Downloaded from www.microbiologyresearch.org by IP: 54.160.113.5 On: Mon, 06 Jun 2016 00:49:47 Short Communication Analysis of sequential hepatitis A virus strains reveals coexistence of distinct viral subpopulations Mauro Costa-Mattioli, 1 Esteban Domingo 2 and Juan Cristina 2,3 Correspondence Juan Cristina cristina@cin.edu.uy 1 Department of Biochemistry and McGill Cancer Center, McGill University, Montreal, Quebec, Canada H3G 1Y6 2 Centro de Biologı ´a Molecular ‘Severo Ochoa’ (CSIC-UAM), Universidad Auto ´ noma de Madrid, Cantoblanco, 28049 Madrid, Spain 3 Laboratorio de Virologı ´a Molecular, Centro de Investigaciones Nucleares, Facultad de Ciencias, Igua ´ 4225, 11400 Montevideo, Uruguay Received 26 June 2005 Accepted 4 October 2005 Hepatitis A virus (HAV) is a hepatotropic member of the family Picornaviridae. Despite a remarkable antigenic stability, recent results have shown that HAV exists in vivo and in cell culture as distributions of genetically related, non-identical variants, referred to as quasispecies. To gain insight into HAV evolution over time in a specific geographical region, genotype I consensus sequences from strains isolated in France in consecutive years were studied. Phylogenetic neighbour-joining method and a non-hierarchical partition analysis, designed to analyse viral quasispecies, indicate that at least five distinct subpopulations of HAV were identified in the course of the disease episode. Strikingly, over time, different subpopulations cycled in dominance. The coexistence of distinct subpopulations whose frequency varies with time is consistent with quasispecies dynamics, and suggests that variation in the dominant HAV population may provide HAV adaptability without being reflected in significant antigenic variation. Hepatitis A virus (HAV) is a hepatotropic member of the family Picornaviridae (van Regenmortel et al., 2000). Despite an overall physical and epidemiological similarity to entero- viruses, the structure of HAV, its tissue tropism and genetic distance from other members of the family Picornaviridae, indicate that HAV is unique within this family (Ticehurst et al., 1989; Palmenberg, 1989; Wimmer & Murdin, 1991; reviewed by Costa-Mattioli et al., 2003). Hepatitis A viruses have been classified into four human (I, II, III and VII) and three simian (IV, V and VI) genotypes (Robertson et al., 1992; Costa-Mattioli et al., 2003). Recent evidence has suggested that, as other RNA viruses, HAV exists in vivo as distributions of closely related variants referred to as quasi- species (Sanchez et al., 2003a). Quasispecies dynamics is characterized by the continuous generation of variant viral genomes, competition among them and selection of the fittest mutant distributions in any given environment. Understanding the principles that shape the evolution of viral quasispecies is becoming increasingly important to be able to model disease progression and to design preventive and therapeutic strategies to control viral disease (Domingo et al., 2001). The complexities of genetic data obtained from RNA virus quasispecies populations may not be accurately described by any single analytical tool (Baccam et al., 2001). Over time, RNA virus evolution is conditioned by perturbations of population equilibrium, which may not be equal among individual hosts, and therefore, multiple viral sublineages may rapidly be established that differ in the number of rounds of replication (and history of environmental perturbations), and may co-circulate in the same geographical area. To study HAV evolution over time in a specific geographical region, we have analysed the highly variable region, VP1, of HAV strains genotype I. The strains studied were isolated in France from 1983 to 2001 (Costa-Mattioli et al., 2002; see also Supplementary Table 1 available in JGV Online). We used two methods, one based on phylogenetic distance, neighbour-joining (NJ; Saitou & Nei, 1987), and the other is a non-hierarchical method developed to study closely related components of mutant spectra of viral quasispecies (PAQ; Baccam et al., 2001). Nucleotide sequences of the entire VP1-coding region were aligned using the CLUSTAL W program (Thompson et al., 1994). The program PAQ (Baccam et al., 2001) was adapted to compare consensus HAV VP1 sequences and to group HAV VP1 genes that were most similar. The program uses the Hamming distance (number of nucleotide differences) to measure the distances between VP1 gene sequences, and a A figure of the phylogenetic analysis of the complete VP1 region using the p-distance model and the UPGMA method, and a table showing French HAV strains examined in this study are available as supplementary material in JGV Online. 0008-1286 G 2006 SGM Printed in Great Britain 115 Journal of General Virology (2006), 87, 115–118 DOI 10.1099/vir.0.81286-0