Resolving ambiguities in genetic typing of human enterovirus species C clinical isolates and identification of enterovirus 96, 99 and 102

1 Polio and Picornavirus Laboratory Branch, Division of Viral Diseases, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, 1600 Clifton Road NE, Mailstop G17, Atlanta, GA 30333, USA 2 Institute of Public Health, Dhaka, Bangladesh Correspondence Betty A. Brown bzb2{at}cdc.gov Molecular methods, based on sequencing the region encoding the VP1 major capsid protein, have recently become the gold standard for enterovirus typing. In the most commonly used scheme, sequences more than 75 % identical (>85 % amino acid identity) in complete or partial VP1 sequence are considered to represent the same type. However, as sequence data have accumulated, it has become clear that the ‘75 %/85 % rule’ may not be universally applicable. To address this issue, we have determined nucleotide sequences for the complete P1 capsid region of a collection of 53 isolates from the species Human enterovirus C (HEV-C), comparing them with each other and with those of 20 reference strains. Pairwise identities, similarity plots and phylogenetic reconstructions identified three potential new enterovirus types, EV96, EV99 and EV102. When pairwise sequence comparisons were considered in aggregate, there was overlap in percentage identity between comparisons of homotypic strains and heterotypic strains. In particular, the differences between coxsackievirus (CV) A13 and CVA17, CVA24 and EV99, and CVA20 and EV102 were difficult to discern, largely because of intratypic sequence diversity. Closer inspection revealed the minimum intratypic values and maximum intratypic values varied by type, suggesting that the rules were at least consistent within a type. By plotting VP1 amino acid identity vs nucleotide identity for each sequence pair and considering each type separately, members of each type were fully resolved from those of other types. This study suggests that a more stringent value of 88 % VP1 amino acid identity is more appropriate for routine typing and that other criteria may need to be applied, on a case by case basis, where lower values are seen. Present address: Lawrence Livermore National Laboratory, 7000 East Avenue L-452, Livermore, CA 94551, USA. The GenBank/EMBL/DDBJ accession numbers for the sequences described in this paper are DQ995633–DQ995648, EF015008–EF015040, EF026081, EF015886, EF555644, and EF555645 (Supplementary Table S1). Supplementary material is available with the online version of this paper.