title: A nested PCR approach for unambiguous typing of pestiviruses infecting cattle.
abstract: An atypical pestivirus ('Hobi'-like pestivirus, putative bovine viral diarrhoea 3, BVDV-3) was identified firstly in contaminated foetal calf serum batches and isolated subsequently from an outbreak of respiratory disease in a cattle herd in Italy. The isolation of the novel pestivirus from animals affected clinically posed concerns about the validity of BVDV eradication programs, considering that 'Hobi'-like pestivirus (BVDV-3) is undetected or mistyped by the molecular diagnostic tools currently employed. In this paper, the development of a nested PCR (nPCR) assay for unambiguous typing of all bovine pestiviruses is reported. The assay consisted of a first-round amplification using an oligonucleotide pair which binds to conserved sequences located in the 5' untranslated region and capsid gene, followed by a heminested PCR using virus-specific forward primers. The assay performances were evaluated analytically, showing good sensitivity and specificity. By analysis of 100 BVDV-positive samples typed using a nPCR assay discriminating ruminant pestiviruses, five samples recognised previously as BVDV-2 were not typed when submitted to the new assay (n=2) or reacted as 'Hobi'-like pestivirus BVDV-3 (n=3). Sequence analysis of the first-round amplification products showed that the untyped strains were border disease viruses, whereas the other three strains were true 'Hobi'-like viruses. The development of a molecular assay able to identify simultaneously all bovine pestiviruses known currently will help warrant biosafety of live vaccines and other biological products and assess the molecular epidemiology of 'Hobi'-like pestivirus, thus leading to the improvement of the eradication programs through unambiguous typing of pestiviruses infecting cattle.
authors: Department of Veterinary Public Health, Faculty of Veterinary Medicine of Bari, Strada per Casamassima Km 3, 70010 Valenzano, Bari, Italy. n.decaro@veterinaria.uniba.it
authors: Nicola
authors: N
authors: Decaro
authors: Rossana
authors: R
authors: Sciarretta
authors: Maria Stella
authors: MS
authors: Lucente
authors: Viviana
authors: V
authors: Mari
authors: Francesca
authors: F
authors: Amorisco
authors: Maria Loredana
authors: ML
authors: Colaianni
authors: Paolo
authors: P
authors: Cordioli
authors: Antonio
authors: A
authors: Parisi
authors: Rossella
authors: R
authors: Lelli
authors: Canio
authors: C
authors: Buonavoglia
mesh_terms: D000818:Animals; D002417:Cattle / Q000821:virology*; D002418:Cattle Diseases / Q000175:diagnosis / Q000821:virology*; D001908:Diarrhea Viruses, Bovine Viral / Q000145:classification* / Q000235:genetics*; D007558:Italy; D018182:Pestivirus Infections / Q000175:diagnosis / Q000662:veterinary* / Q000821:virology*; D016133:Polymerase Chain Reaction / Q000379:methods; D012680:Sensitivity and Specificity