Bovine viral diarrhea virus (BVDV), a Pestivirus belonging to the family Flaviviridae, rapresents an economically important pathogen worldwide. BVDV can be divided into two species and classified in two biotypes, cytopathic (CP) or noncytopathic (NCP). Due to viral establishment before maturation of the fetal immune system, viral proteins are regarded as self-antigens and the calf remains immunotolerant to BVDV. BVDV-1 is widely distributed in Italy while BVDV-2 has been detected occasionally. In this study we investigated the viral variability in different body compartments and described the quasispecies diversity whitin an immunotollerant (PI) calf. The virus was isolated from a PI calf identified during a serological investigation in 2014. The full genome sequence was obtained by blood and used as reference genome. Primers were designed targeting three regions: the highly variable E2, the NS2-NS3 genes, containing the molecular biotype determinants as it is cleaved in two components in CP strains, and NS5. Viral RNA was extracted from different organs, retrotranscribed in single stranded cDNA and amplified. PCR fragments were processed for NGS amplicon sequencing with Nextera XT protocol and sequenced using Illumina MiSeq platform. The reads were analyzed with a resequencing approach in order to evaluate the tissue compartmentalization. Relationships among samples were evaluated by phylogenetic and network analyses. Even if the consensus sequences obtained by each tissue were highly similar, quasispecies was described evaluating the presence and the frequency of variants (SNPs). No clear accumulation of mutations from the NCP to CP strain was observed. Among all analyzed tissues, the thymus and the nervous tissues show the highest number of variants suggesting a different viral evolution. 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.
BVDV2 VIRAL COMPARTMENTALIZATION IN PI INFECTED CALF
Barbara Colitti;Maria Teresa Capucchio;NOGAROL, Chiara;Luigi Bertolotti;Sergio Rosati
2018-01-01
Abstract
Bovine viral diarrhea virus (BVDV), a Pestivirus belonging to the family Flaviviridae, rapresents an economically important pathogen worldwide. BVDV can be divided into two species and classified in two biotypes, cytopathic (CP) or noncytopathic (NCP). Due to viral establishment before maturation of the fetal immune system, viral proteins are regarded as self-antigens and the calf remains immunotolerant to BVDV. BVDV-1 is widely distributed in Italy while BVDV-2 has been detected occasionally. In this study we investigated the viral variability in different body compartments and described the quasispecies diversity whitin an immunotollerant (PI) calf. The virus was isolated from a PI calf identified during a serological investigation in 2014. The full genome sequence was obtained by blood and used as reference genome. Primers were designed targeting three regions: the highly variable E2, the NS2-NS3 genes, containing the molecular biotype determinants as it is cleaved in two components in CP strains, and NS5. Viral RNA was extracted from different organs, retrotranscribed in single stranded cDNA and amplified. PCR fragments were processed for NGS amplicon sequencing with Nextera XT protocol and sequenced using Illumina MiSeq platform. The reads were analyzed with a resequencing approach in order to evaluate the tissue compartmentalization. Relationships among samples were evaluated by phylogenetic and network analyses. Even if the consensus sequences obtained by each tissue were highly similar, quasispecies was described evaluating the presence and the frequency of variants (SNPs). No clear accumulation of mutations from the NCP to CP strain was observed. Among all analyzed tissues, the thymus and the nervous tissues show the highest number of variants suggesting a different viral evolution. 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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