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Recent studies(1–4) suggested that antibody which recognizes a region, namely HVR1 of the HCV envelope protein, is able to neutralize the infectivity of HCV. However, because the change of amino acid sequence in the HVR1 is rapid, HCV could escape from humoral immunoglobulin, and the infection can persist. The ability to produce humoral immunoglobulin is consistently lower in early infancy than in adulthood. Thus, the pattern of genetic drift of HCV toward an escape from the immunosurveillance system might differ from that in an adult case. However, the data needed to confirm this possibility have not been available. Based on the above findings, we investigated the sequential change of the HCV E2/NS1 gene in an infant who was transfused with blood containing HCV on the 6th d after the birth.

METHODS

Patient. The detailed clinical course of this patient has been described previously(5). The patient suffered cerebellar hemorrhage, marked hydrocephalus, and hemophilia A. He was accidentally transfused with 160 mL of blood, obtained from his maternal grandfather, containing the HCV on the 6th d after birth, and he then developed chronic viremia. The grandfather had no history of hepatitis or blood transfusion but had a tattoo. The patient's father and mother were HCV antibody-negative. The infant's blood samples were obtained at 3 d after transfusion, and also at various times after transfusion as shown in Figure 1. The grandfather's blood samples were obtained on the day of transfusion. Blood samples were immediately separated into serum and subdivided, and then stored at -30 °C until evaluation. The infant's HCV RNA level was determined by branched DNA probe assay (Quantiplex HCV-RNA Version 1.0)(6), his C-22 antibody level by an immunoradiometric assay (Ortho HCV core-antibody IRMA test)(7), and his ALT by the method of Wroblewski-Karmen at various times after transfusion. The HCV genotype was determined by the method of Okamoto et al.(8), except that a C gene sequence was typed by PCRs using four type-specific primers individually instead of a mixture of those primers. The grandfather's HCV RNA level was 0.7 Meq/mL on the day of transfusion. At 23 d after transfusion, the blood HCV RNA level of the infant showed an increase from below 0.5 Meq/mL at 3 d to 11.0 Meq/mL. Thereafter, the RNA level decreased, falling below 0.5 Meq/mL from mo 12 to 14. At mo 15, however, it rose again. The C-22 antibody level was 17 U at 1 mo, which was almost the same as immediately after the transfusion. At 3 mo it increased to 35 U; at 6 mo it decreased to 25 U and continued to rise, and at 12 mo the C-22 antibody level reached a maximum of 130 U and then decreased slightly. Although the ALT level increased slightly at 3-6 mo (maximum ALT level; 43 U/L), it fell at 11 mo and then leveled off. Two different HCV types [type II(1b) and III (2a)] were observed in both grandfather and infant. Parentheses indicate the HCV genotype subgroup proposed by Simmonds(9).

Figure 1
figure 1

Changes in serum ALT, anti-C-22 antibody status, and HCV RNA levels. The C-22 antibody assay procedure consists of a two-step immunoradiometric assay using polystyrene beads coated with recombinant c22-3. The radioactivity of the antigen-coated polystyrene bead is measured using a gamma counter. Based on the radiation counts/min associated with the amount of core antibody in the sample, the quantity of core antibody is translated from the standard curve, which is constructed from five different points of core antibody titer. The titer of core antibody is expressed in arbitrary units.

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Assays. The arrows in Figure 1 denote the times when stored serum samples were used for HCV E2/NS1 gene analysis. For cDNA synthesis and PCR amplification, RNA was extracted from 100μL of serum by means of the acid guanidiniumphenol-chloroform method, then dissolved in 30 μL of 10 mM Tris-HCl (pH 7.5). Ten microliters of RNA solution were used for cDNA synthesis and PCR amplification in the conditions described earlier(10), which were similar to the conditions described by Okada et al.(11), using a thermal cycler (model PJ2000, Perkin Elmer, CA). The primers for the first PCR were: 5′-CGCATGGCATGGGACATGAT-3′ (sense; nucleotides 949-968 of HCV-J6 strain, a type III HCV subgroup)(12) and 5′-GGAGTGAAGCAATACACTGG-3′ and 5′-GGGGTGAAACAATACACCGG-3′ (antisense; nucleotides 1519-1538). The primers for nested PCR were: 5′-GGGACATGATGATGATCAACTGG-3′ (sense; nucleotides 959-978) and 5′-GTGAAGGAATTCACTGGGCCACA-3′ and 5′-GTGAAAGAATTCACCGGGCCGCA-3′ (antisense; nucleotides 1513-1535). Cycles were adjusted to 35 for the first PCR and to 30 for the second PCR, respectively. Amplified cDNA fragments were cut from an agarose gel and purified using glass powder (EASYTRAP, Takara Shuzo Co., Japan). After digestion with EcoRI and BclI, cDNA fragments were cloned into the EcoRI-BamHI site of plasmid vector (pUC119) as described previously(10). Nucleotide sequences were determined by the dideoxy method, with a Taq Dye Deoxy Terminator Cycle Sequencing Kit (Applied Biosystems, Japan), using 373A fluorescent DNA sequencer (Applied Biosystems, Japan).

The amino acid sequences were deduced from the corresponding nucleotide sequences that span nucleotides 1135 through 1461 (327 bp). The overall frequency of homology, as well as the frequency of homology of the nucleotide sequence for the following parts, was estimated between the cDNA clone from the grandfather and the infant. Region a spans nucleotides 1150 through 1224(amino acids 384-408), which corresponds to the HVR1 of the E2/NS1 gene, region b nucleotides 1225 through 1419 (amino acids 409-473), and region c nucleotides 1420 through 1446 (amino acids 474-482), which corresponds to HVR2. Nucleotides were numbered from the first residue of the single open reading frame of HCV-J6 strain.

RESULTS

The overall nucleotide sequences of all cDNA clones from the grandfather and the infant were more homologous to the sequence of HCV J6 than that of HCV-BK, a type II HCV subgroup(13). The frequency of homology of nucleotide sequences for each sequenced region a, b, and c within the cDNA clone of the grandfather were 79.5 ± 9.6%, 96.8 ± 2.4%, and 99.5 ± 1.4%, respectively.

Figure 2 shows variations in amino acid sequence focusing on HVR1 and 2. The variable amino acid sequence of HVR1 was observed from clone to clone in the grandfather on the transfusion day. In the infant, the three types of amino acid sequences of HVR1, which were identical to that of the grandfather, were observed for cDNA clones at 3 d. Moreover, amino acid sequences of HVR1 for 23 d were identical or closely related to the other amino acid sequences of the grandfather. A highly variable amino acid sequence of HVR1 was observed in cDNA clones at 3 mo and also at 6 mo. Once again, the amino acid sequence of HVR1 at 16 mo, which was similar to that of minor cDNA clone at 6 mo, was more closely related to that of one of the grandfather's cDNA clones. In contrast to HVR1, the change of amino acid sequences of HVR2 was not observed throughout the patient's infancy.

Figure 2
figure 2

Sequences for NS1/E2 HVR1 and 2 obtained from the grandfather and infant on various days after transfusion. The consensus sequence for the predicted amino acids of the grandfather is shown by single letters on the top line. A dotted line indicates identical residue to the consensus. Numbers in parentheses denote the number of cDNA clones. HVR1 and 2 are bracketed within rectangles.

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To estimate the rate of variation in the HCV E2/NS1 gene of the infant on any given day, one among 8 cDNA clones of the grandfather, which had the highest homology for 327 bp to each cDNA clone of the infant, was selected as a source HCV. The difference in nucleotide sequence, as well as amino acid sequence, between the cDNA clone of the infant, and this source cDNA clone was calculated. The HVR1 showed a higher variation than the other two regions. Most nucleotide substitutions occurred with an alteration in amino acid as shown in Table 1. To rule out the influence of different posttransfusion times, the rate of amino acid substitutions observed in HVR1 was calculated as the number of amino acid differences observed per month. The average rate of amino acid substitutions in the HVR1 at 3 and 23 d, and 3, 6, and 16 mo was 18, 3.8, 5.2, 2.1, and 0.5, respectively. This showed that a higher variation of amino acid sequence in HVR1 occurred within a shorter duration of infection but was not correlated to HCV RNA levels.

Table 1 Rate of change in region-dependent nucleotide and predicted amino acid differences between the grandfather and the infant
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DISCUSSION

Although the quasispecies nature of HVR1 in chronic hepatitis C adult patients is consistent(4, 11), the cDNA clones from the grandfather also showed a highly variable HVR1 sequence. In this study, we tried to estimate the variation in HVR1 of the HCV E2/NS1 gene with age in the blood-transfused hepatitis C infant. We speculated that all cDNA clones of the infant were induced exclusively only from the 8 cDNA clones of the grandfather. The possible existence of a minor virus that could not be subcloned in the grandfather's samples could not be completely ruled out. But, if comparisons were made at two time points separated by longer intervals, for example, between the grandfather on the transfusion day and the infant at 16 mo, the HVR1 amino acid sequence from the infant was very similar to one from the grandfather with only 4 amino acid changes observed. This alteration rate of amino acid in HVR1 is 0.3/mo, a value similar to that of adult patients(4, 14) who showed 0.5-1.7/mo during the chronic state of hepatitis. However, this result did not reflect the actual alteration rate of HVR1 in the infant. To obtain more precise information, we analyzed the HVR1 in the infant at intervals of a few months. The results showed a higher alteration rate of amino acids in HVR1 throughout infancy as shown in the Table 1 and Figure 2.

Although there was a discrepancy of HCV genotype determined by PCR with type-specific primer and by homology search of nucleotide sequence, it led us to the following speculation. First, typing HCV by PCR with type-specific primers resulted in the misjudging of a mixed type of type II and III subgroup for a type III subgroup. This possibility is also suggested in a previous report by Wakamiya(15). Second, the primers used selectively amplified type III HCV subgroups in PCRs; but the exact reason for the discrepancy is unknown.

Because of repeated intracranial hemorrhages, occasional transfusion with the father's blood and injections of coagulation factor VIII were undertaken. The effects from the blood and the coagulation factor on HCV antibody and HCV RNA do not have to be taken into account because the father did not suffer from HCV infection and the coagulation factors were produced after 1993. The present result leads to the following speculation. First, this is the first report, to our knowledge, on the pattern of sequential change in the HCV E2/NS1 gene in an infant suffering from blood-transfused hepatitis C. Second, the infant showed a relatively low C-22 antibody level until 6 mo. Although immunologic attack on virus-infected cells by cytotoxic T cells may result in elevation of ALT values, the infant's ALT level increased slightly at 3-6 mo(maximum ALT level, 43 U/L). These data suggest that the immunosurveillance system against HCV and the cytopathic effect on hepatocytes may be weak. Contrary to the above possibility, observation of the frequent alteration of the amino acid sequence in the HVR1 suggests that the constant selection of HCV by the immunosystem or the escape from immunosurveillance occurred even in early infancy. Third, the alteration in amino acid sequence was remarkable but unsequential. This pattern was different from that seen in adults(3, 4, 14) with acute hepatitis C or in our mother-to-infant HCV-infected cases (unpublished data) that showed a sequential alteration in amino acids, a tendency in keeping with the development of the chronic phase. Okamoto et al.(16) indicate that when extensively diluted infective material harboring a mixed population of quasispecies E2/NS1 HVR1 was injected into chimpanzees, the resulting injection was the product of a single strain. The precise mechanisms of this difference are not understood at this time, possibly because a large volume of HCV was inoculated at 6 d after birth in this case. But further study is necessary to confirm a pattern of genetic drift of HCV to escape from the immunosurveillance system during early infancy.