Bovine viral diarrhea virus (BVDV), a Pestivirus in the family Flaviviridae, is an economically important pathogen of cattle worldwide. The primary propagators of the virus are immunotolerant persistently infected (PI) cattle, which shed large quantities of virus throughout life. However, up to now very little was known regarding the viral population structure in these animals.The goal of this study was to describe the extent and distribution of BVDV variability in multiple body compartments of naturally infected PI cattle derived from a Western Canadian beef herd using next generation sequencing techniques.
Materials and Methods
A cow-calf herd with a no vaccination policy experienced an introduction of BVDV in their BVDV naïve herd during, or just shortly following the 2013 breeding season. This season is 2 months during summer as is common in Alberta. Due to this short timeframe the fetuses were in approximately the same state of their development when they became infected with BVDV. In the fall of 2014 a cohort of 26 PI calves were identified, all likely infected from the same source.
The calves were purchased, euthanized, and a variety of fluids and tissues including serum, mesenteric lymphnodes and obex were collected. PCR amplification of the viral genome was followed by sequencing on an Illumina MiSeq. Genomes were assembled using MIRA against a reference strain and variant profiles were constructed at the nucleotide and amino acid levels. These profiles were compared to identify putative host- and tissue-specific mutations.
Parts of the genome that are known to accommodate rapid changes (E2, NS2-3) also had a large variability in the studied PIs.Surprisingly, we found a substantial variability in certain regions of NS5b, a region generally considered as very conserved. Whether this variability is a characteristic of this specific virus strain or a more general finding in PI-derived BVDV using our analysis techniques warrants further - investigation. Variant analysis shows hot-spots of variability in the E2, NS3, and NS5B regions of the genome. These areas tend to differ from the group mean variant profile more often than other genomic regions.
There appears to be a stretch of very variable amino acid positions at position 1710-1750 of the amino acid sequence. These mutations include a variety of frame shifts and single amino acid changes. Although not previously documented, given that these mutations are occurring in the NS3 gene it is likely that they can contribute to biotypic conversion from noncytopathic to cytopathic strains and the subsequent induction of fatal mucosal disease.
Further work to understand the phenotypic importance of these observations is needed, but it is clear that BVDV exhibits a remarkable variability within and between PI animals.
A major driver of evolutionary change of viruses is the immune system, however, despite the absence of an acquired immune response against BVDV in these PI cattle, there are strong indications of viral variability that is of clinical and epidemiological importance. Our results demonstrates that the virus proteins can be modified in their amino acid structure leading to new strains and changes in their pathogenic properties. Understanding the extend and nature of these genetic changes will enable us to improve our responses through optimization of future vaccine development.