2018
DOI: 10.1186/s13567-018-0603-1
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Bottlenecks in the transmission of porcine reproductive and respiratory syndrome virus (PRRSV1) to naïve pigs and the quasi-species variation of the virus during infection in vaccinated pigs

Abstract: This paper describes the results of two experiments regarding porcine reproductive and respiratory syndrome virus (PRRSV1): the first one studied the existence of bottlenecks in an experimental one-to-one model of transmission in pigs; while the second analysed the differences between viral quasi-species in vaccinated pigs that developed shorter or longer viraemias after natural challenge. Serum samples, as well as the initial inoculum, were deep-sequenced and a viral quasi-species was constructed per sample. … Show more

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Cited by 12 publications
(12 citation statements)
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“…The inoculum strain PEDV Calaf‐1 was sequenced using the Illumina Miseq Platform applying the protocol described for RNA viruses by Cortey et al., (2019). The method applied did not include any PCR amplification step.…”
Section: Methodsmentioning
confidence: 99%
“…The inoculum strain PEDV Calaf‐1 was sequenced using the Illumina Miseq Platform applying the protocol described for RNA viruses by Cortey et al., (2019). The method applied did not include any PCR amplification step.…”
Section: Methodsmentioning
confidence: 99%
“…After the amplification, the RT‐PCR products were purified using the Ilustra GFX PCR DNA and Gel Band Purification Kit (GE Healthcare) and were equimolar mixed, fragmented and sequenced in an external service (AC‐Gen), by next‐generation sequencing (NGS) using the Ion Torrent technology. The remaining 14 PEDV and the SeCoV complete genomes were obtained from the total RNA extraction, without any amplification step, applying a RNA virus‐specific tailor‐made NGS protocol (Cortey, Arocena, Pileri, Martín‐Valls, & Mateu, 2018; Vidal et al., 2018). NGS runs were performed by the Genomics Bioinformatics Service (SGB) of the UAB using an Illumina Miseq Platform.…”
Section: Methodsmentioning
confidence: 99%
“…To establish whether the passage in eggs introduced any viral mutations and therefore a potential bias, two oral swab samples from each virus strain with high viral titers were directly deep-sequenced before and after a 48-72 h passage. Viral quasi-species from original viral stocks and from egg allantoic fluid were inferred as previously described ( 52 ). Briefly, i) total RNA was extracted from allantoic fluid with TRIZOL LS (Ambion) and directly deep-sequenced without using primers; (ii) the genomic library was constructed for Illumina NGS using a commercial protocol and reagents (Thermo Fischer); (iii) MiSeq run of 2x250 bp was performed at Servei de Genòmica i Bioinformàtica (SGB, IBB, Barcelona); (iv) low quality reads (QC > 20) were trimmed using Trimmomatic ( 37 ); (v) quality reads were mapped against the corresponding HPAIV strain (H5N8 or H7N1) using the Burrows-Wheeler Aligner applying the BWA-MEM algorithm for long reads ( 53 ); (vi) variants were annotated with SnpSift ( 54 ) to determine the frequency of each nucleotide at each position of the reference genome; and (vii) viral quasi-species were constructed in fasta format.…”
Section: Methodsmentioning
confidence: 99%