Evolution of hepatitis C viral quasispecies and hepatic injury in perinatally infected children followed prospectively

Abstract

Perinatal infection with hepatitis C virus (HCV) is characterized by a wide range of alanine aminotransferase (ALT) levels. The mechanisms responsible for this variability are unknown. We examined whether the evolution of the HCV quasispecies was associated with different ALT profiles in perinatally infected children. Sequences within HCV envelope 1 and 2 genes, inclusive of the hypervariable region 1, the viral load, and the nascent humoral immunity were analyzed in serial serum samples from 12 perinatally infected children prospectively followed for a median of 53 months. These patients were selected to represent two different ALT patterns during the first year of life: 6 had high levels (maximum values ranging from 4.2 to 30 times the normal upper limit), and 6 had normal or slightly elevated levels (<2 times the normal upper limit). Two patterns of viral evolution were identified according to the ALT profiles. Biochemical evidence of hepatic injury was invariably associated with a mono-or oligoclonal viral population, whereas mild or no liver damage correlated with the early emergence of a heterogeneous viral quasispecies. Consistent with selective immune pressure, amino acid changes occurred almost exclusively within the hypervariable region 1 and were temporally associated with antibody seroconversion; at this time, the difference in genetic diversity between the two groups was highly significant (P ‫؍‬ 0.002). The two patterns of viral evolution persisted over time and did not correlate with viral load or genotype. Our study demonstrates that, in perinatally infected children, the evolution of HCV quasispecies correlates with hepatic injury.perinatal hepatitis C virus infection ͉ genetic variability ͉ alanine aminotransferase levels M aternal-infant transmission is the dominant route for acquisition of hepatitis C virus (HCV) infection in children in developed countries, with an estimated transmission rate of Ϸ5% (1-3). Age and mode of infection are assumed to influence the progression of HCV disease in adults, but limited data are available on the natural history of perinatally acquired HCV infection (4, 5). Recent studies suggest that 80% of these children develop chronic infection (6-8), a rate similar to that documented in adults (9) but higher than that reported in children with posttransfusion HCV infection (10). Persistent HCV infection in infants and children is associated with minimal or mild liver damage and very rarely with advanced liver disease (5,(11)(12)(13)(14). However, perinatal HCV infection is associated with a wide range of aminotransferase levels during the first year of life (5, 7). Whereas some infants reach serum concentrations of alanine aminotransferase (ALT) compatible with acute hepatitis, others show normal or slightly elevated levels (5, 7). The mechanisms responsible for the different ALT profiles seen in perinatal HCV infection are unknown but are likely the result of a complex interplay between viral factors and host immunity. One of the most effective strategies enacted ...

Genetic and catalytic efficiency structure of an HCV protease quasispecies†

“…Nested PCR was then performed with a 5Ј oligonucleotide (HCVproL2) encoding an EcoRI site, residues 21 to 34 of NS4, and a dipeptide linker, Gly-Gly, along with residues 2 to 8 of NS3 (residues 3411 to 3431 of the HCV-J strain) (5Ј-GGGTTGAATTCTATG-GCTCCTATTGGATCTGTTGTTATTGTTGG AA-GAATTATTTTGTCTGGAAGAGGAGGACCTAT-CACGGCCTACTCCCAA-3Ј) and a 3Ј oligonucleotide (HCV proR) that was complementary to residues 3930 to 3950 of the HCV-J strain (amino acid residues 175 to 181 of NS3) and encoded an in-frame stop codon flanked by an XhoI site (5Ј-GGGAGGGGCTCGAGTCAA-GACCGCATAGTAGTTTCCAT-3Ј). The PCR products were digested with EcoRI and XhoI and ligated to pBSK-(Stratagene) to generate a ␤gal-HCV NS3 2-181 / 4 [21][22][23][24][25][26][27][28][29][30][31][32][33][34] protease fusion protein (pHCVNS3 2-181 /4 [21][22][23][24][25][26][27][28][29][30][31][32][33][34] protease). To ensure that multiple HCV NS3/4 protease templates were present in each analyzed quasispecies, for each sample, 40 different PCR amplifications were performed and pooled before cloning.…”

“…The catalytic efficiency of the different HCV NS3/4 proteases was determined using a bacteriophage lambda ( )-based genetic screen as previously described. 26,27 Escherichia coli JM109 cells containing plasmid pcI.HCVcro 27 that included the NS4B/NS5A (NEDCSTPCSGSWLRDVW) or the NS5A/NS5B (ASEDVVCCSMSYTWTGA) cleavage site were then transformed with plasmid pH-CVNS3 2-181 /4 [21][22][23][24][25][26][27][28][29][30][31][32][33][34] protease. The resulting cells were grown overnight at 30°C in the presence of 0.2% maltose, harvested by centrifugation, and resuspended to an optical density at 600 nm of 2.0 per ml in 10 mM MgSO 4 .…”

“…The resulting cells were grown overnight at 30°C in the presence of 0.2% maltose, harvested by centrifugation, and resuspended to an optical density at 600 nm of 2.0 per ml in 10 mM MgSO 4 . To induce the expression of HCV NS3 2-181 /4 [21][22][23][24][25][26][27][28][29][30][31][32][33][34] protease, cells (200 l) were incubated in 1 ml of Luria-Bertani medium containing 12.5 g tetracycline, 20 g ampicillin, 0.2% maltose, 10 mM MgSO 4 , and 1 mM isopropyl-␤-D-thiogalactopyranoside (IPTG) for 1 hour. Thereafter, cell cultures were infected with 10 7 PFU of phage.…”

“…After incubation at 37°C for 6 hours, the resulting phage plaques were counted to score growth. In the experiments in which E. coli cells express the wild-type HCV I389/NS3-3 NS3/4 protease obtained from the subgenomic replicon, 33,34 phage replicated up to 6000-fold more efficiently than in cells that did not express the HCV NS3 2-181 /4 [21][22][23][24][25][26][27][28][29][30][31][32][33][34] construct.…”

“…18,19 Early evolution of the viral quasispecies was shown to predict the clinical outcome of acute hepatitis C. 20,21 Likewise, the long-term viral evolution was shown to correlate with the severity of liver disease. 22,23 The HCV quasispecies has been well characterized for different viral genomic regions. The highest degrees of amino acid diversity within an infected individual (10%) 20 has been observed in variable regions of viral envelope genes (E1 and E2).…”