Beyond the whole genome consensus: unravelling of PRRSV phylogenomics using next generation sequencing technologies

Abstract

The highly heterogeneous porcine reproductive and respiratory syndrome virus (PRRSV) is the causative agent responsible for an economically important pig disease with the characteristic symptoms of reproductive losses in breeding sows and respiratory illnesses in young piglets. The virus can be broadly divided into the European and North American-like genotype 1 and 2 respectively. In addition to this intra-strains variability, the impact of coexisting viral quasispecies on disease development has recently gained much attention; owing very much to the advent of the next-generation sequencing (NGS) technologies. Genomic data produced from the massive sequencing capacities of NGS have enabled the study of PRRSV at an unprecedented rate and details. Unlike conventional sequencing methods which require knowledge of conserved regions, NGS allows de novo assembly of the full viral genomes. Evolutionary variations gained from different genotypic strains provide valuable insights into functionally important regions of the virus. Together with the advancement of sophisticated bioinformatics tools, ultra-deep NGS technologies make the detection of low frequency co-evolving quasispecies possible. This short review gives an overview, including a proposed workflow, on the use of NGS to explore the genetic diversity of PRRSV at both macro- and micro-evolutionary levels.

Keywords: PRRSV; Phylogenomics; Quasispecies; Ultra-deep next generation sequencing.

Fig. 1

Phylogenetic analysis of whole genome PRRSV strains. Whole genome sequences from 336 PRRSV isolates currently available in the Genbanks were aligned with the program MUSCLE. The unrooted maximum likelihood phylogenetic tree with 500 bootstrap replications was constructed using MEGA5 and the tree drawn with iTOL. Accession number of each sequence precedes the isolate's name. The two main clades separate the virus into the European genotype 1 and North-American-like/Asian genotype 2.

Fig. 2

Proposed workflow for NGS analysis of PRRSV. The workflow includes a wet laboratory section where samples are prepared and sequenced; and a bioinformatics analysis section where raw sequencing data are pre-processed before undergoing various downstream analyses.

Fig. 3

Sequence Entropy of whole genome PRRSV strains. Multiple whole genome sequence alignment of PRRSV genotype 1 and 2 was generated with MUSCLE and their respective entropy calculated with the molecular evolutionary program, Hyphy. The plots were constructed with 100 nt sliding window. In addition to common reported regions of variability, strains from PRRSV genotype 1 also exhibit higher degree of heterogeneity at the 3′ end of the genome.