Molecular characterization of hepatitis a virus isolates from a transcontinental shellfish-borne outbreak

Abstract

One hundred eighty-four serologically confirmed cases of hepatitis A were reported in eastern Spain in 1999. A matched case-control study implicated imported coquina clams complying with European Union shellfish standards as the source of infection; this implication was confirmed by the detection by reverse transcription-PCR of hepatitis A virus (HAV) RNA in shellfish samples. In spite of the recognized low variability of HAV, genetic characterization of the complete capsid region of virus isolates from patient serum samples revealed the existence of both synonymous and nonsynonymous variants. Two antigenic variants were detected, one in a discontinuous epitope defined by monoclonal antibody K3-4C8 and a second in a linear VP1 epitope of the virus. In spite of these antigenic variants, all isolates were assigned to genotype IB, providing further evidence that the outbreak originated from a common source, although multiple strains were likely to be involved.

FIG. 1.

Age distribution (years) of patients with hepatitis A in the Valencia outbreak.

FIG. 2.

Alignment of a 127-nt fragment from the 5′NCR of 30 serum samples from hepatitis A patients. The first three sequences were obtained from GenBank (accession no. M14707, M16632, and X83302). The number of samples included in each group of sequences is shown in parentheses. Two shellfish sample sequences showed 100% homology with groups labeled with an asterisk. Nucleotide variants are depicted in bold. ATT., attenuated; IRES, internal ribosome entry site.

FIG. 3.

Phylogenetic tree based on the sequences of the 5′ NCR shown in Fig. 2. Letters corresponding to the same groups as those depicted in Fig. 2 are shown in parentheses. Symbols: circle, shellfish samples; asterisk, samples with a valine-to-isoleucine change at position 72 of protein VP3; dagger, samples with a valine-to-alanine change at position 63 of protein VP0; double dagger, samples with a methionine-to-valine change at position 28 of protein VP1.

FIG. 4.

Nucleotide diversity (Pi) in the capsid genomic region, expressed as the average number of nucleotide substitutions per site (18). Rectangles represent known HAV epitope coding regions: black, immunodominant site including discontinuous epitopes in VP3 and VP1 (24, 26); white, H7c27 epitope (26); gray, linear epitopes in VP3 and VP1 (5, 13).

FIG. 5.

Effect of the Iso-for-Val substitution at amino acid 72 of VP3 on virus recognition by MAbs K3-4C8 and K2-4F2, as measured by an ELISA. Recognition of the virus isolate from 50 μl of a 10% stool suspension was compared with that of approximately 50,000 TCID₅₀ of strain HM-175. Results are expressed as the mean and standard deviation.

FIG. 6.

Phylogenetic tree based on the nucleotide sequence of the VP1/2A region (nt 3024 to 3191). Letters corresponding to the same groups as those depicted in Fig. 2 and 3 are shown in parentheses. Symbols: asterisk, samples with a valine-to-isoleucine change at position 72 of protein VP3; dagger, samples with a valine-to-alanine change at position 63 of protein VP0; double dagger, samples with a methionine-to-valine change at position 28 of protein VP1.