A search for RNA insertions and NS3 gene duplication in the genome of cytopathic isolates of bovine viral diarrhea virus

Calves born persistently infected with non-cytopathic bovine viral diarrhea virus (ncpBVDV) frequently develop a fatal gastroenteric illness called mucosal disease. Both the original virus (ncpBVDV) and an antigenically identical but cytopathic virus (cpBVDV) can be isolated from animals affected by mucosal disease. Cytopathic BVDVs originate from their ncp counterparts by diverse genetic mechanisms, all leading to the expression of the non-structural polypeptide NS3 as a discrete protein. In contrast, ncpBVDVs express only the large precursor polypeptide, NS2-3, which contains the NS3 sequence within its carboxy-terminal half. We report here the investigation of the mechanism leading to NS3 expression in 41 cpBVDV isolates. An RT-PCR strategy was employed to detect RNA insertions within the NS2-3 gene and/or duplication of the NS3 gene, two common mechanisms of NS3 expression. RT-PCR amplification revealed insertions in the NS2-3 gene of three cp isolates, with the inserts being similar in size to that present in the cpBVDV NADL strain. Sequencing of one such insert revealed a 296-nucleotide sequence with a central core of 270 nucleotides coding for an amino acid sequence highly homologous (98 percent) to the NADL insert, a sequence corresponding to part of the cellular J-Domain gene. One cpBVDV isolate contained a duplication of the NS3 gene downstream from the original locus. In contrast, no detectable NS2-3 insertions or NS3 gene duplications were observed in the genome of 37 cp isolates. These results demonstrate that processing of NS2-3 without bulk mRNA insertions or NS3 gene duplications seems to be a frequent mechanism leading to NS3 expression and BVDV cytopathology.

Caracterización genómica de la cepa cubana de efecto citopático ligero / Genomic characterization of the Cuban strain of light cytopathic effect

Durante la epidemia de neuropatía ocurrida en Cuba en los años 1992/1994 se aislaron del líquido cefalorraquídeo de pacientes agentes virales relacionados antigénicamente con los virus Coxsackie. Para establecer una función de estos virus en la etiopatogenia de la enfermedad se seleccionaron las cepas 47/93 IPK identificada como Coxsackie A9 y la cepa 44/93 IPK de efecto citopático ligero (ECP-L) y se realizó un estudio de sus características antigénicas mediante Western Blot. Se comprobó la relación antigénica entre ambas cepas y se demostró la ausencia de proteínas estructurales en su forma nativa en los agentes de ECP-L. A partir de estos resultados se plantea la posibilidad de que la persistencia sea un mecanismo por el cual estos virus participen en la etiopatogenia de la neuropatía epidémica en Cuba