BVDV strain MRI103 loses diversity rapidly in culture

The pestivirus bovine viral diarrhoea virus (BVDV) is an important pathogen of cattle with significant impacts on animal health and welfare. The wide host range of this virus may lead to formation of virus reservoirs in other ruminant or wildlife species, presenting a concern for the long-term success of BVDV eradication campaigns. It is likely that the quasispecies nature of this RNA virus facilitates interspecies transmission by providing genetic plasticity. Understanding the spectrum of sequence variants present in BVDV persistently infected (PI) animals is therefore essential for studies of BVDV transmission. To analyse quasispecies diversity without amplification bias, we extracted viral RNA from serum of a PI cow, and from cell culture fluid after three passages of the same virus in culture, to produce cDNA without amplification. Sequencing of this material using Illumina 250bp paired-read technology produced full-length consensus sequences from both sources and demonstrated the quasispecies diversity of this BVDV type 1a field strain within serum and after culture. We report the distribution and diversity of over 800 single nucleotide polymorphisms and provide evidence for a loss of diversity after only three passages in cell culture implying that cultured viruses cannot be used to understand quasispecies diversity and may not provide reliable molecular markers for source tracing or transmission studies. Additionally, both serum and cultured viruses could be sequenced as a set of 25 overlapping PCR amplicons that demonstrated the same consensus sequences and the presence of many of the same quasispecies variants. The observation that aspects of the quasispecies structure revealed by massively parallel sequencing are also detected after PCR and Sanger sequencing suggests that this approach may be useful for small or difficult to analyse samples.