Genetic typing of bovine viral diarrhoea virus in cattle on Irish farms

Abstract

Bovine viral diarrhoea (BVD) is a pestivirus infection of cattle and other domesticated and free living animals. Two genotypes, bovine viral diarrhoea virus type 1 (BVDV-1) and bovine viral diarrhoea virus type 2 (BVDV-2) are recognised with 17 subgroups of BVDV 1 and 3 subgroups of BVDV 2. However, little is known about the genotypes of BVDV circulating in Irish cattle herds and the aim of this project was to characterise the BVD virus types archived at the Irish Equine Centre. A database of all BVDV positive diagnostic samples archived at the Irish Equine Centre was compiled. This consisted of information relating to in excess of one thousand blood and milk samples submitted for analysis between 2011 and 2014. A randomized panel of 375 virus positive samples representative of location, type (beef or dairy), gender, age, breed, year and month of detection was selected for genotyping. Total nucleic acid was extracted from all samples and the RNA concentration was measured using a biophotometer. Forward and reverse primers were synthesised to target two regions consistently conserved among all pestiviruses, a highly conserved 288bp portion of the 5’ untranslated region (UTR) and a more variable 428bp segment of the N-terminal autoprotease (Npro) gene. A one-step reverse transcriptase polymerase chain reaction (RT-PCR) method was specifically optimized to amplify both targets. Amplified products were gel purified, sequenced and aligned with 5’UTR and Npro gene sequences representative of all known BVDV genotypes and sub-genotypes. The genotype and subgenotype was established for 325 field viruses and one commercial vaccine viral strain based on the analysis of at least one genomic region. BVDV-1a was the prominent subgenotype (n=317) with BVDV-1b (n=6), BVDV-1d (n=1) and BVDV-1e (n=1) also observed. A number of nucleotide sequence alignments and phylogenetic trees were constructed to represent the relationship between Irish field viruses and reference strains representative of all known BVDV genotypes and subgenotypes. Evidence of both herd specific clustering and circulation of multiple strains within herds was observed; however no evidence of county specific clustering was observed. A number of completely conserved nucleotide regions were observed and the 5’UTR was found to be more conserved than the Npro fragment analysed. The results of this study provide a detailed and comprehensive insight into the genetic diversity of BVDV in circulation within Irish cattle herds and will act as a baseline reference for future BVDV genotyping studies