Bovine viral diarrhea virus (BVDV), a Pestivirus belonging to the family Flaviviridae, rapresents an economically important pathogen of cattle worldwide. BVDV can be divided into two species (1 and 2), and classified in two biotypes, cytopathic (CP) or noncytopathic (NCP) basing on its ability to show cytopathic effect. BVDV-1 is widely distributed in Italy while BVDV-2 have been detected occasionally. Due to viral establishment before maturation of the fetal immune system, viral proteins are regarded as self-antigens and the PI calf remain immunotolerant to the BVDV strain, allowing viral replication in all tissues and excretions without host detection. For this reason PI are considered primary propagators of the virus as they continuously shed large quantities of virus. The aim of this study is to investigate the viral variability in different body compartments and describe the quasispecies diversity of a PI cattle through analysis of three regions of viral genome. The virus was isolated in a previous work from a PI calf identified during a serological investigation in an outbreak of BVDV2 in Italy in 2014. Blood and tissue samples were collected during regular slaughter. The full genome sequence was obtained from cell colture with NGS sequencing using blood serum for the infection. Primers were designed in order to amplify three regions described in literature as more interesting: the highly variable E2 region, the region encompassing NS2-NS3 genes and NS5 region. Viral RNA was extracted from different organs, retrotranscribed in single stranded cDNA and used as template for PCR amplifications with Platinum HiFi Taq DNA polymerase. Amplified PCR fragments were processed for NGS amplicon sequencing with Nextera XT protocol. Paired-end sequencing was performed using Illumina MiSeq platform. The reads generated were analyzed with a resequencing approach (Geneious software) using the blood full genome sequence as reference in order to evaluate the compartmentalization in different tissues. Relationships among samples were evaluated by phylogenetic and network analyses. Even if the consensus sequences obtained by all the samples were highly similar, quasispecies was described evaluating the presence and the frequency of variants among all the reads. The results showed a high number of different variable sites, characterizing each tissue and proving the existence of different evolutionary processes within them. Interestingly the largest part of the identified variable positions showed non-synonymous substitutions. Variable sites suggest that viral population is more heterogeneous than expected, involving especially amino acids changes. The quasispecies analyses within PI cattle highlight the complex dynamics of BVDV pathogenesis and compatmentalization into the host and can increase the knowledge about viral evolution of BVDV in PI animals.
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