers, suggesting that they had experienced GBV-C infections. Neither of them had any known risk for blood-borne viral infections. Thus, their GBV-C-infected partners are the probable sources of infection. It is possible that the twin girl developed E2 antibodies to her brother's virus despite being persistently negative for GBV-C RNA. If so, the persisting salivary GBV-C in two family members may have caused this transient infection. Thus, sexual, and possibly also social, contacts may help to explain the high prevalence of GBV-C E2 antibodies outside risk groups.