Genotyping rotavirus RNA from archived rotavirus-positive rapid test strips

Abstract

Genotyping circulating rotaviruses before and after introduction of rotavirus vaccine is useful for evaluating vaccine-associated changes in genotype distribution. We determined frequency of rotavirus genotypes among 61 rotavirus-positive children hospitalized in Israel during the 2005-06 rotavirus season. Accurate molecular epidemiologic data were recovered from affinity-concentrated rotavirus immobilized in rotavirus-positive bands from air-dried, diagnostic rotavirus rapid test strips (dipstick) stored at room temperature from 1 week to 5 years. G genotypes were identical for 21 paired dipsticks and suspensions, whereas dipsticks or suspensions detected an additional G genotype in 2 samples. RNA sequences from 7 pairs were identical. Phylogenetic analysis suggested previously unreported G2 sublineages and G9 lineages. The ease with which dipsticks can be stored at local facilities and transported to central reference laboratories can reverse increasing difficulties in obtaining geographically representative stool samples and expand surveillance to regions lacking adequate laboratory facilities.

Figure

Neighbor-joining phylogenetic trees for viral protein (VP) 7 G1, G2, G3, and G9 genotypes of hospitalized children in Israel, including sequences recovered from archived rotavirus dipsticks. Representative isolates for lineages and sublineages of VP7 genotypes G1 (A), G2 (B), G3 (C), and G9 (D) were chosen from Phan et al. (15) Page and Steele (16), Wang et al. (17), and Martinez-Laso et al. (18), respectively, and the sequences were downloaded from the European Molecular Biology Laboratory/GenBank/DNA Data Bank of Japan. These sequences were aligned with Israeli sequences by using the Sequencher program (Genecodes, Ann Arbor, MI, USA) and truncated to the longest segment common to all sequences in the alignment; 515 nt for G1, 547 nt for G2, 274 nt for G3, and 207 nt for G9. Each of the 4 phylogenetic trees was prepared by using ClustalX (13) for data bootstrapped 1,000× and was analyzed with NJplot (14). Whole numbers indicate bootstrap values for branches; fractional numbers indicate genetic distances. The genotype, lineage, and, where relevant, sublineage of each isolate appears in brackets after the name of the isolate: for example, KY3303[G2.2a] is VP7 genotype G 2, lineage 2, sublineage a for isolate KY3303. A letter at the end of the name of the Israeli sequences indicates the source of the RNA (D for dipstick or S for fecal suspension). D&S appears when the sequences were identical. The GenBank accession numbers for all sequences in this figure appear in Table A1. Scale bars indicate percent of nucleotide substitutions per site.