全基因组系统发育分析是对牛病毒性腹泻病毒 1 (BVDV-1) 和 BVDV-2 进行分型的最合适策略,但对许多实验室来说并不可行。因此,BVDV 分离株/毒株经常根据单个基因组区域(主要是 5' 非翻译区 (UTR))的分析进行亚型分类。然而,这种方法可能导致病毒分类不准确和/或缺乏统计支持。在此,我们描述了新型引物组,其扩增子可以轻松测序并用于 BVDV 亚型分型。最初,对先前被描述为最适合 BVDV 亚型分型的靶标的基因组区域进行了分析,以设计高覆盖率引物。对假定的扩增子进行了计算机分析,以确定它们是否适合重现 118 个 BVDV-1 和 88 个 BVDV-2 完整/近完整基因组 (CNCG) (GenBank) 的系统发育分类。该分析还考虑了可通过引物 HCV90-368、324-326 和 BP189-389 (5'UTR) 扩增的区域,这些引物已用于 BVDV 诊断和/或分类。在确认我们的引物扩增子与CNCG的分析之间的一致性后,我们优化了 RT-PCR 并评估了其扩增 BVDV 分离株/菌株的性能(BVDV-1 的 n = 35;BVDV-2 的 n = 33) 。在 BVDV 亚型分型的潜在目标中,我们为 NS3-NS4A (BVDV-1)(526 bp 扩增子)和 NS5B (BVDV-2)(728 bp)设计了高覆盖率引物。基于这些区域的分类完全再现了所有 CNCG 的子类型。另一方面,基于来自引物 HCV90-368、324-326 和 BP189-389 的推定扩增子的亚型分型显示出与 CNCG 分析相关的不一致。 NS3-NS4A 和 NS5B 引物还允许扩增所有测试的 BVDV 分离株/毒株。最后,我们建议在未来 BVDV 的系统发育和流行病学研究中使用这些引物。
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Novel genomic targets for proper subtyping of bovine viral diarrhea virus 1 (BVDV-1) and BVDV-2
Whole-genome phylogenetic analysis, the most suitable strategy for subtyping bovine viral diarrhea virus 1 (BVDV-1) and BVDV-2, is not feasible for many laboratories. Consequently, BVDV isolates/strains have been frequently subtyped based on analysis of single genomic regions, mainly the 5’ untranslated region (UTR). This approach, however, may lead to inaccurate and/or poorly statistically supported viral classification. Herein, we describe novel primer sets whose amplicons may be easily sequenced and used for BVDV subtyping. Initially, genomic regions previously described as the most suitable targets for BVDV subtyping were analyzed for design of high-coverage primers. 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) (GenBank). This analysis was also performed considering the region amplifiable by primers HCV90-368, 324-326 and BP189-389 (5’UTR), which have been used for BVDV diagnosis and/or classification. After confirming the agreement between the analyses of our primers’ amplicon versus the CNCGs, we optimized the RT-PCRs and evaluated their performance for amplification of BVDV isolates/strains (n = 35 for BVDV-1; n = 33 for BVDV-2). Among the potential targets for BVDV subtyping, we designed high-coverage primers for NS3-NS4A (BVDV-1) (526 bp amplicon) and NS5B (BVDV-2) (728 bp). The classification based on these regions fully reproduced the subtyping of all CNCGs. On the other hand, subtyping based on the putative amplicons from primers HCV90-368, 324-326 and BP189-389 showed disagreements in relation the CNCG analysis. The NS3-NS4A and NS5B primers also allowed the amplification of all BVDV isolates/strains tested. Finally, we suggest the use of these primers in future phylogenetic and epidemiological studies of BVDVs.