Vertical Transmission of the Hepatitis C Virus to Infants of Anti-Human Immunodeficiency Virus-Negative Mothers: Molecular Evolution of Hypervariable Region 1 in Prenatal and Perinatal or Postnatal Infections

Longitudinal study.

In our study 2,263 anti-HIV antibody-negative pregnant women were screened for anti-HCV antibodies between June 1992 and June 1995; anti-HCV antibody positivity was found in 56 cases (2.4%), and 33 women were enrolled in a prospective longitudinal study of HCV transmission. Their ages ranged between 19 and 42 years (mean age, 28 years); 20 women (60%) had an acknowledged history of intravenous drug use, 1 (3%) was a health care worker with professional exposure (3%), 4 (12%) had an acknowledged history of anti-HCV antibody-positive sexual partners, and 8 (24%) had no risk factor. No woman was in interferon therapy. Only 2 pregnant women were diagnosed as chronic hepatitis sufferers based on histological findings by liver biopsy, and 26 were asymptomatic. Four women breast fed up to 10 to 12 months, and two breast fed up to 30 days. All but 3 of 33 babies were delivered vaginally.

Serum samples were stored at −80°C within 3 h of collection in a day hospital. Testing for anti-HCV antibodies was done by a commercially available third-generation enzyme immunoassay (EIA; Abbott); positive results obtained by EIA were confirmed by a third-generation recombinant immunoblot assay (RIBA) (Ortho Diagnostic), and serum alanine aminotransferase (ALT) levels were determined. The test for anti-E2 antibodies was carried out by HCV E2 immunoglobulin G (IgG) kit EIA (Nuclear Laser Medicine) in cases of transmission of the infection.

Total RNA was extracted from 100 μl of serum by using the guanidine thiocyanate method and was detected by reverse transcriptase PCR and nested PCR by using two sets of oligonucleotide primers deduced from the highly conserved 5′ untranslated region of the HCV genome (1-3).

Quantification of HCV RNA in serum was performed by the Amplicor HCV Monitor (Roche Diagnostic Systems, Branchburg, N.J.), and HCV genotypes were determined through a line probe assay (INNO-LiPA; Innogenetics, Nucear Laser Medicine).

A 447-bp sequence of the E2 region encompassing HVR1 and HVR2 of the HCV genome (from nucleotide [nt] 1324 to nt 1771) was amplified by heminested reverse transcriptase PCR. E2 cDNA was obtained with an E2A antisense primer (5′-TTCATCCAGGTGCASCCGSAA-3′; nt 1986 to 1966) and amplified with E2A and E2S (5′-CCYGGTTGCTCYTTYTCTATCTT-3′; nt 848 to 870) primers. A second amplification was performed with E2A and E2NA (5′-GGTGGGTAGTGCCAGCAATA-3′; nt 1813 to 1794) primers.

Crude PCR products were used as templates for a cycle sequencing reaction with the Thermo Sequenase cycle kit (Amersham, Little Chalfont, United Kingdom) and an infrared IRD 800-labeled E2-specific sequencing MB primer (5′-GCATGGCTTGGGATATGATG; nt 1291 to 1310). Termination products were electrophoresed and analyzed in a model 4000L Licor DNA sequencer (Molecular Biotechnology) (18).

Computer analysis of the sequence data was performed with Chromas, version 1.51 (Technelysium Pty. Ltd.). Nucleotide and deduced amino acid sequences were aligned with the CLUSTALW program, and synonymous and nonsynonymous nucleotide mutations were evaluated. Mother-child distances were computed by DNADIST of the Philip, version 3.57, package according to the Kimura model.

The t test for equality of means of two independent samples and the Wilcoxon signed rank test were used for statistical analysis (program SPSS, version 8.0, for Windows).

At birth, serum samples from mother-child pairs showed similar antibody titers in EIAs and similar RIBA profiles. HCV RNA was detected in serum samples at delivery of all mothers but four. Both viremia and the HCV genotype were determined for 29 HCV RNA-positive mothers. HCV genotypes 1a (35.2%), 3a (47%), and 4c (17.6%) were most represented in the intravenous drug user risk group, and 1b and 2c were most represented in the group with no risk factors (sporadic hepatitis) risk group (57.1 and 28.5%, respectively).

The EIA serological follow-up study of 31 noninfected newborns showed the clearance of anti-HCV antibodies within 9 months in 50% of the babies; in five children anti-HCV antibodies were detectable up to 15 months (14.7%). No infant was anti-HCV antibody positive at 18 months.

There was a correlation between anti-HCV antibody titers in mothers and the time of serum reversion in their babies: multiple anti-HCV antibody reactivity, as determined by immunoblotting, in sera of mothers was associated with a longer persistence of passively acquired anti-HCV antibodies in their children (P < 0.0005), and, in particular, there was a direct correlation between the anti-C22 titers of mothers and the time of serum reversion in their infants (P < 0.005). Abnormal ALT levels in the serum of infants did not generally correspond to the persistence of anti-HCV antibodies or HCV RNA; only two babies (C13 and C32) acquired the HCV infection, as shown by persistent viremia with HCV RNA levels of up to 1.2 × 10⁶ copies/ml and production of specific anti-HCV antibodies (Fig. 1 and 2) A t test for equality of means of two independent samples detected a significant correlation between high levels of viremia (>10⁶ copies/ml) and frequency of vertical transmission (P < 0.0005). No infant born to HCV RNA-negative mothers had fluctuating or persistent viremia.

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FIG. 1.

Longitudinal evaluation of viremia, ALT levels, and production of IgG anti-C33 antibodies in a serial serum sample from C13.

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FIG. 2.

Longitudinal evaluation of viremia, ALT levels, and production of IgG anti-C22 and anti-NS3 antibodies in a serial serum sample from C32.

Clinical evolution of acquired infection.

C32 exhibited highly abnormal ALT levels starting from 6 months, and C13 showed a slight alteration of ALT levels starting from 9 months (Fig. 1 and 2). The HCV genotypes in C13 and C32 were 2c and 1b, respectively, and those in mother-infant pairs were identical. RIBA was indeterminate for C13 and M13, the only pair which expressed positivity for the anti-C22 antibody alone.

A longitudinal study was performed to follow up the two cases (C13 and C32, born to M13 and M32, respectively) of vertical transmission up to 5 to 6 years of age in order to evaluate the evolution of the HCV infection. Both HCV-infected babies were breast fed for 12 months.

In C13 the titer of HCV RNA at the birth was 4.1 × 10⁴ copies/ml. The viral genome in C32 was absent at birth and became detectable at 6 months of age, with a titer of 5.1 × 10⁴ copies/ml. HCV RNA was detected only in the colostrum (<1.0 × 10³ copies/ml) of M32.

Both C13 and C32 showed persistent viremia during the entire longitudinal study. The production of specific anti-C33 antibodies, absent in the mother, occurred at 9 months of age in C13, and specific production of anti-C22 and anti-C33 antibodies was observed in C32 at 15 and 18 months of age, respectively. Both mother and child were anti-E2 antibody negative (Nuclear Laser Medicine) at the time of birth, and only C13 showed seroconversion (anti-E2 positive) during a follow-up (Fig. 1).

The highest HCV RNA titer in C13, 1.3 × 10⁶ copies/ml, was at 9 months; after the production of specific anti-HCV antibodies, the HCV RNA titer decreased to low levels (≤1.0 × 10³ copies/ml) up to 4 years of age. Except for a brief period (at 55 months of age) when HCV RNA was not detectable, C13 has shown a fluctuating viremia to date. Borderline ALT levels were observed throughout the follow-up period (Fig. 1). Except for a slight splenomegaly, C13 has shown no important clinical symptoms of infection.

C32 was not viremic at birth; HCV RNA was detected at 6 months of age. Viremia fluctuated throughout the follow-up period and peaked (1.2 × 10⁶ copies/ml) at 18 months of age. In contrast to C13, C32 has persistently altered ALT levels (250 to 350 U/liter). After the production of specific anti-C33 IgG antibodies at 18 months of age, ALT levels remained elevated and viremia began to decrease; the HCV RNA titer remained low between 45 and 54 months of age but increased thereafter (Fig. 2). C32 showed no clinical symptoms of infection.

Comparison of E2 region encompassing HVR sequences in mother-infant pairs.

Sequencing of a portion of the E2 region allowed us to evaluate the mutation rate in HVR1 by comparing serial samples of C13 and C32 with those of their mothers at delivery.

Deduced amino acid sequence alignments of the M13 sample at delivery and nine samples of C13 are shown in Table 1 Nonsynonymous amino acid mutations occurred, particularly in HVR1, starting from 9 months of age.

Deduced amino acid sequence alignments of the M32 sample at delivery and of eight C32 samples are shown in Table 2 There are two distinct HVR (HVR1 and HVR2) typical of HCV genotype 1b in the C32-M32 pair. Starting from 46 months of age, nonsynonymous amino acid mutations occurred in HVR1 but not in HVR2.

The DNA distance matrix for the nucleotide sequence of the newborn began to differ from that for the mother at 9 months in C13 and at 46 months in C32, with values of 0.0791 and 0.025, respectively, for HVR1 at 47 to 48 months of age (Tables 1 and 2).

Discussion.

Our data show that, in HCV RNA-positive, HIV-negative mothers, HCV infection is transmitted to 6.8% of newborns.

Although intravenous drug use itself, as well as coinfection with HIV, may be a major predisposing factor in perinatal transmission of HCV, none of the 20 drug-addicted women of our cohort study transmitted the infection to their babies; moreover, we found no link between active liver disease in mothers and enhanced risk of viral transmission in the absence of nursing (9, 16). Although the mothers of both infected babies were asymptomatic and didn't belong to any risk group, they had high levels of circulating HCV RNA at delivery (≥10⁶ copies/ml) and breast fed up 10 months.

Genotypes 1a and 3a were more frequent among the intravenous drug users of our cohort study than among other groups, but none of these genotypes was transmitted.

Although HCV RNA can be present in breast milk, most studies reveal that infection through breast feeding is apparently infrequent. In this study in only one case was breast feeding possibly responsible for HCV transmission.

According to our results, C32 possibly acquired HCV infection via perinatal or postnatal transmission by nursing (data not shown), as suggested by several factors: (i) absence of HCV RNA in serum at birth, (ii) ALT level increase and presence of HCV RNA at 6 months of age, (iii) delayed production of anti-C22 (at 15 months of age) and anti-C33 (at 18 months of age) IgG antibodies, and (iv) presence of HCV RNA in the M32 colostrum (1.0 × 10³ copies/ml).

The presence of HCV RNA at birth (1.0 × 10⁴ copies/ml) in C13 suggests fetal infection; this assumption is supported by quasispecies analysis of the core E1 region in a previous study, where nucleotide variability was considerably greater at the time of birth than 6 months after birth (11). Our results reveal interesting differences in the serological profiles of the two infants; the presence of anti-E2 antibodies could explain the high variability of HVR1 in C13, a region found within the E2 envelope protein which was shown to be a major site for the genetic evolution of HCV after the onset of hepatitis and which might escape the host immune surveillance system (14).

By sequencing from crude PCR products of the E2 region in sequential serum samples from HCV-infected newborns, it was found that nonsynonymous mutations occurred in C13 and C32; their greatest frequency was detected after 3 months of age in C13 and 2 years after specific seroconversion in C32.

Differences in the HVR1 distance matrices for C13 and C32 with respect to those for their mothers could reflect different types of vertical transmission (intrauterine transmission or perinatal or postnatal transmission), different genotypes, and different serological profiles. Different selective pressures acting on HCV in C13 and C32 may have produced different clinical outcomes, although they were both asymptomatic at the end of the study (6).

Long-term follow-up of HCV-infected newborns is clearly necessary to evaluate the impact of these aspects over time.

We conclude that HCV infection can be vertically transmitted by HIV-negative HCV RNA-positive mothers with asymptomatic infection it was significant in our statistical study that a high level of HCV RNA (>1.0 × 10⁶ copies/ml) in serum is an important risk factor (P < 0.0005). Further investigation is necessary to understand the role of the type of delivery (vaginal or caesarean), breast feeding, and familial contact in HCV vertical transmission, and only by studying more cases of vertical transmission will it be possible to correlate routes of transmission with HVR1 variability.

Compared to traditional methods of examining viral complexity involving sequencing of viral quasispecies, too cumbersome to be applied to large numbers of individuals and in a diagnostic routine, the sequencing of HVR1 with crude PCR products represents a simple, economic routine diagnostic tool, which also provides important information on persistent infections and on the evolution of vertically acquired viruses. To date, the frequencies of the different types of HCV infection (prenatal, perinatal, and postnatal) transmitted vertically are still not known; we suggest that the new procedure be used to address this topic.

The study of the effects of neonatal infection on the evolution of the HCV envelope protein could help elucidate some aspects of intrahost virus evolution: the early detection of the HCV variant may be considered the starting sequence for a particular host, allowing easier and better analysis of sequence diversification over time and clarification of viral pathogenicity mechanisms (4). The evolutionary analysis of virus populations in HCV-infected newborns (i.e., in a host with immature humoral and cytotoxic immune responses) may provide insight into the natural history of HCV infection, viral pathogenic potential, and virus-host interplay.