Nucleotide variability and translation efficiency of the 5' untranslated region of hepatitis A virus: update from clinical isolates associated with mild and severe hepatitis

Abstract

Mutations in the internal ribosome entry site (IRES) of hepatitis A virus (HAV) have been associated with enhanced in vitro replication and viral attenuation in animal models. To address the possible role of IRES variability in clinical presentation, IRES sequences were obtained from HAV isolates associated with benign (n = 8) or severe (n = 4) hepatitis. IRES activity was assessed using a bicistronic dual-luciferase expression system in adenocarcinoma (HeLa) and hepatoma (HuH7) cell lines. Activity was higher in HuH7 than in HeLa cells, except for an infrequently isolated genotype IIA strain. Though globally low, significant variation in IRES-dependent translation efficiency was observed between field isolates, reflecting the low but significant genetic variability of this region (94.2% +/- 0.5% nucleotide identity). No mutation was exclusive of benign or severe hepatitis, and variations in IRES activity were not associated with a clinical phenotype, indirectly supporting the preponderance of host factors in determining the clinical presentation.

FIG. 1.

Phylogenetic analysis of studied and reference sequences (n = 35); the tree was constructed from a Kimura two-parameter genetic distance matrix using the neighbor-joining method followed by a bootstrap test (1,000 replicates). (A) 5′ UTR sequences (nucleotides 63 to 738, according to HM-175 strain numbering). (B) VP1-2A sequences (210 nucleotides).

FIG. 2.

Localization of variable positions of genotype I isolates in IRES secondary structures as described by Brown et al. (5). Each variable position is framed on the IRES structure. Isolate codes are highlighted. The substitution is depicted according to HM-175 numbering. “del” indicates a nucleotide deletion; an underlined substitution indicates an increase of bonds in a base pair, and an italicized substitution indicates a decrease; an asterisk indicates a mismatch.

FIG. 3.

IRES activity measured after transitory transfection of pCREL vectors in HuH7 and HeLa cells, expressed in arbitrary units (AU). Vertical bars represent standard deviations of the means from triplicate measures. (A) CRH (negative control), HRV (human rhinovirus), HM-175 strain (G1RW), HM-175/18f (G1RC), and studied clinical isolates (G1, G2, or G3 for genotype I, II, or III and B, S, or F for benign, severe, or fulminant hepatitis, respectively). (B) CRH (negative control); HM-175 (G1RW); HM-175 with T255C and T278C mutations, present in the G1B5 strain (RW-B5); HM-175 with G324 and C372 mutations (RW-FUJ).