Potential novel N-glycosylation patterns associated with the emergence of new genetic variants of PRRSV-2 in the U.S.

Glycosylation of proteins is a post-translational process where oligosaccharides are attached to proteins, potentially altering their folding, epitope availability, and immune recognition. In Porcine reproductive and respiratory syndrome virus-type 2 (PRRSV-2), positive selection pressure acts on amino acid sites potentially associated with immune escape through glycan shielding. Here, we describe the patterns of potential N-glycosylation sites over time and across different phylogenetic lineages of PRRSV-2 to better understand how these may contribute to patterns of coexistence and emergence of different lineages. We screened 19,179 PRRSV GP5 sequences (2004-2021) in silico for potential N-glycosylated sites. The emergence of novel combinations of N-glycosylated sites coincided with past PRRSV epidemics in the U.S. For lineage L1A, glycosylation at residues 32, 33, 44, 51, and 57 first appeared in 2012, but represented >62% of all L1A sequences by 2015, coinciding with the emergence of the L1A 1-7-4 strain that increased in prevalence from 8 to 86% of all L1A sequences from 2012 to 2015. The L1C 1-4-4 strain that emerged in 2020 also had a distinct N-glycosylation pattern (residues 32, 33, 44, and 51). From 2020 to 2021, this pattern was responsible for 44-47% of the L1C sequences, contrasting to <5% in years prior. Our findings support the hypothesis that antigenic evolution contributes to the sequential dominance of different PRRSV strains and that N-glycosylation patterns may partially account for antigenic differences amongst strains. Further studies on glycosylation and its effect on PRRSV GP5 folding are needed to further understand how glycosylation patterns shape PRRSV occurrence

Porcine Reproductive and Respiratory Syndrome (PRRSV2) Viral Diversity within a Farrow-to-Wean Farm Cohort Study

Describing PRRSV whole-genome viral diversity data over time within the host and withinfarm is crucial for a better understanding of viral evolution and its implications. A cohort study was conducted at one naïve farrow-to-wean farm reporting a PRRSV outbreak. All piglets 3–5 days of age (DOA) born to mass-exposed sows through live virus inoculation with the recently introduced wildtype virus two weeks prior were sampled and followed up at 17–19 DOA. Samples from 127 piglets were individually tested for PRRSV by RT-PCR and 100 sequences were generated using Oxford Nanopore Technologies chemistry. Female piglets had significantly higher median Ct values than males (15.5 vs. 13.7, Kruskal–Wallis p < 0.001) at 3–5 DOA. A 52.8% mortality between sampling points was found, and the odds of dying by 17–19 DOA decreased with every one unit increase in Ct values at 3–5 DOA (OR = 0.76, 95% CI 0.61–0.94, p = 0.01). Although the within-pig percent nucleotide identity was overall high (99.7%) between 3–5 DOA and 17–19 DOA samples, ORFs 4 and 5a showed much lower identities (97.26% and 98.53%, respectively). When looking solely at ORF5, 62% of the sequences were identical to the 3–5 DOA consensus. Ten and eight regions showed increased nucleotide and amino acid genetic diversity, respectively, all found throughout ORFs 2a/2b, 4, 5a/5, 6, and 7.This study was funded by Boehringer Ingelheim Vetmedica Inc., PO 6101538739; the Swine Health Information Center (SHIC) as the funding agency for MSHMP; and by the Swine Disease Eradication Center (SDEC) at the University of Minnesota.info:eu-repo/semantics/publishedVersio

Porcine Reproductive and Respiratory Syndrome (PRRSV2) Viral Diversity within a Farrow-to-Wean Farm Cohort Study

Describing PRRSV whole-genome viral diversity data over time within the host and within-farm is crucial for a better understanding of viral evolution and its implications. A cohort study was conducted at one naïve farrow-to-wean farm reporting a PRRSV outbreak. All piglets 3-5 days of age (DOA) born to mass-exposed sows through live virus inoculation with the recently introduced wild-type virus two weeks prior were sampled and followed up at 17-19 DOA. Samples from 127 piglets were individually tested for PRRSV by RT-PCR and 100 sequences were generated using Oxford Nanopore Technologies chemistry. Female piglets had significantly higher median Ct values than males (15.5 vs. 13.7, Kruskal-Wallis p < 0.001) at 3-5 DOA. A 52.8% mortality between sampling points was found, and the odds of dying by 17-19 DOA decreased with every one unit increase in Ct values at 3-5 DOA (OR = 0.76, 95% CI 0.61-0.94, p = 0.01). Although the within-pig percent nucleotide identity was overall high (99.7%) between 3-5 DOA and 17-19 DOA samples, ORFs 4 and 5a showed much lower identities (97.26% and 98.53%, respectively). When looking solely at ORF5, 62% of the sequences were identical to the 3-5 DOA consensus. Ten and eight regions showed increased nucleotide and amino acid genetic diversity, respectively, all found throughout ORFs 2a/2b, 4, 5a/5, 6, and 7

Phylogenetic Structure and Sequential Dominance of Sub-Lineages of PRRSV Type-2 Lineage 1 in the United States

The genetic diversity and frequent emergence of novel genetic variants of porcine reproductive and respiratory syndrome virus type-2 (PRRSV) hinders control efforts, yet drivers of macro-evolutionary patterns of PRRSV remain poorly documented. Utilizing a comprehensive database of >20,000 orf5 sequences, our objective was to classify variants according to the phylogenetic structure of PRRSV co-circulating in the U.S., quantify evolutionary dynamics of sub-lineage emergence, and describe potential antigenic differences among sub-lineages. We subdivided the most prevalent lineage (Lineage 1, accounting for approximately 60% of available sequences) into eight sub-lineages. Bayesian coalescent SkyGrid models were used to estimate each sub-lineage’s effective population size over time. We show that a new sub-lineage emerged every 1 to 4 years and that the time between emergence and peak population size was 4.5 years on average (range: 2–8 years). A pattern of sequential dominance of different sub-lineages was identified, with a new dominant sub-lineage replacing its predecessor approximately every 3 years. Consensus amino acid sequences for each sub-lineage differed in key GP5 sites related to host immunity, suggesting that sub-lineage turnover may be linked to immune-mediated competition. This has important implications for understanding drivers of genetic diversity and emergence of new PRRSV variants in the U.S

The PRRSV-2 Saga: Evolutionary and Epidemiological Dynamics of Porcine Reproductive and Respiratory Syndrome Virus 2 in the United States

University of Minnesota Ph.D. dissertation. 2024. Major: Veterinary Medicine. Advisor: Kimberly VanderWaal. 1 computer file (PDF); 188 pages.Porcine reproductive and respiratory syndrome (PRRS) has inflicted substantial economic losses on the US swine industry over the past three decades, driven by the main etiological agent, PRRSV-2, which continuously evolves and spreads despite control efforts. Enhancing disease control measures necessitates an understanding of evolutionary dynamics of PRRSV. By leveraging virus genetic data and bioinformatics tools, this dissertation aims to unravel how PRRSV-2 has adapted, persisted, and disseminated within the U.S. Chapter 1 provides a background of the disease, the virus itself, and the existing knowledge gaps. Chapter 2 employs nationwide PRRSV-2 genetic and geographic data to uncover the patterns of disease spread and the dynamics of the virus population within the U.S. In Chapter 3, we conduct an in-depth investigation into between-farm transmission of an emerging PRRSV-2 sub-lineage within a specific, swine-dense region, using genetic and animal movement data. Chapter 4 utilizes data from the largest active PRRS monitoring program in the U.S. to forecast the potential emerging variants. Finally, in Chapter 5, we pinpoint the origin of a novel PRRSV-2 variant through an advanced analysis of whole-genome sequences.Chapter 2 revealed a cyclical pattern of sub-lineages contributing to the overall PRRSV-2 population and a shift across time in major hotspots for inter-regional spread. In Chapter 3, we narrow our focus to intra-regional spread by applying molecular epidemiological tools to construct farm-to-farm transmission networks for an emerging PRRSV-2 sub-lineage. These networks allowed us to examine factors contributing to between-farm spread and highlighted the significance of live animal movement, while recognizing that most transmission events remained unexplained. Both Chapters 2 and 3 characterize the periodic emergence of novel genetic variants of PRRSV-2, and anticipating such emergence events could aid in more strategic disease control. Chapter 4 demonstrated the utility of phylogenetic branching patterns and putative antigenic differences as early indicators of variant emergence. Finally, in Chapter 5, we expand the discussion of variant emergence from the ORF5 gene to the whole genome perspective. Analysis of whole-genome sequences unveiled a recombinant ancestor for an emerging variant of concern and emphasized the role of genomic recombination in PRRSV-2 evolution. Ultimately, our findings address novel insights into PRRSV-2 evolution and epidemiology at various geographic scales, providing beneficial guidance for targeted and early-response PRRS mitigation strategies in the U.S.Pamornchainavakul, Nakarin. (2024). The PRRSV-2 Saga: Evolutionary and Epidemiological Dynamics of Porcine Reproductive and Respiratory Syndrome Virus 2 in the United States. Retrieved from the University Digital Conservancy, https://hdl.handle.net/11299/262879

Potential Novel N-Glycosylation Patterns Associated with the Emergence of New Genetic Variants of PRRSV-2 in the U.S

Glycosylation of proteins is a post-translational process where oligosaccharides are attached to proteins, potentially altering their folding, epitope availability, and immune recognition. In Porcine reproductive and respiratory syndrome virus-type 2 (PRRSV-2), positive selection pressure acts on amino acid sites potentially associated with immune escape through glycan shielding. Here, we describe the patterns of potential N-glycosylation sites over time and across different phylogenetic lineages of PRRSV-2 to better understand how these may contribute to patterns of coexistence and emergence of different lineages. We screened 19,179 PRRSV GP5 sequences (2004&ndash;2021) in silico for potential N-glycosylated sites. The emergence of novel combinations of N-glycosylated sites coincided with past PRRSV epidemics in the U.S. For lineage L1A, glycosylation at residues 32, 33, 44, 51, and 57 first appeared in 2012, but represented &gt;62% of all L1A sequences by 2015, coinciding with the emergence of the L1A 1-7-4 strain that increased in prevalence from 8 to 86% of all L1A sequences from 2012 to 2015. The L1C 1-4-4 strain that emerged in 2020 also had a distinct N-glycosylation pattern (residues 32, 33, 44, and 51). From 2020 to 2021, this pattern was responsible for 44&ndash;47% of the L1C sequences, contrasting to &lt;5% in years prior. Our findings support the hypothesis that antigenic evolution contributes to the sequential dominance of different PRRSV strains and that N-glycosylation patterns may partially account for antigenic differences amongst strains. Further studies on glycosylation and its effect on PRRSV GP5 folding are needed to further understand how glycosylation patterns shape PRRSV occurrence

Porcine Reproductive and Respiratory Syndrome (PRRSV2) Viral Diversity within a Farrow-to-Wean Farm Cohort Study

Describing PRRSV whole-genome viral diversity data over time within the host and within-farm is crucial for a better understanding of viral evolution and its implications. A cohort study was conducted at one naïve farrow-to-wean farm reporting a PRRSV outbreak. All piglets 3–5 days of age (DOA) born to mass-exposed sows through live virus inoculation with the recently introduced wild-type virus two weeks prior were sampled and followed up at 17–19 DOA. Samples from 127 piglets were individually tested for PRRSV by RT-PCR and 100 sequences were generated using Oxford Nanopore Technologies chemistry. Female piglets had significantly higher median Ct values than males (15.5 vs. 13.7, Kruskal–Wallis p p = 0.01). Although the within-pig percent nucleotide identity was overall high (99.7%) between 3–5 DOA and 17–19 DOA samples, ORFs 4 and 5a showed much lower identities (97.26% and 98.53%, respectively). When looking solely at ORF5, 62% of the sequences were identical to the 3–5 DOA consensus. Ten and eight regions showed increased nucleotide and amino acid genetic diversity, respectively, all found throughout ORFs 2a/2b, 4, 5a/5, 6, and 7

Phylogenetically Distinct Near-Complete Genome Sequences of Porcine Reproductive and Respiratory Syndrome Virus Type 2 Variants from Four Distinct Disease Outbreaks at U.S. Swine Farms over the Past 6 Years

Porcine reproductive and respiratory syndrome virus (PRRSV) continues to mutate, causing disruptive PRRS outbreaks in farms that lead to reproductive failure and respiratory disease-associated mortality. We present four new PRRSV type 2 variants in the United States belonging to four distinct orf5 sublineages within lineage 1