BVDV-1 is a RNA virus (Pestivirus). BVDV in persistently infected animals may exist in two biotypes: non-cytopathic (ncp) and cytopathic (cp), with the latter causing fatal mucosal disease. Mutations leading from ncp to cp biotype include nucleotide substitutions or insertions of viral or cellular sequences. Since the viral population is mixed, detection of such mutation from NGS data is a bioinformatic challenge. Here we present a bioinformatic approach to detect such mutations. Supernatants of five isolates were processed as follows: RNA extraction, cDNA and ds-cDNA synthesis, and finally Nextera XT library preparation protocol. Part of the samples were also processed according to NEBNext Ultra RNA protocol. Libraries were sequenced on MiSeq. After adapter trimming, de novo assembly was performed with Trinity, generating contigs, that were characterized by a blast search. BVDV contigs were further blasted against Bos genus sequences, and bovine contigs were blasted against the drafted strain-specific viral genome. Read mapping against the generated contigs (bwa) was used to detect nucleotide substitutions. A full genome sequence has been retrieved for all the 5 samples, plus additional shorter contigs. In one sample, 3 contigs showed a 77-251bp insertion of the bovine ubiquitin C gene. In all the samples, a variable number of SNPs were detected in different gene regions. Most of the SNPs were detected in the NS2/NS3 region. In some cases, BVDV short contigs were composed by reassorted viral sequences. Blast analysis of short contigs led to the identification of probable insertions of both bovine and viral sequences. Furthermore, since BVDV mutation rate is high, using a strain-specific draft genome as a reference in NGS data analysis allows better results, such as SNP identification, since using a different reference increases variability, and thus tangle a correct identification of ncp/cp substitutions

Bovine viral diarrhea virus 1 (BVDV-1) mutation detection by NGS in virus pairs / F. Cerutti, C. Caruso, C. Luzzago, S. Lauzi, P.L. Acutis, L. Masoero, S. Peletto. ((Intervento presentato al 13. convegno International Conference on Molecular Epidemiology and Evolutionary Genetics of Infectious Disease tenutosi a Antwerp nel 2016.

Bovine viral diarrhea virus 1 (BVDV-1) mutation detection by NGS in virus pairs

C. Luzzago;S. Lauzi;
2016

Abstract

BVDV-1 is a RNA virus (Pestivirus). BVDV in persistently infected animals may exist in two biotypes: non-cytopathic (ncp) and cytopathic (cp), with the latter causing fatal mucosal disease. Mutations leading from ncp to cp biotype include nucleotide substitutions or insertions of viral or cellular sequences. Since the viral population is mixed, detection of such mutation from NGS data is a bioinformatic challenge. Here we present a bioinformatic approach to detect such mutations. Supernatants of five isolates were processed as follows: RNA extraction, cDNA and ds-cDNA synthesis, and finally Nextera XT library preparation protocol. Part of the samples were also processed according to NEBNext Ultra RNA protocol. Libraries were sequenced on MiSeq. After adapter trimming, de novo assembly was performed with Trinity, generating contigs, that were characterized by a blast search. BVDV contigs were further blasted against Bos genus sequences, and bovine contigs were blasted against the drafted strain-specific viral genome. Read mapping against the generated contigs (bwa) was used to detect nucleotide substitutions. A full genome sequence has been retrieved for all the 5 samples, plus additional shorter contigs. In one sample, 3 contigs showed a 77-251bp insertion of the bovine ubiquitin C gene. In all the samples, a variable number of SNPs were detected in different gene regions. Most of the SNPs were detected in the NS2/NS3 region. In some cases, BVDV short contigs were composed by reassorted viral sequences. Blast analysis of short contigs led to the identification of probable insertions of both bovine and viral sequences. Furthermore, since BVDV mutation rate is high, using a strain-specific draft genome as a reference in NGS data analysis allows better results, such as SNP identification, since using a different reference increases variability, and thus tangle a correct identification of ncp/cp substitutions
mag-2016
Settore VET/05 - Malattie Infettive degli Animali Domestici
Bovine viral diarrhea virus 1 (BVDV-1) mutation detection by NGS in virus pairs / F. Cerutti, C. Caruso, C. Luzzago, S. Lauzi, P.L. Acutis, L. Masoero, S. Peletto. ((Intervento presentato al 13. convegno International Conference on Molecular Epidemiology and Evolutionary Genetics of Infectious Disease tenutosi a Antwerp nel 2016.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2434/651697
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