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

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.

Enterovirus 68 is associated with respiratory illness and shares biological features with both the enteroviruses and the rhinoviruses.

Enterovirus (EV) 68 was originally isolated in California in 1962 from four children with respiratory illness. Since that time, reports of EV68 isolation have been very uncommon. Between 1989 and 2003, 12 additional EV68 clinical isolates were identified and characterized, all of which were obtained from respiratory specimens of patients with respiratory tract illnesses. No EV68 isolates from enteric specimens have been identified from these same laboratories. These recent isolates, as well as the original California strains and human rhinovirus (HRV) 87 (recently shown to be an isolate of EV68 and distinct from the other human rhinoviruses), were compared by partial nucleotide sequencing in three genomic regions (partial sequencing of the 5'-non-translated region and 3D polymerase gene, and complete sequencing of the VP1 capsid gene). The EV68 isolates, including HRV87, were monophyletic in all three regions of the genome. EV68 isolates and HRV87 grew poorly at 37 degrees C relative to growth at 33 degrees C and their titres were reduced by incubation at pH 3.0, whereas the control enterovirus, echovirus 11, grew equally well at 33 and 37 degrees C and its titre was not affected by treatment at pH 3.0. Acid lability and a lower optimum growth temperature are characteristic features of the human rhinoviruses. It is concluded that EV68 is primarily an agent of respiratory disease and that it shares important biological and molecular properties with both the enteroviruses and the rhinoviruses.

Molecular epidemiology and type-specific detection of echovirus 11 isolates from the Americas, Europe, Africa, Australia, southern Asia and the Middle East.

Echovirus 11 (E11) is among the most commonly isolated human enteroviruses. To examine the range of genetic variation within the E11 serotype, we determined the complete VP1 sequences for 53 geographically dispersed E11 strains isolated in 16 countries from 1953 to 2001. E11 sequences were monophyletic with respect to all other enterovirus serotypes. The sequences clustered into four monophyletic genogroups, A-D; members of each genogroup differed from one another by <20%. Isolates in different genogroups differed from one another by 19-28%. The E11 prototype strain, USA/CA53-Gregory, was the sole member of genogroup B. All recent US isolates were members of one of two discrete lineages within genogroup D. The well-characterized E11 antigenic variant, USA/CA63-Silva, was also a member of genogroup D. Members of genogroups A and C were antigenically similar to USA/CA53-Gregory, as measured by neutralization with anti-Gregory and anti-Silva antisera. Only USA/CA63-Silva was neutralized more efficiently by the anti-Silva antiserum; other genogroup D viruses were Gregory-like or intermediate in their neutralization phenotype. Recent non-US isolates were distributed in genogroups A, C and D. Sequence similarities among genogroup D isolates from North America, Europe, Asia, Australia and North Africa demonstrate that an E11 strain can spread rapidly over a wide geographic area. The aligned sequences were used to develop an E11-specific RT-PCR assay, using degenerate, inosine-containing primers, to amplify all members of all genogroups.