Genomic variation in macrophage-cultured European porcine reproductive and respiratory syndrome virus Olot/91 revealed using ultra-deep next generation sequencing

Abstract

Background: Porcine Reproductive and Respiratory Syndrome (PRRS) is a disease of major economic impact worldwide. The etiologic agent of this disease is the PRRS virus (PRRSV). Increasing evidence suggest that microevolution within a coexisting quasispecies population can give rise to high sequence heterogeneity in PRRSV.

Findings: We developed a pipeline based on the ultra-deep next generation sequencing approach to first construct the complete genome of a European PRRSV, strain Olot/9, cultured on macrophages and then capture the rare variants representative of the mixed quasispecies population. Olot/91 differs from the reference Lelystad strain by about 5% and a total of 88 variants, with frequencies as low as 1%, were detected in the mixed population. These variants included 16 non-synonymous variants concentrated in the genes encoding structural and nonstructural proteins; including Glycoprotein 2a and 5.

Conclusion: Using an ultra-deep sequencing methodology, the complete genome of Olot/91 was constructed without any prior knowledge of the sequence. Rare variants that constitute minor fractions of the heterogeneous PRRSV population could successfully be detected to allow further exploration of microevolutionary events.

Figure 1

Schema of the strategy used to analyse the next generation sequencing data. Raw data were quality filtered before being either mapped to a reference genome or assembled de novo. The filtered reads were then mapped back onto the newly constructed genome to search for rare variants from the quasispecies population.

Figure 2

PRRSV genomic variant spectra. (A) Note that the read depth is plotted on a log scale. The black solid curve depicts the coverage of the mapped Olot/91. The green bars show the locations of the Olot/91's variants when compared to the reference LV genome while the red bars are mixed variants in the viral population. Darker bars are variants that led to non-synonymous changes. (B) Genomic organisation of PRRSV. B- and T-cell epitopes distributed across the ORFs were represented with black horizontal bars.

Figure 3

Variant validation using Sanger sequencing and realtime PCR. (A) Minor peaks representative of the quasispecies' genotypes could be seen under the highlighted major peaks from Olot/91. *The 3% low frequency variant can be further confirmed by the raw chromatogram (Additional file 2: Figure S2). (B) Variants from the main Olot/91 and its associated quasispecies resulted in different Ct curves.