Whole Genome Analysis of Two Novel Type 2 Porcine Reproductive and Respiratory Syndrome Viruses with Complex Genome Recombination between Lineage 8, 3, and 1 Strains Identified in Southwestern China

Abstract

Recombination among porcine reproductive and respiratory syndrome viruses (PRRSVs) is thought to contribute to the emergence of new PRRSV variants. In this study, two newly emerged PRRSV strains, designated SCcd16 and SCya17, are isolated from lung tissues of piglets in Southwestern China. Genome comparative analysis reveals that SCcd16/SCya17 exhibit 93.1%/93.2%, 86.9%/87.0%, 85.3%/85.7%, and 83.6%/82.0% nucleotide similarity to PRRSVs JXA1, VR-2332, QYYZ and NADC30, respectively. They only exhibit 44.8%/45.1% sequence identity with LV (PRRSV-1), indicating that both emergent strains belong to the PRRSV-2 genotype. Genomic sequence alignment shows that SCcd16 and SCya17 have the same discontinuous 30-amino acid (aa) deletion in Nsp2 of the highly pathogenic Chinese PRRSV strain JXA1, when compared to strain VR-2332. Notably, SCya17 shows a unique 5-nt deletion in its 3’-UTR. Phylogenetic analysis shows that both of the isolates are classified in the QYYZ-like lineage based on ORF5 genotyping, whereas they appear to constitute an inter-lineage between JXA1-like and QYYZ-like lineages based on their genomic sequences. Furthermore, recombination analyses reveal that the two newly emerged PRRSV isolates share the same novel recombination pattern. They have both likely originated from multiple recombination events between lineage 8 (JXA1-like), lineage 1 (NADC30-like), and lineage 3 (QYYZ-like) strains that have circulated in China recently. The genomic data from SCcd16 and SCya17 indicate that there is on going evolution of PRRSV field strains through genetic recombination, leading to outbreaks in the pig populations in Southwestern China.

Keywords: evolutionary; lineage; porcine reproductive and respiratory syndrome virus; recombination.

Figure 1

The results of the IFA. (a) PAMs inoculated with SCcd16; (b) PAMs inoculated with SCya17; (c) positive control cells inoculated with SCwhn09CD; (d) uninfected negative controls for PAMs. Scale bar = 10 μm.

Figure 2

Phylogenetic trees based on ORF5 (a) and full-length genomic sequence (b) of SCcd16 and SCya17 isolates with 40 PRRSV reference strains available in GenBank. The two isolates in this study are labeled with “red triangle”. The representative strains are labeled with “black squares”. The phylogenetic tree is constructed by using the N-J method (1000 bootstrap) in MEGA6. Numbers along branches are bootstrap values. Scale bar indicates nucleotide substitute per site.

Figure 3

The sequence alignment of Nsp2, GP5, and 3′-UTR. (a) Two discontinuous amino acid deletions (481 and 533–562) in Nsp2 of SCcd16 and SCya17 (blue regions). Three discontinuous amino acid deletions (322–432, 483, and 504–522) in Nsp2 of NADC30 (gray regions); (b) multiple alignment of GP5 amino acid sequences of SCcd16 and SCya17 and seventeen PRRSV reference strains. Black boxes indicate the regions of signal peptide, two hypervariable regions (HVRs), and three transmembrane domains (TMs). Gray areas indicate the amino acid residues in the decoy epitope, primary neutralizing epitope (PNE), two T cell epitopes and the 3rd B cell epitope; (c) five continuous nucleotides deletion in 3′-UTR at positions 12 to 16 of SCya17 (gray region).

Figure 4

The predicted secondary structures of 3′-UTR sequences of SCcd16 (a) and SCya17 (b) are presented. The positions of the first and last nucleotide of both the 5′ and 3′-UTR are indicated. Schematic representation of secondary structures predicted by Mfold (http://mfold.rit.albany.edu/?q=mfold/RNA-Folding-Form) under default folding conditions and modified using RNAviz (http://rnaviz.sourceforge. net/).

Figure 5

Recombination analysis of strain SCcd16 and SCya17. Analysis is made use of a sliding window of 200-bp window and a 20-bp step. The y-axis indicates the percentage similarity between the query sequence and the reference sequences. (a) Genome scale similarity comparisons of SCcd16 (query) with JXA1 (blue), NADC30 (red), FJFS (QYYZ-like, green) and VR-2332 (gray); (b) genome scale similarity comparisons of SCya17 (query) with JXA1 (blue), NADC30 (red), XJzx1-2015 (QYYZ-like, green) and VR-2332 (gray). The supposed recombination regions are shown with different colors, and the recombination breakpoints are marked at the bottom with nucleotide sites and viral genome structure referenced to VR-2332; (c) phylogenetic trees based on different regions of SCcd16; (d) phylogenetic trees based on different regions of SCya17.