Alvos genômicos para subtipagem de vírus da diarreia viral bovina 1 (BVDV-1) e BVDV-2

Abstract

Whole-genome phylogenetic analysis, the most suitable strategy for subtyping bovine viral diarrhea vírus 1 (BVDV) and BVDV-2, is not feasible for many laboratories. Consequently, BVDV isolates/strains have been frequently subtyped by the analysis of single genomic regions, mainly the 5' untranslated region (5'UTR). This approach, however, may lead to inaccurate and/or poorly statistically supported viral classifications. Herein, we describe novel primer sets whose amplicons may be easily sequenced and used for subtyping BVDV-1 and BVDV-2. Initially, genomic regions of BVDV-1 and BVDV-2, previously described as the most suitable targets for bovine pestivirus subtyping, were analyzed for the design of high-coverage primers. Then, the putative amplicons were analyzed in silico for their suitability to reproduce the phylogenetic classification of 118 (BVDV-1) and 88 (BVDV-2) complete/near complete genomes (CNCGs) available on GenBank. This analysis was also performed considering the region amplifiable by primers HCV90-368, largely used used for classification/subtyping of bovine pestiviruses. After confirming agreement between the analyses performed with the putative amplicons from our primers versus those from the CNCGs, we optimized the RT-PCR assays and evaluated their performance in the amplification of several BVDV isolates/strains from Brazil, Argentina and the United States. Among potential targets for bovine pestivirus subtyping, we designed high-coverage primers for NS3-NS4A (BVDV-1) (526bp amplicon) and NS5B (BVDV-2) (728bp). The phylogenetic classification based on these regions fully reproduced the subtyping of all CNCGs analyzed in the study. On the other hand, subtyping based on the putative amplicon from primers HCV90-368 showed four (BVDV-1) and twelve (BVDV-2) disagreements in relation to the CNCG classification. The NS3-NS4A and NS5B primers also allowed the amplification of all BVDV isolates/strains analyzed here. Finally, considering the phylogenetic analyses from our putative amplicons, as well as the performance of RT-PCR assays, we suggest the use of these primers in future phylogenetic and epidemiological studies of BVDV-1 and BVDV-2.