Three nested RT-PCR assays were developed to permit sensitive typing of enteroviruses directly from clinical samples. These assays amplified short fragments from different genomic regions codifying for three proteins: VP2, VP1, and RNA polymerase. Given that enteroviruses have a high rate of degeneration within target codons among serotypes, the primers used consisted of mixed base and deoxyinosine residues. These techniques detected at 0.03-0.003 TCID50 of prototype Poliovirus 1 and Echovirus 30. They were used to characterize the enteroviral RNA detected in 18 CSF, stool, and throw swab samples and in 8 enterovirus isolates from patients with several syndromes. Phylogenetic analysis in each independent sequenced region grouped the enterovirus into four clusters, enabling genetic classification. A comparative study was performed among the 26 sequences obtained after direct sequencing of products with those available in the nucleotide databases. The efficiency of each assay for enterovirus identification was evaluated by both distance (Clustal) and similarity (M-NW) indices. Comparative results obtained independently in the three regions showed the highest yield of correlation between nucleotide sequences of all prototype serotypes and the analyzed genotypes in the VP1 region (26/26, 100% Clustal; 22/26, 85% M-NW). Conversely, the VP2 region failed to identify some of the circulating enteroviruses (17/26, 65% Clustal; 16/26, 62% M-NW). Using the RNA polymerase region, sequences from samples and isolates were associated with prototype strains whenever these were available (20/21, 95% Clustal; 12/21, 57% M-NW). These assays were useful for molecular identification of enterovirus directly from samples even when isolation was not possible.

No abstract

The family Picornaviridae is a large and diverse group of viruses that infect humans and animals. Picornaviruses are among the most common infections of humans and cause a wide spectrum of acute human disease. This study began as an investigation of acute flaccid paralysis (AFP) in a small area of eastern Bolivia, where surveillance had identified a persistently high AFP rate in children. Stools were collected and diagnostic studies ruled out poliovirus. We tested stool specimens from 51 AFP cases and 34 healthy household or community contacts collected during 2002–2003 using real-time and semi-nested reverse transcription polymerase chain reaction assays for enterovirus, parechovirus, cardiovirus, kobuvirus, salivirus and cosavirus. Anecdotal reports suggested a temporal association with neurological disease in domestic pigs, so six porcine stools were also collected and tested with the same set of assays, with the addition of an assay for porcine teschovirus. A total of 126 picornaviruses were detected in 73 of 85 human individuals, consisting of 53 different picornavirus types encompassing five genera (all except Kobuvirus). All six porcine stools contained porcine and/or human picornaviruses. No single virus, or combination of viruses, specifically correlated with AFP; however, the study revealed a surprising complexity of enteric picornaviruses in a single community.

No abstract