Genomic variations in echovirus 30 persistent isolates recovered from a chronically infected immunodeficient child and comparison with the reference strain

Abstract

Seven sequential isolates of echovirus type 30 (EV30) were recovered over 22 months from a child with severe combined immune deficiency syndrome. The nucleotide sequences of the 5' halves of the genomes (4,400 nucleotides) of the first (S1) and last (S7) isolates were determined and compared with that of the EV30 Bastianni reference strain, also determined in this study. In genome regions P1 and P2, 101 variations were identified between the two isolates. Synonymous differences far outnumbered nonsynonymous differences. Amino acid changes affected both capsid and nonstructural polypeptides (particularly 2B). The VP1 nucleotide sequences of the seven isolates were determined to analyze genome evolution during the chronic infection. In the phylogenetic tree, the seven isolates were directly related to the prototype strain in an individual monophyletic group, strongly suggesting that the chronic infection in the child arose from a single persistent EV30 isolate. Four lineages were observed in the persistent isolates. Isolates S2, S4, S5, and S6 were close relatives of one another, whereas isolates S1 and S3 formed individual lineages. Isolate S7, distantly related to all other isolates, formed the fourth lineage. These findings suggest the quasispecies nature of the genomes of the seven sequential EV30 isolates. Grouping of persistent isolates on the basis of replicative capacities was consistent with phylogenetic relationships. Overall, the results indicate that genetically related EV30 variants with different replicative capacities coexisted in a carrier state, probably in the gastrointestinal tract, during the infection of the child.

FIG. 1

Phylogenetic relationships of EV30 persistent isolates to one another. Pairwise maximum likelihood distances were estimated by the Tamura-Nei model (38) from the VP1 sequences by using all informative nucleotide sites (199 in 876). The tree was reconstructed by the quartet puzzling method (37), and the reliability value of each internal branch indicates (in percent) how often the corresponding cluster was found among the 1,000 intermediate trees. In 210 quartets analyzed, only 7 (3.3%) were unresolved. Branch length was drawn to the indicated scale. EV30 was used as the outgroup to root the trees.

FIG. 2

Duration of chronic EV30 infection estimated from nucleotide substitution rates in VP1-coding sequence. The proportion of nucleotide differences at all sites (diamonds), at synonymous sites (squares), and at the third codon positions (triangles) were calculated for each pairwise comparison with the MEGA computer program (23). Nucleotide substitutions were extrapolated back to zero substitution.

FIG. 3

Time course of virus production in MRC5 cells. MRC5 cells were seeded at 2 × 10⁵ per 35-mm culture dish and were cultured for 4 days before inoculation. Data are the means of two independent experiments, and virus titers were determined in duplicate for each experiment.