Mixed infection of a child care provider with hepatitis A virus isolates from subgenotypes IA and IB revealed by heteroduplex mobility assay

Phylogenetic analysis based on a 168 base segment encompassing the putative VP1/2A junction of the hepatitis A virus (HAV) genome has enabled the classification of HAV isolates into seven genotypes (I-VII). Genotype I, which includes the vast majority of the human HAV isolates, has been divided further into subgenotypes IA and IB. An heteroduplex mobility assay was designed with amplification products from the VP1/2A junction region, and used as a genotyping method able to discriminate HAV isolates belonging to IA, IB and non-I genotypes. The method was used to successfully genotype 48 samples (16 IA and 32 IB). However, one HAV RNA positive serum sample (AUX-23), collected from a 15 year old female employed at a child care center located in Rio de Janeiro, Brazil, showed an unusual pattern. PCR products from sample AUX-23 gave rise to heteroduplex bands when mixed with IA products as well as with IB products, suggesting the presence of HAV isolates from both subgenotypes in the serum. PCR products from sample AUX-23 were then cloned and 20 clones were analyzed by heteroduplex mobility assay. Eleven were subgenotype IA and 9 were IB. Three clones of each subgenotype were then sequenced to confirm the results. These data constitute the first report of mixed infection of a single individual with different HAV isolates.