Measuring How Recombination Re-shapes the Evolutionary History of PRRSV-2: A Genome-Based Phylodynamic Analysis of the Emergence of a Novel PRRSV-2 Variant

Abstract

While the widespread and endemic circulation of porcine reproductive and respiratory syndrome virus type 2 (PRRSV-2) causes persistent economic losses to the U.S. swine industry, unusual increases of severe cases associated with the emergence of new genetic variants are a major source of concern for pork producers. Between 2020 and 2021, such an event occurred across pig production sites in the Midwestern U.S. The emerging viral clade is referred to as the novel sub-lineage 1C (L1C) 1-4-4 variant. This genetic classification is based on the open reading frame 5 (ORF5) gene. However, although whole genome sequence (WGS) suggested that this variant represented the emergence of a new strain, the true evolutionary history of this variant remains unclear. To better elucidate the variant's evolutionary history, we conducted a recombination detection analysis, time-scaled phylogenetic estimation, and discrete trait analysis on a set of L1C-1-4-4 WGSs (n = 19) alongside other publicly published WGSs (n = 232) collected over a 26-year period (1995–2021). Results from various methodologies consistently suggest that the novel L1C variant was a descendant of a recombinant ancestor characterized by recombination at the ORF1a gene between two segments that would be otherwise classified as L1C and L1A in the ORF5 gene. Based on analysis of different WGS fragments, the L1C-1-4-4 variant descended from an ancestor that existed around late 2018 to early 2019, with relatively high substitution rates in the proximal ORF1a as well as ORF5 regions. Two viruses from 2018 were found to be the closest relatives to the 2020-21 outbreak strain but had different recombination profiles, suggesting that these viruses were not direct ancestors. We also assessed the overall frequency of putative recombination amongst ORF5 and other parts of the genome and found that recombination events which leave detectable numbers of descendants are not common. However, the rapid spread and high virulence of the L1C-1-4-4 recombinant variant demonstrates that inter-sub-lineage recombination occasionally found amongst the U.S. PRRSV-2 might be an evolutionary mechanisms that contributed to this emergence. More generally, recombination amongst PRRSV-2 accelerates genetic change and increases the chance of the emergence of high fitness variants.

Refining PRRSV-2 genetic classification based on global ORF5 sequences and investigation of their geographic distributions and temporal changes

“…In addition, we acknowledge that ORF5 only occupies ~4% of PRRSV genome, and phylogenetic analyses based on ORF5 sequences may not necessarily reflect PRRVS-2 evolution in other genomic regions, which also shapes PRRSV-2 genetic diversity. Interestingly, in a recent publication ( 44 ), analyses show that ORF5-based lineages and sublineages are relatively stable when extrapolated to the whole genome; sequences belonging to the same ORF5-based lineage remain largely clustered together when phylogenies are built with other portions of the genome ( 44 ). However, it should be noted that this observation may not necessarily always stand, especially when recombination of viruses occurs.…”

“…Nonetheless, ORF5-based lineages are a useful tool to label viruses that are phylogenetically related, though, of course, analysis of whole-genome sequences will broaden the ability to characterize PRRSV-2 genetic diversity and relatedness, including the ability to identify and characterize recombinant PRRSV-2 strains. Recombination between an MLV vaccine virus and a field virus or between two MLV vaccine viruses or between wild-type viruses in different geographic regions has been well documented ( 26 , 34 , 44 – 55 ). Although it makes sense at this stage to use ORF5 sequences for genetic classification because of the availability of large databases and the shorter turnaround time to determine ORF5 sequences, genetic classification based on whole-genome sequences should be explored in the future when more whole-genome sequences become available.…”

A recombinant porcine reproductive and respiratory syndrome virus type 2 field strain derived from two PRRSV-2-modified live virus vaccines

“…Moreover, whenever possible, reducing the commingling of piglets infected with distinct PRRSV strains can help reduce the emergence of new recombinant strains. These are examples of measures that could help reduce the opportunity presented by emerging virulent PRRSV recombinant strains such as the L1C variant RFLP 1-4-4 strain that emerged in the USA in recent years ( 48 , 49 ).…”

Phylogenetic-based methods for fine-scale classification of PRRSV-2 ORF5 sequences: a comparison of their robustness and reproducibility

“…A previous study used the inclusion criteria of >98% nucleotide identity to any of other sequences to define this clade. While this clade has a distinct recombination profile in genomic regions outside of the ORF5 gene, viruses belonging to this variant have >98% nucleotide identity at the whole genome level and form a distinct monophyletic clade within phylogenetic trees based on whole genomes or the ORF5 gene (36). This clade does not receive a distinguishing label in lineage classification, RFLP-typing, or the combination of both.…”