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The repeated emergence of new genetic variants of PRRSV-2, the virus that causes porcine reproductive and respiratory syndrome (PRRS), reflects its rapid evolution and the failure of previous control efforts. Understanding spatiotemporal heterogeneity in variant emergence and spread is critical for future outbreak prevention. Here, we investigate how the pace of evolution varies across time and space, identify the origins of sub-lineage emergence, and map the patterns of the inter-regional spread of PRRSV-2 Lineage 1 (L1)-the current dominant lineage in the U.S. We performed comparative phylogeographic analyses on subsets of 19,395 viral ORF5 sequences collected across the U.S. and Canada between 1991 and 2021. The discrete trait analysis of multiple spatiotemporally stratified sampled sets ( n = 500 each) was used to infer the ancestral geographic region and dispersion of each sub-lineage. The robustness of the results was compared to that of other modeling methods and subsampling strategies. Generally, the spatial spread and population dynamics varied across sub-lineages, time, and space. The Upper Midwest was a main spreading hotspot for multiple sub-lineages, e.g., L1C and L1F, though one of the most recent emergence events (L1A(2)) spread outwards from the east. An understanding of historical patterns of emergence and spread can be used to strategize disease control and the containment of emerging variants.

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.

Using georeferenced phylogenetic trees, phylogeography allows researchers to elucidate interactions between environmental heterogeneities and patterns of infectious disease spread. Concordant with the increasing availability of pathogen genetic sequence data, there is a growing need for tools to test epidemiological hypotheses in this field. In this study, we apply tools traditionally used in ecology to elucidate the epidemiology of foot-and-mouth disease virus (FMDV) in Uganda. We analyze FMDV serotype O genetic sequences and their corresponding spatiotemporal metadata from a cross-sectional study of cattle. We apply step selection function (SSF) models, typically used to study wildlife habitat selection, to viral phylogenies to show that FMDV is more likely to be found in areas of low rainfall. Next, we use a novel approach, a resource gradient function (RGF) model, to elucidate characteristics of viral source and sink areas. An RGF model applied to our data reveals that areas of high cattle density and areas near livestock markets may serve as sources of FMDV dissemination in Uganda, and areas of low rainfall serve as viral sinks that experience frequent reintroductions. Our results may help to inform risk-based FMDV control strategies in Uganda. More broadly, these tools advance the phylogenetic toolkit, as they may help to uncover patterns of spread of other organisms for which genetic sequences and corresponding spatiotemporal metadata exist.

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.