Figure 1: Original agarose gel photograph obtained from second amplification step of VIDISCA. Numbering of the 16 second-stage PCR products is shown above the first and last three lanes. The product in lane 4 was sequenced and showed a parechovirus as described in the text. A 100 bp marker is used on the gel (500 bp band enhanced, 400, 300, 200, 100 bp).

Mentions: During routine diagnostic work on patients with acute enteritis in a municipal health service, a stool sample from a 30 year-old female kitchen worker with acute enteritis displayed a cytopathic effect (CPE) on cultured African Green Monkey Kidney (GMK) cells. The CPE resembled that of enteroviruses, including rounding and blebbing, shrinking, and detachment of cells from the monolayer. The virus isolate could be passaged to uninfected cells but showed no detectable neutralisation if subcultured with several different pools of polyclonal anti-enterovirus sera. RT-PCR for Norovirus and Enterovirus, PCR for Adenovirus, and antigen-EIA for Astro- and Rotavirus were negative on the supernatant and on the original patient material. The unknown isolate was termed BNI-788st. In order to type it, supernatant was subjected to VIDISCA, with an additional ultracentrifugation step as opposed to the original protocol [8]. In the second amplification stage, one of 16 PCR reactions yielded a distinct amplification product (Figure 1). Sequencing showed a 188 nucleotide DNA fragment that was homologous in a nucleotide BLAST search with the capsid (P1) protein region of HPeV strains. It should be noted here that no specific Parechovirus diagnostics (serotyping of cell culture, RT-PCR) had been done because these viruses are known to occur almost exclusively in children, and this patient was an adult.

de Souza Luna LK, Baumgarte S, Grywna K, Panning M, Drexler JF, Drosten C - Virol. J. (2008)

Bottom Line: In this study we used VIDISCA, a virus identification method which has not yet been widely applied, on such an untyped virus isolate.We found a human parechovirus (HPeV) type 1 (strain designation: BNI-788st).We found that the novel strain was likely the result of recombination between structural protein genes of an ancestor of contemporary HPeV1 strains and nonstructural protein genes from an unknown ancestor, most closely related to HPeV3.In contrast to the non-structural protein genes of other HPeV prototype strains, the non-structural protein genes of BNI-788st and HPeV3 prototype strains did not co-segregate in bootscan analysis with that of other prototype strains.HPeV3 nonstructural protein genes may form a distinct element in a pool of circulating HPeV non-structural protein genes.

Affiliation: Clinical Virology Group, Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany. lkluna@yahoo.com

Abstract: Enteritis is caused by a spectrum of viruses that is most likely not fully characterised. When testing stool samples by cell culture, virus isolates are sometimes obtained which cannot be typed by current methods. In this study we used VIDISCA, a virus identification method which has not yet been widely applied, on such an untyped virus isolate.We found a human parechovirus (HPeV) type 1 (strain designation: BNI-788st). Because genomes of contemporary HPeV1 were not available, we determined its complete genome sequence. We found that the novel strain was likely the result of recombination between structural protein genes of an ancestor of contemporary HPeV1 strains and nonstructural protein genes from an unknown ancestor, most closely related to HPeV3. In contrast to the non-structural protein genes of other HPeV prototype strains, the non-structural protein genes of BNI-788st and HPeV3 prototype strains did not co-segregate in bootscan analysis with that of other prototype strains.HPeV3 nonstructural protein genes may form a distinct element in a pool of circulating HPeV non-structural protein genes. More research into the complex HPeV evolution is required to connect virus ecology with disease patterns in humans.