US-UK COLLAB: DRIVERS OF DIVERSITY AND TRANSMISSION OF CO-CIRCULATING VIRAL LINEAGES IN HOST META-POPULATIONS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: AFRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 1019891
• Grant No.: 2019-67015-29918
• Cumulative Award Amt.: $2,395,000.00
• Proposal No.: 2019-03034
• Project Start Date: Aug 15, 2019
• Project End Date: Aug 14, 2024
• Grant Year: 2019
• Program Code: [A1222]- Ecology and Evolution of Infectious Diseases

Recipient Organization
UNIV OF MINNESOTA
(N/A)
ST PAUL,MN 55108

Performing Department
Veterinary Population Medicine

Non Technical Summary
Genetic variation and the presence of multiple co-circulating genetic strains, or "lineages," is the norm for many pathogens, particularly for rapidly evolving viruses.Interactions between different lineages can occur if infection by one lineage confers partial cross-immunity to a related lineage, thus creating the potential for immune-mediated competition amongst co-circulating lineages. Partial cross-immunity may help explain epidemiological patterns such as fluctuations in the frequency of different viral lineages through time as well as immune-mediated selection and emergence of new genetic variants. This study focuses on the transmission dynamics of genetically diverse pathogens, particularly in the context of limited cross-immunity, immune-mediated selection, and host population structure. Using a highly diverse virus,porcine reproductive and respiratory syndrome virus (PRRSV), which is hyperendemic in pig populations in the U.S., this project investigates how continual viral evolution and variable cross-immunity among genetic variants drives co-circulation of different viral lineages, and in turn shapes the distribution of disease across host populations.Given the rich data available in livestock host-pathogen systems, this project will provide new insight into multi-strain ecological theory.In this study, the researchers will first evaluate how partial immunity influences evolution within and between hosts using an experimental approach, where quasi-species evolution will be tracked via next generation sequencing. Second, commercial swine farms with variable levels of immunity (from either past infection or vaccination) will be monitored longitudinally to quantify viral evolution and invasion success of new PRRSV introductions in field settings. Third, using a dataset of exceptional spatiotemporal resolution available through the Morrison Swine Health Monitoring Project, the researchers will quantify how spatial heterogeneities in cross-immunity (based on previous infection and/or vaccination) and population connectivity influence co-circulation and interactions among viral lineages. Finally, mathematical and computational models will be used to elucidate how spatial and temporal variation in cross-immunity influences evolutionary dynamics and the persistence and sequential dominance of different co-circulating PRRSV lineages.The researchers will apply innovative analytical tools, such as phylodynamic and network modeling, to analyze how spatial heterogeneity in cross-immunity may shape the spread of different PRRSV lineages. These analytical approaches also provide a framework for researching landscapes of cross-immunity in other host-pathogen systems.This project also exploreshow immunity-driven factors, such as vaccination or prior infection, may impose selective pressures and alter transmission dynamics for multi-lineage pathogens.Thus, this study advances our understanding of the evolution, transmission and persistence of highly diverse viruses, and will bring new insights for dynamics of other human and animal pathogens.

Animal Health Component

50%

Research Effort Categories

Basic

50%

Applied

50%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
311	3510	1170	50%
311	3510	1101	20%
311	3510	1090	20%
311	3510	2090	10%

Knowledge Area
311 - Animal Diseases;

Subject Of Investigation
3510 - Swine, live animal;

Field Of Science
2090 - Statistics, econometrics, and biometrics; 1090 - Immunology; 1101 - Virology; 1170 - Epidemiology;

Keywords

evolution

immunity

multi-strain dynamics

phylodynamics

quasi-species

Goals / Objectives
Our study will focus on the transmission dynamics of genetically diverse pathogens, particularly in the context of limited cross-immunity, immune-mediated selection, and host population structure. For genetically diverse pathogens, ecological interactions between different genetic lineages can occur if infection by one lineage confers partial cross-immunity to a related lineage, thus creating the potential for immune-mediated competition amongst co-circulating lineages. Partial cross-immunity is thought to be a factor that helps explain epidemiological patterns such as cyclic fluctuations in the frequency of different viral lineages through time as well as immune-mediated selection and emergence of new genetic variants. In addition, host population structure can generate spatial variation in herd immunity that could influence the invasion success and geographic spread of new variants. Despite the importance of multi-lineage dynamics in shaping disease spread and evolution, these concepts have rarely been applied to non-human pathogens. Given the rich data available in livestock host-pathogen systems, our project provides new insight into multi-lineage ecological theory. Using a highly diverse virus,porcine reproductive and respiratory syndrome virus (PRRSV), which is hyperendemic in pig populations in the U.S., we investigate how continual viral evolution and variable cross-immunity among genetic variants drives co-circulation of different viral lineages, and in turn shapes disease distributions across host metapopulations. Our overarching goal is todetermine how evolutionary dynamics, landscape of variable cross-immunity, and connectivity of the host population determines the distribution, co-circulation, and maintenance of genetic diversity of PRRSV lineages.We will investigate this question through four specific goals:Evaluate how partial immunity influences evolution within and between individualsObjective 1a: Use anin vivoexperimental approach to investigate the extent to which heterologous immunity drives viral evolution within-hosts and throughout between-animal infection chains using NGSObjective 1b: Conduct anin vitroexperimental study to track quasi-species evolution and theimpacts of humoral response to vaccination on viral evolution through identifying viral mutants generated in PAM virus cultures under antibody pressure.Objective 1c: Quantify immunological epitopes (i.e., antigenic determinant/antibody binding sites) important for mediating cross-immunityObjective 1d: Developwithin/between host model to forward-simulate evolutionary processes to test contrasting hypotheses about whether within-host immune selection drives quasi-species evolution vs. partial cross-immunity creates susceptibility bottlenecksAssess the impact of cross-immunity on within-herd viral evolution and invasion success of new PRRSV introductions into host populationsObjective 2a: Conduct longitudinal monitoring of farms to quantify evolution and turnover of lineages within a population in field settingsObjective 2b: Quantify the extent of genetic dissimilarity between outbreak strains to the immune profile of the herd (vaccine strain or previous outbreak strain)Objective 2c: Calculate the maximum relative abundance achieved by the new variant within the viral population as a metric of its invasion success.Objective 2d: Conduct survival analyses on the time elapsed from the original outbreak to the new introduction in order to assess whether genetically dissimilar PRRSV variants are able to invade the population earlier.Quantify how landscapes of variable cross-immunity and population connectivity influence co-circulation and interactions among viral lineagesObjective 3a: Construct temporal network autocorrelation models (TNAMs) to analyze the effect of a farm's previous infection by different lineages (or vaccination) on the occurrence of outbreaks of a particular lineage.Objective 3b:Perform phylogenetic GLM analyses to assess drivers of viral migration rates (i.e., transitions) between locationsObjective 3c: Develop, validate, and apply phylodynamic methods thatincorporate the time-varying effect of the immune profile in the source and recipient localesInvestigate the conditions in which cross-immunity landscapes lead to evolution, persistence, and sequential dominance of co-circulating lineagesObjective 4a: Quantify temporal dynamics of PRRSV lineagesObjective 4b: Develop discrete multi-strain network modelObjective 4c: Develop multi-strain model with evolutionObjective 4d: Fit models to observed data to compare hypotheses about maintenance of viral diversity

Project Methods
Aim 1. Evaluate how partial immunity influences viral evolution within and between hosts1.1 EffortsExperiments and lab work will be performed at UMN (led by Rovira/Schroeder/Cheeran), and the data analysis will be performed jointly by the RI and UMN (led by Schroeder/Lycett).1.2 Expected Results and EvaluationIf within-host immune selection drives quasi-species evolution, then we expect NGS to reveal greater genetic diversity/phylogenetic distance relative to the initial inoculum in vaccinated pig infection chains than in non-vaccinated contexts.If partial cross-immunity creates susceptibility bottlenecks in between-animal infection chains, then we expect to see greater genetic divergence across entire vaccinated infection chains relative to non-vaccinated chains, but no difference in quasi-species evolution within single hosts after infection is established.In addition, we expect to see more non-synonymous mutations and resulting epitopes in vaccinated hosts and chains than in non-vaccinated contexts.MethodsTracking quasi-species evolutionin vivoduring experimental infections: Virus evolution will be assessed post-challenge in vaccinated/unvaccinated pigs.Following established protocols, piglets will be infected at day 28 via intramuscular injection. Post-inoculation, sera samples will be collected at 7 and 14 days. Day 14 sample will serve as the intramuscular inoculum for the next piglet.Total RNA will be extracted and sequenced from serum samples. We will also quantify the epitope immune pressure imposed by the antibody responses in vaccinated vs non-vaccinated pigs.Tracking quasi-species evolutionin vitro: Impacts of humoral response to vaccination on viral evolution will be assessedin vitroby identifying viral mutants generated in PAM virus cultures under antibody pressure. Experimental groups include: vaccinated serum, non-vaccinated serum, and no serum, with each done in triplicate. RNA will be analyzed by NGS to determine viral quasi-species selected by antibody pressure imposed by vaccinated, non-vaccinated, or no serum.1.4 Data analysisWe will reconstruct viral haplotypes, and also use techniques such as Phyloscanner to assess within- and between-host viral diversity by creating phylogenetic trees of sets of reads/contigs from all the animals in the chains. Additionally, we will use a detailed within/between host model to generate simulated sequence data and perform forward simulations.By fitting simulation models with different assumptions to the experimental data, we will be able assess which hypotheses is most supported: within-host immune selection drives quasi-species evolution vs. partial cross-immunity creates susceptibility bottlenecks.Aim 2: Impact of cross-immunity in field settingsEffortsLongitudinal monitoring of farms and lab work be led by UMN (Corzo/Schroeder), and data analysis will be performed jointly by RI and UMN (Corzo/Schroeder/Lycett/Doeschl-Wilson).2.2 Expected Results and EvaluationWe expect more rapid viral evolution in herds with partial cross-immunity (either through vaccination, LVI or previous natural infection) than in fully susceptible herds, and that populations with partial cross-protection will exhibit more non-synonymous mutations.If new outbreaks occur primarily through the introduction of new variants, then the length of time between sequential outbreaks will be longer if both outbreaks are caused by the same genetic lineage.Genetically dissimilar viruses may be able to invade earlier and achieve higher relative abundance within the viral population due to limited cross-protection.2.3 MethodsFarms (3 of each management type) will be enrolled in the study and sampled monthly for 30 months. Piglets are the age group that maintain PRRSV endemicity within a farm. Therefore, we will focus on sampling processing fluids from piglets. Samples will be screened via RT-PCR, and positive samples will undergo NGS as described in Aim 1.2.4 Data analysisTo compare the evolutionary dynamics of PRRSV based on the immune profile of the farm (vaccinated, LVI, or clean), data analysis will be performed as described in Aim 1 to assess phylogenetic patterns, molecular evolution, andsynonymous/non-synonymous mutations. In addition, if new variants are introduced in any of the farms, we will quantify the degree of genetic dissimilarity and calculate the maximum relative abundance achieved by the new variant within the viral population as a metric of its invasion success.Aim 3.Quantify how landscapes of cross-immunity and population connectivity influence co-circulation and interactions among viral lineages3.1 EffortsNetwork analysis will be led by VanderWaal (UMN) and phylodynamic modeling by Lycett (RI), with input in both cases from swine experts from RI and UMN (Corzo/Rovira/Doeschl-Wilson).3.2 Expected results and EvaluationWe expect thatgenetic diversity is influenced by geography (genetic drift-enhancing genetic differences across space) and population connectivity (reducing genetic differences across space).We further hypothesize that a given viral lineage is less likely to disseminate into areas where herd cross-immunity is high (through vaccination or previous infection).Diversifying selection is expected to be observed in viral clades closely related to commercial modified-live vaccines.3.3 Data analysisNetwork autocorrelation models:For each lineage, we will construct temporal network autocorrelation models (TNAMs) to analyze the effect of a farm's previous infection by different lineages (or vaccination) on the occurrence of outbreaks of a particular lineage.Phylodynamic models:Bayesian phylodynamic models will be inferred using BEAST. Frequencies and timings of invasions and emergence of new phylogenetic clades will be estimated, along with inference of the distribution of clades and viral genetic diversity through time in different locations.Since the immune profile (at each location) is a time varying multivariate quantity, we will infer time-varying rates matrices, and then estimate the time-varying contributions of the potential covariates.Aim 4. Investigate the conditions in which cross-immunity landscapes lead to evolution, persistence, and sequential dominance of co-circulating lineages4.1 OverviewUMN (VanderWaal/Craft) and RI (Kao/Lycett/Doeschl-Wilson) will develop mathematical simulation models jointly, with input from team members leading Aims 1-3, and RI will guide model fitting with their team's expertise in ABC-SMC.4.2 Expected results and EvaluationWe will test two contrasting hypotheses about the maintenance of multiple lineages within the population: a) persistence of multiple strains can be explained by heterogeneity in population structure and frequency dependent selection based on cross-immunity alone; or b) emergence of new genetic types due to continuous evolution is necessary to maintain multi-lineage dynamics.Cycles of sequential dominance of lineages follow a predictable patternUse of vaccination leads to emergence and/or dominance of non-vaccine lineages4.3 MethodsWe will use a partial immunity model with ≥4 strains to model the co-circulation of multiple lineages within metapopulations/networks. Models will be adopted for metapopulation or network-based contexts. The base model will be a multi-lineage model with no evolution, but based on results from previous aims, we will configure a stochastic individual-based model which can generate phylogenetic trees and sequence evolution This model will be fit to data on frequency of breaks at the farm-level (Aim 2 and 3) and periodicity, spatial distribution, and sequential dominance of different lineages at the landscape-level using ABC-SMC methodology. We will obtain feedback on model development from industry partners as quarterly webinars, and the model code will also be made available publicly.

Progress 08/15/19 to 08/14/24

Outputs
Target Audience:- Scientists and epidemiologists • Government and policy making agencies • Project participants from study regions for the longitudinal sampling study • Swine veterinarians and producers • Industry players (swine production companies, pharmaceutical companies, biosecurity companies etc.) Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project provided training opportunities to nine different post-docs,four graduate students, and one DVM student. The project also supported six different professional scientific staff members. All trainees received one-on-one mentoring with their supervisors, participated in monthly EEID research team meetings, and presented as graduate and departmental seminars in the College of Veterinary Medicine. These trainees had opportunities to present at national and international conferences, including submission of 55+ abstracts at conferences such is the International Society of Veterinary Epidemiology and Economics (ISVEE), Conference for Research Workers in Animal Diseases (CRWAD), North American PRRS Symposium, Epidemics, etc.In addition, several post-docs attended workshops for professional development through the SISMID program (Summer Institute in Statistics and Modeling in Infectious Diseases).Students and post-docs also had the opportunity to engage in international exchanges at the Roslin Institute, University of Edinburgh. How have the results been disseminated to communities of interest? Scientific dissemination:This project resulted in 21+ published/submitted peer-reviewed manuscripts, and at least 55+ presentations at conferences. Stakeholder dissemination:Many of these conferences we presented at have both an academic and industry audience.For example, the majority of attendees at both the American Association of Swine Veterinarians (3 presentation) and the Allen D. Leman Swine Conference (12 presentations) are practicing swine veterinarians, scientific and technical representatives of vaccine manufacturing companies, though there are some faculty and graduate students at these events as well.Additionally, we contributed 13 lay articles toNational Hog Farmer, an online magazine geared for swine producers. We also wrote 14 "Science pages" for the Morrison Swine Health Monitoring Project, which reaches >200 industry professionals, and were invited guests to speak about our research on four industry podcasts (>3.2k subscribers). Webtools and R Packages(see Other Products) We also have made advances of this research more accessible to end-users by creating the PRRSLoom webtool for end-users to classify their own sequences into phylogenetic lineages described by this work.The webtool contains a variety of additional educational information to help end-users interpret the genetic classifications.In parallel, we developed a new R Package to analyze quasi-species diversity for long-read sequences.This was necessary, as we discovered that nearly all quasi-species analysis pipelines are based on short-read Illumina sequencing and are not appropriate for long-read data. This pipeline was rigorously tested on PRRSV as well as four other RNA viruses, and an R package was created so it can be used by other researchers aiming to conduct quasi-species analysis with long-read sequencing technology. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Impact statement: This project aimed to understand multi-strain dynamics of RNA viruses, focusing on PRRSV-2 in commercial pig production.One of the key Changes in Action arising from this project was the widespread adoption of the lineage-based nomenclature proposed by our research team to describe the multiple co-circulating lineages within the United States and globally. This not only has allowed us to better describe PRRSV-2 multi-strain dynamics present in the U.S., but adoption of this nomenclature has improved communication between research labs, diagnostic labs, disease monitoring programs, and field veterinarians. All major diagnostic laboratories now employ lineages as part of everyday reporting.While the research advances and outputs of this project are numerous, one significant Change in Knowledge arising from this project that we were able to experimentally demonstrate that vaccine-use can drive PRRSV-2 evolution. To our knowledge, our work provides the first causal demonstration of this phenomenon.A review paper written for a multi-disciplinary audience was published in Nature Ecology and Evolution outlining key concepts underpinning multi-strain dynamics from immunological, evolutionary and methodological perspectives. Objective 1:Evaluate how partial immunity influences evolution within and between individuals Sub-objectives of this aim includedin vivoandin vitroexperiments to investigate the extent to which heterologous immunity drives viral evolution within-hosts and throughout between-animal infection chains, and quantification of immunological epitopes (i.e., antigenic determinant/antibody binding sites) important for mediating cross-immunity. Over the 84-day pig-to-pig infection chain experiment, genomes from unvaccinated pigs remained genetically similar to the challenge virus (mean distance to inoculum = 0.5%) and to each other (mean within-group distance = 0.3%). In contrast, vaccinated groups were more genetically different from the initial inoculum (mean distance to inoculum = 2.8-3.6%). 15-40% of mutations occurred in immunologic epitopes. Phylogenetic analysis showed samples from unvaccinated pigs clustered near the tree's root (the original inoculum), while those from vaccinated groups formed ladder-like structures that were progressively more distant from the inoculum, indicating directional evolution under selection pressure. This selection was also clear in vaccinated groups at the viral quasispecies level. Many of the sites found to have mutated during the infection chain experiment parallel those found in larger-scale analyses of antigenic evolution conducted by our team, suggesting that antigenic selection pressures and immune-mediated competition may indeed mediate the turnover in the dominant lineage of PRRSV-2 through time. In addition, we conducted anin vitroanalysis of the immunogenicity of Epitope A. ELISA results showed that antibody responses to epitope A were dominant and sequence-specific in vaccinated animals. These data suggest differential induction of antibody responses to GP5 epitope A by vaccine strains and field isolates, which likely results from alternate signal peptide cleavage in the two vaccine strains studied here. An R package developed for the needs of our analysis was made publicly available on CRAN and is submitted for publication at Genome Biology.The package fills a need for better bioinformatic tools to study viral quasi-species evolution, as evidenced by the fact that it has already been downloaded 1000 times. Objective 2.Assess the impact of cross-immunity on within-herd viral evolution and invasion success of new PRRSV introductions into host populations. To achieve this aim, we longitudinally tracked genetic diversity on sow farms.We generated whole genome sequences from sow farms experienced repeated outbreaks caused by the same apparent strain of the virus. For each farm, we compared the amino acid changes that occurred throughout the course of the outbreak, and found that several specific amino acid changes (associated with Epitope C in ORF5) occur at high frequency, suggesting immune evasion is occurring in natural populations at the micro-evolutionary scale. We conducted temporal network autocorrelation analyses to evaluate the effect of previous exposures on PRRS occurrence on farms. From these analyses, several time-varying exposures were observed to influence PRRS occurrence on farms. These included prior exposure to PRRS 6 months before, frequency of animal shipments, and direct and indirect farm contacts via animal movements. The methods developed under this objective can also be applied to other studies, while the findings of the study will help guide decisions on disease prevention and management. Objective 3:Quantify how landscapes of variable cross-immunity and population connectivity influence co-circulation and interactions among viral lineages. We conducted phylodynamic analyses on drivers of spatial spread within- and between-regions.Within a region, we found that the spatial spread of PRRSV was most linked to the movement of feeder-aged pigs moving from nursery sites to finishing sites, as opposed to the movement of weaned pigs or breeding age pigs.This work shows the implications of PRRS amplification that likely occurs in nursery sites, and highlights the need for better biosecurity associating with this age-class.We also conducted a phylogeographic analysis of each of the main sub-lineages of PRRS in the U.S., highlighting their region of emergence and regional hotspots for spread.We evaluated whether immunological selection pressures differed by region, but found no evidence of this. We further trained a machine learning model to predict the emergence potential of different clades of PRRSV-2 across time.One of the most important predictors in the model was the dissimilarity of the clade (at the amino acid level) from the most prevalent contemporary strains, suggesting that antigenic distinctiveness may contribute to emergence potential Objective 4:Investigate the conditions in which cross-immunity landscapes lead to evolution, persistence, and sequential dominance of co-circulating lineages. Our research has demonstrated that a new PRRSV sub-lineage has emerged every 3-4 years and over the past decade and that all of these contemporary viruses belong to lineage 1 (which we sub-divided into 7 sub-lineages). Changes in specific motifs have also been shown to correlate with lineage turnover and these motifs appear to be associated with gain or loss of N-glycosylation sites associated with immune response. This area of research is ongoing but key findings so far suggest that GP5 epitope turnover occurs over time and these changes in epitopes also cluster with specific lineages and sub lineages.From the knowledge obtained in objectives 1a, 3a-c and 4a, we developed multi-strain models using old and new data to answer questions on cross-immunity, dominance, and co-circulation of old and emerging PRRSV. Specifically using an individual-based stochastic metapopulation model ("DSPS: Discrete Spatial Phylo Simulator" development lead at Roslin, UK), to model PRRSV transmission in pigs, we have implemented tracking mutations to the viral sequence and their impact on reinfection of hosts by novel strains. Thus, we can model response to vaccination and immune escape of the virus. Additionally, we can modify model parameters at specific points of the simulation to reflect the implementation of time dependent control measures.The model developed here has been extended to other multi-strain pathogens, such as SARS-CoV-2.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Jing Huang, Venkatramana D. Krishna, Igor A. D. Paploski, Kimberly VanderWaal, Declan C. Schroeder, Maxim C-J. Cheeran. Porcine antibody response to epitope A on PRRSV GP5 and its role in virus neutralization. NAPRRS/NC229: International Conference of Swine Viral Diseases. Chicago, IL. December 2024.
• Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Jing Huang, Venkatramana D. Krishna, Igor A. D. Paploski, Kimberly VanderWaal, Declan C. Schroeder, Maxim C-J. Cheeran. Porcine antibody response to epitope A on porcine reproductive and respiratory syndrome virus glycoprotein 5 and its role in virus neutralization. Allen D. Leman Swine Conference, Saint Paul, MN. September 2024.
• Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Jing Huang, Venkatramana D. Krishna, Alexander Kennedy, Igor A. D. Paploski, Dennis N. Makau, Kimberly VanderWaal, Declan C. Schroeder, Maxim C-J. Cheeran. Differential Antibody Responses to PRRSV Glycoprotein 5 by Vaccine and Field Strains. NAPRRS/NC229: International Conference of Swine Viral Diseases. Chicago, IL. November 2023.
• Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Jing Huang, Venkatramana D. Krishna, Alexander Kennedy, Igor A. D. Paploski, Dennis N. Makau, Kimberly VanderWaal, Declan C. Schroeder, Maxim C-J. Cheeran. Differential Antibody Responses to PRRSV Glycoprotein 5 by Vaccine and Field Strains. Allen D. Leman Swine Conference, Saint Paul, MN. September 2023.
• Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Jing Huang, Venkatramana D. Krishna, Igor A. D. Paploski, Kimberly VanderWaal, Declan C. Schroeder, Maxim C-J. Cheeran. Porcine antibody response to epitope A on porcine reproductive and respiratory syndrome virus glycoprotein 5 and its role in virus neutralization. International Porcine Respiratory and Reproductive Syndrome Symposium. Yantai, China. August 2024.
• Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Jing Huang, Venkatramana D. Krishna, Igor A. D. Paploski, Kimberly VanderWaal, Declan C. Schroeder, Maxim C-J. Cheeran. Differential Antibody Responses to Glycoprotein 5 Epitope A and Its Effect on PRRSV Neutralization in Swine. 20th Annual Institute for Molecular Virology Symposium. Minneapolis, MN. May 2024.
• Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Jing Huang, Venkatramana D. Krishna, Nkechi Odogwu, Igor A. D. Paploski, Dennis N. Makau, Nakarin Pamornchainavakul, Julia P. Baker, Qian Liu, Tatiana Lenskaia, Kimberly VanderWaal, Declan C. Schroeder, Maxim C-J. Cheeran. GP5-specific antibody response to porcine reproductive and respiratory syndrome virus challenge in vaccinated swine. Allen D. Leman Swine Conference, Saint Paul, MN. September 2022.
• Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Jing Huang, Venkatramana D. Krishna, Nkechi Odogwu, Igor A. D. Paploski, Dennis N. Makau, Nakarin Pamornchainavakul, Julia P. Baker, Qian Liu, Tatiana Lenskaia, Kimberly VanderWaal, Declan C. Schroeder, Maxim C-J. Cheeran. GP5-Specific Antibody Response to Porcine Reproductive and Respiratory Syndrome Virus Challenge in Vaccinated Swine. NAPRRS/NC229: International Conference of Swine Viral Diseases. Chicago, IL. December 2022.
• Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Rachel Bradley, Venkatramana D. Krishna, Maria Pieters, Jianping Wang, and Maxim C-J. Cheeran Development of antigen based diagnostic ELISA for Mycoplasma hyopneumoniae. Lenam Swine Health Conference, Saint Paul MN September 2021
• Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Clarissa Pellegrini Ferreira, Igor A. D. Paploski, Jing Huang, Maxim C-J Cheeran, Kim VanderWaal, Declan C. Schroeder. Garbage in, garbage out  a lesson learned from whole genome sequencing of PRRSV. Leman Swine Health Conference, Saint Paul MN September 2024.
• Type: Peer Reviewed Journal Articles Status: Published Year Published: 2021 Citation: Makau DN, Paploski IAD, VanderWaal K. Temporal stability of swine movement networks in the U.S. Prev Vet Med. 2021 May 3;191:105369. doi: 10.1016/j.prevetmed.2021.105369. Epub ahead of print. PMID: 33965745.
• Type: Peer Reviewed Journal Articles Status: Published Year Published: 2024 Citation: PAPLOSKI, IGOR A.D.; KIESEL, GABRIELA; MAKAU, DENNIS N.; PAMORNCHAINAVAKUL, NAKARIN; BAKER, JULIA P.; KIKUTI, MARIANA; CORZO, CESAR A.; VANDERWAAL, KIMBERLY.. Weathering the storm: Extreme weather events and their association with PED and PRRS occurrence. VETERINARY MICROBIOLOGY, DOI: 10.1016/j.vetmic.2024.110299, 2024.
• Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Baker JP, Rovira A, Vanderwaal K. Repeat Offenders: PRRSV clinical re-breaks from a whole genome perspective. American Association of Swine Veterinarians Annual Meeting. Nashville, TN. February 2024.
• Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Baker JP, Rovira A, Vanderwaal K. Repeat Offenders: PRRSV clinical re-breaks from a whole genome perspective. International Pig Veterinary Society Annual Meeting. Leipzig, Germany. June 2024
• Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Baker JP, Paploski IAD, Rovira Albert, Guggenbiller D, Kaptur R, VanderWaal K. Switching immune target: applying MJPRRS classifications to characterize how PRRSV GP5-epitope C changes over time. International PRRS Symposium. Yantai, China. August 2024
• Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Baker JP, Gutierrez AH, Pamornchainavakul N, De Groot AS, VanderWaal K. Computationally predicted T-cell epitope trends for 30 years of wild-type PRRSV-2 strains from the USA. NAPRRS/NC229: International Conference of Swine Viral Diseases. Chicago, IL. December 2024.
• Type: Other Journal Articles Status: Under Review Year Published: 2024 Citation: Baker JP, Rovira A, Vanderwaal K. Repeat Offenders: PRRSV clinical re-breaks from a whole genome perspective. Veterinary Microbiology. 2024.
• Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Baker JP, Rovira A, Vanderwaal K. Repeat Offenders: understanding PRRSV clinical rebreaks. NAPRRS/NC229: International Conference of Swine Viral Diseases. Chicago, IL. December 2023.
• Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Baker JP, Paploski IAD, Rovira Albert, Guggenbiller D, Kaptur R, VanderWaal K. Shifting immune target: how PRRSV GP5-epitope C changes over time. Leman Swine Conference. Saint Paul, MN. September 2022.
• Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Baker JP, Paploski IAD, Rovira Albert, Guggenbiller D, Kaptur R, VanderWaal K. Switching immune target: applying MJPRRS classifications to characterize how PRRSV GP5-epitope C changes over time. NAPRRS/NC229: International Conference of Swine Viral Diseases. Chicago, IL. December 2022.
• Type: Other Journal Articles Status: Other Year Published: 2024 Citation: Nakarin Pamornchainavakul, Declan Schroeder, Kimberly VanderWaal et al. QoALa: a comprehensive workflow for viral quasispecies diversity comparison using long-read sequencing data, 18 July 2024, PREPRINT (Version 1) available at Research Square [https://doi.org/10.21203/rs.3.rs-4637890/v1]
• Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Nakarin Pamornchainavakul, Igor Paploski, Dennis Makau, Cesar Corzo, and Kim VanderWaal. Estimating farm-level R's for PRRSV using sequence-based transmission trees. Allen D. Leman Swine Conference: Research Highlights. September 2020 Conference Papers and Presentations 2020
• Type: Other Journal Articles Status: Published Year Published: 2024 Citation: VANDERWAAL, KIMBERLY; PAMORNCHAINAVAKUL, NAKARIN; KIKUTI, MARIANA; LINHARES, DANIEL C.L.; TREVISAN, GIOVANI; ZHANG, JIANQIANG; ANDERSON, TAVIS K.; ZELLER, MICHAEL; ROSSOW, STEPHANIE; HOLTKAMP, DERALD J.; MAKAU, DENNIS N.; CORZO, CESAR A.; PAPLOSKI, IGOR A.D. Phylogenetic-based methods for fine-scale classification of PRRSV-2 ORF5 sequences: a comparison of their robustness and reproducibility. FRONTIERS IN VIROLOGY, DOI: 10.3389/fviro.2024.1433931, 2024
• Type: Peer Reviewed Journal Articles Status: Published Year Published: 2024 Citation: PAMORNCHAINAVAKUL, NAKARIN; KIKUTI, MARIANA; PAPLOSKI, IGOR A.D.; CORZO, CESAR A.; VANDERWAAL, KIMBERLY. Predicting Potential PRRSV-2 Variant Emergence through Phylogenetic Inference. TRANSBOUNDARY AND EMERGING DISEASES, DOI: 10.1155/2024/7945955, 2024.
• Type: Peer Reviewed Journal Articles Status: Published Year Published: 2023 Citation: YIM-IM, WANNARAT; ANDERSON, TAVIS K.; PAPLOSKI, IGOR A.D.; VANDERWAAL, KIMBERLY; GAUGER, PHILLIP; KRUEGER, KAREN; SHI, MANG; MAIN, RODGER; ZHANG, JIANQIANG. Refining PRRSV-2 genetic classification based on global ORF5 sequences and investigation of their geographic distributions and temporal changes. MICROBIOLOGY SPECTRUM, DOI: 10.1128/spectrum.02916-23M, 2023.
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Nakarin Pamornchainavakul, Igor Paploski, Dennis Makau, Cesar Corzo, and Kim VanderWaal. Estimating farm-level reproductive numbers for PRRSV using sequence-based transmission tree analysis. Conference of Research Workers in Animal Diseases (CRWAD). December 2020 Conference Papers and Presentations 2020
• Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Nakarin Pamornchainavakul, Igor Paploski, Dennis Makau, Cesar Corzo, and Kim VanderWaal. Estimating farm-level reproduction numbers for PRRSV using sequence-based transmission tree analysis. The 52nd AASV Annual Meeting, February 2021 Conference Papers and Presentations 2021
• Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Nakarin Pamornchainavakul, Igor Paploski, Dennis Makau, Cesar Corzo, and Kim VanderWaal. Integrating genetic and animal movement data to reconstruct transmission networks. Allen D. Leman Swine Conference: Research Highlights. September 2021 Conference Papers and Presentations 2021
• Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: N. Pamornchainavakul; IAD. Paploski; D. Makau; C. Corzo; K. VanderWaal. Revealing the invisible links and modes of between-farm PRRSV transmission using genetic-based network analysis. Epidemics 8 - 8th International Conference on Infectious Disease Dynamics. December 2021 Conference Papers and Presentations 2021
• Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Nakarin Pamornchainavakul, Igor Paploski, Dennis Makau, Cesar Corzo, and Kim VanderWaal. Revealing invisible links and modes of between-farm PRRSV transmission using genetic-based network analysis. Conference of Research Workers in Animal Diseases. December 2021 Conference Papers and Presentations 2021
• Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Nakarin Pamornchainavakul, Mariana Kikuti, Igor A. D. Paploski, Dennis N. Makau, Albert Rovira, Cesar A. Corzo, Kimberly VanderWaal. Genomic recombination and the epidemiological emergence of novel PRRSV-2 variants: a genome-based phylodynamic approach. The International Symposium of Veterinary Epidemiology and Economics (ISVEE) 16. August 2022 Conference Papers and Presentations 2022
• Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Nakarin Pamornchainavakul, Igor AD Paploski, Dennis N Makau, Mariana Kikuti, Albert Rovira, Samantha Lycett, Cesar A Corzo, and Kimberly VanderWaal. Mapping hotspots for emergence and inter-regional spread of contemporary PRRSV sub-lineages in the United States using Phylogeography. The International Symposium of Veterinary Epidemiology and Economics (ISVEE) 16. August 2022 Conference Papers and Presentations 2022
• Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Nakarin Pamornchainavakul, Dennis N. Makau, Igor A. D. Paploski, Mariana Kikuti, Samantha Lycett, Cesar A. Corzo, Kimberly VanderWaal. Early indicators of the emergence potential of PRRSV-2 variants based on phylogenetic structure. Allen D. Leman Swine Conference: Research Highlights. September 2022 Conference Papers and Presentations 2022
• Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Nakarin Pamornchainavakul, Mariana Kikuti, Igor AD Paploski, Cesar A Corzo, and Kimberly VanderWaal. Predicting PRRSV-2 Variant Emergence: Insights from a Decade of Genomic Analysis. Allen D. Leman Swine Conference: Research Highlights. September 2023 Conference Papers and Presentations 2023
• Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: N. Pamornchainavakul, M. Kikuti, I.A.D. Paploski, C.A. Corzo, and K.VanderWaal. Predicting PRRSV-2 Variant Emergence: Insights from a Decade of Genomic Analysis. North American PRRS Symposium. December 2023 Conference Papers and Presentations 2023
• Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: N. Pamornchainavakul, M. Kikuti, I.A.D. Paploski, C.A. Corzo, and K.VanderWaal. Predicting PRRSV-2 Variant Emergence: Insights from a Decade of Genomic Analysis. 27th International Pig Veterinary Society Congress. June 2024 Conference Papers and Presentations 2024
• Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Kimberly VanderWaal. PRRSV-2 Variant Classification: A Dynamic Nomenclature for Enhanced Monitoring and Surveillance. Poster presentation, NAPRRS 2024, Chicago, IL.
• Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Nakarin Pamornchainvakul, Igor A.D. Paploski , Dennis N. Makau, Julia Baker, Jing Huang, Clarissa P. Ferreira, Cesar A. Corzo, Albert Rovira, Maxim CJ Cheeran, Declan C Schroeder, and Kimberly VanderWaal. Experimental Evidence of Vaccine-driven Evolution of PRRSV-2. Allen D. Leman Swine Conference: Research Highlights. September 2024 Conference Papers and Presentations 2024
• Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Nakarin Pamornchainvakul, Igor A.D. Paploski , Dennis N. Makau, Julia Baker, Jing Huang, Clarissa P. Ferreira, Cesar A. Corzo, Albert Rovira, Maxim CJ Cheeran, Declan C Schroeder, and Kimberly VanderWaal. Experimental Evidence of Vaccine-driven Evolution of PRRSV-2. North American PRRS Symposium. December 2024 Conference Papers and Presentations 2024
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2025 Citation: Nakarin Pamornchainvakul, Igor A.D. Paploski , Dennis N. Makau, Julia Baker, Jing Huang, Clarissa P. Ferreira, Cesar A. Corzo, Albert Rovira, Maxim CJ Cheeran, Declan C Schroeder, and Kimberly VanderWaal. Experimental evidence of vaccine-driven evolution of PRRSV-2 in pigs-to-pig infection chains. The 56th AASV Annual Meeting, March 2025 Conference Papers and Presentations 2025
• Type: Other Journal Articles Status: Submitted Year Published: 2024 Citation: Herrera da Silva, Joao P.; Rossow, Stephanie; Paploski, Igor A.D.; Kikuti, Mariana; Corzo, Cesar A.; VanderWaal, Kimberly : Complete genome and recombination analysis of a novel porcine reproductive and respiratory syndrome virus 2 (variant 1H.18) identified in the midwestern U.S. Submitted Microbiology Resource Announcements 2024.
• Type: Other Journal Articles Status: Other Year Published: 2025 Citation: Jing Huang, Venkatramana D. Krishna, Igor A. D. Paploski, Kimberly VanderWaal, Declan C. Schroeder, Maxim C-J. Cheeran. Characterization of GP5-specific response in pigs vaccinated with modified live Porcine reproductive and respiratory syndrome virus vaccines derived from two lineages. Veterinary Sciences (draft finished)
• Type: Other Journal Articles Status: Other Year Published: 2025 Citation: Jing Huang, Igor A. D. Paploski, Kimberly VanderWaal, Declan C. Schroeder, Maxim C-J. Cheeran. Differential Antibody Responses to Epitope A of Glycoprotein 5 in Swine Exposed to Field and Vaccine Strains. Veterinary Immunology and Immunopathology (draft finished)

Progress 08/15/22 to 08/14/23

Outputs
Target Audience:• Scientists and epidemiologists • Government and policy making agencies • Project participants from study regions for the longitudinal sampling study • Swine veterinarians • Industry players (swine production companies, pharmaceutical companies biosecurity companies etc.) Changes/Problems: Objective 2:We originally collected longitudinal samples across 12 months using processing fluid sample types.However, processing fluids proved to be too impure of a sample to achieve whole genome sequencing.Thus, we performed a second study where we retrospectively identified farms experiencing long-term outbreaks caused by the same genetic variant of the virus, with the selection criteria being that farms must have bio-banked viral isolates.We identified 38 viral isolates associated with 19 long-term clinical outbreaks caused by the same apparent viral strain. Whole genome sequences have been obtained from these isolates. We hypothesize that isolates associated with long-term clinical outbreaks bear mutations that allow them to continue to spread and cause clinical disease in populations that were previously exposed to that variant.Thus, for each clinical outbreak, we compared the amino acid changes that occurred throughout the course of the outbreak, and found that several specific amino acid changes (associated with Epitope C in ORF5) occur at high frequency, suggesting immune evasion is occurring. What opportunities for training and professional development has the project provided?This project has provided support for four graduate students and four postdocs. These trainees gained expertise in virology, immunology, bioinformatics, and epidemiology. All had the opportunity to present research at national or international conferences, as well as as gained experience in study design, oral and written scientific communication. How have the results been disseminated to communities of interest?We continue to enhance our partnerships with the industry and other diagnostic institutions. In the past year we have worked with a vaccine company that specializes in protein-based classification of PRRSV to develop strain specific vaccines. Through data sharing we have been able to assist with the classification of PRRSV in different lineages and map temporal changes in PRRSV epitopes over time. We have also held several meetings with interested swine industry players including company veterinarians and vaccine manufacturing companies. These meetings have been good forums for sharing observed trends of PRRSV spread and evolution over the last 10 years. Moreover, vaccine companies have been keen to understand the importance of observed viral phylogenetic divergence in relation to market vaccines and their effectiveness in PRRS management. From these discussions, both swine production companies and pharmaceutical companies have been discussing how the current knowledge would modify vaccine development in the future and employment of different immunization regimes in swine farms. Findings of this project have so far been presented in industry-oriented meetings largely attended by the target audience, the 2019- 2023Allen D. Leman Swine Conference, the 2019-2023Swine Disease Eradication Center meetings and the 26th International pig veterinary society congress, 2022. Additionally, some of the research findings have been disseminated through scientific manuscripts published in peer reviewed journals and other media outlets. Please see outputs for a full list. What do you plan to do during the next reporting period to accomplish the goals?In the remaining period of this grant, we intend to complete the quasi-species analysis associated with the infetion chain experiment (Aim 1) as well as complete the epi-evoltuionarymodeling (Aim 4). Those are the largest pending pieces of this project, as the remaining objectives are either complete or close to complete pending manuscript preparation.

Impacts
What was accomplished under these goals? Objective 1a - Completion status = 90% In the previous reporting period, we completed a 7-month in vivo "infection chain" experiment wherein we follow the evolution of PRRSV across an infection chain consisting of six sequentially infected batches of pigs. Treatments included non-vaccinated, vaccinated with a homologous vaccine, and vaccinated with a non-homology vaccine. Samples collected from this experiment will be used to investigate antibody avidity to epitopes (part of aim 1C), within-host evolution (aim 1a), and parameterize the evolutionary models (aim 1d). Sample sequencing is in-progress. In parallel, we have been developing a novel bioinformatic pipeline to analyze quasi-species diversity for long-read sequences.This was necessary, as we discovered that nearly all quasi-species analysis pipelines are based on short-read Illumina sequencing, which typically has a lower error-rate. The advantage of long-reads is that it allows for haplotype reconstruction (as opposed to simply analyzing single nucleotide polymorphisms -SNPs).That being said, existing pipelines do not adequately address sequencing error and result in unacceptably high levels of data loss.Therefore, we developed a long-read pipeline that minimizes data loss (i.e., rather than throwing out entire reads with low frequency SNPs, these SNPs are replaced by the consensus nucleotide for that position). In addition, we tested the sensitivity of quasi-species diversity metrics to read-depth by systematically down-sampling reads and re-calculating metrics. We identify a minimum threshold of ~100 reads that produce robust and stable quasi-species diversity metrics, and also highlight specific metrics that are particularly sensitive to read depth and should be used with caution for between-sample comparisons. This pipeline was rigorously tested on PRRSV as well as four other RNA viruses, and the tool is being prepared in such a way that it can be used by other researchers that aim to conduct quasi-species analysis with long-read sequencing technology. Objective 1c - Completion statue: 90% We have now published a series of papers and conference proceedings detailing evolutionary changes in antigenic sites and epitopes, highlighted the role they play in immune-mediated evolution within lineages and immune-mediate competition between lineages. We have focused particularly on the ectodomain of the GP5 protein, which harbors three major epitopes as well as multiple N-glycosylation sites. N-glycosylation is an important mechanism of immune evasion, and we document changes in N-glycosylation patterns that appear to be associated with lineage emergence and re-emergence.We also have conducted an evolutionary analysis of how the inferred immune-phenotype of Epitope C (an important epitope for homologous neutralization) evolves under immune pressure; changes in this epitope also to be associated with clade persistence and emergence. Our team also has conducted an in vitro analysis of the immunogenicity of Epitope A. ELISA results showed that antibody responses to epitope A were dominant and sequence-specific in vaccinated animals. However, no significant difference was observed in the antibody response to epitope A between naive and experimentally infected animals. These data suggest differential induction of antibody responses to GP5 epitope A by vaccine strains and field isolates, which likely results from alternate signal peptide cleavage in the two vaccine strains studied here. Signal peptide cleavage analysis suggested that all selected vaccine strains retained epitope A on GP5 (cleaved between aa 26 and 27), while the other three field isolates belonging to different viral lineages (lineage 1C, 1A and 1G) were predicted to undergo cleavage between aa 31 and 32 thereby removing the epitope A from the viral GP5 protein. Further serological studies are needed to determine the prevalence of epitope A-specific antibodies in infected or vaccinated animals that would provide more insights for future PRRSV vaccine design.This work is being prepared for publication Objective 1d - Completion status = 80% We have developed a model that forward simulates evolutionary processes, which allows sequence evolution to be simulated across time between different "demes".Here, a deme can be defined as two animals. Objective 2 - Completion status = 75% - See Changes/Problems Objective 3a - Completion status = 100% Using virus isolate data and animal movement data from farms in two production systems in the US, we have conducted temporal network autocorrelation analyses to evaluate the effect of previous exposures on PRRS occurrence on farms. This manuscript has been published. From these analyses, several time-varying exposures were observed to influence PRRS occurrence on farms. These included prior exposure to PRRS 6 months before, frequency of animal shipments, and direct and indirect farm contacts via animal movements. The methods developed under this objective can also be applied to other studies, while the findings of the study will help guide decisions on disease prevention and management. Objective 3b - Completion status= 100% We have now conducted phylodynamic analyses on drivers of spatial spread within- and between- regions, which have been published in two separate manuscripts.Within a region, we found that the spatial spread of PRRSV was most linked to the movement of feeder-aged pigs moving from nursery sites to finishing sites, as opposed to the movement of weaned pigs or breeding age pigs.This work shows the implications of PRRS amplification that likely occurs in nursery sites, and highlights the need for better biosecurity associating with this age-class.We also conducted a phylogeographic analysis of each of the main sub-lineages of PRRS in the U.S., highlighting their region of emergence and regional hotspots for spread.We evaluated whether immunological selection pressures differed by region, but found no evidence of this. Objective 4a - Completion status = 100% Analysis of temporal dynamics of PRRS populations in the US has been done using multiple datasets and published. These data have included sequence data from Morison Swine Health Monitoring Project, the UMN Veterinary Diagnostic Laboratory and other collaborating VDLs. From the analysis it is evident that a new PRRSV lineage emerge every 3-4 years and over the past decade there most of the dominant viruses have belonged to lineage 1. Changes in specific motifs have also been shown to correlate with lineage turnover and these motifs appear to be associated with gain or loss of N-glycosylation sites associated with immune response. Changes in specific epitopes in GP5 is also suspected to occur in PRRSV as different strains emerge. This area of research is ongoing but key findings so far suggest that GP5 epitope turnover occurs over time and these changes in epitopes also cluster with specific lineages and sub lineages. Objective 4b & 4c - Completion status = 80% From the knowledge obtained in objectives 1a, 3a-c and 4a, we are developing multi-strain models using old and new data to answer questions on cross-immunity, dominance, and co-circulation of old and emerging PRRSV. Specifically using an individual-based stochastic metapopulation model ("DSPS: Discrete Spatial Phylo Simulator" development lead at Roslin, UK), to model PRRSV transmission in pigs, we have implemented tracking mutations to the viral sequence and their impact on reinfection of hosts by novel strains. Thus, we can model response to vaccination and immune escape of the virus. Additionally, we can modify model parameters at specific points of the simulation to reflect the implementation of time dependent control measures.The model framework and implementation of all desired features in the code has been completed, and simulations are still ongoing and expected to be complete in December 2023.

Publications

• Type: Journal Articles Status: Published Year Published: 2023 Citation: PAMORNCHAINAVAKUL, NAKARIN; MAKAU, DENNIS N.; PAPLOSKI, IGOR A.D.; CORZO, CESAR A.; VANDERWAAL, KIMBERLY. Unveiling invisible farm-to-farm PRRSV-2 transmission links and routes through transmission tree and network analysis. EVOLUTIONARY APPLICATIONS, DOI: 10.1111/eva.13596, 2023.
• Type: Journal Articles Status: Published Year Published: 2023 Citation: KIKUTI, MARIANA; VILALTA, CARLES; SANHUEZA, JUAN; PAMORNCHAINAVAKUL, NAKARIN; KEVILL, JESSICA; YANG, MY; PAPLOSKI, IGOR A.D.; LENSKAIA, TATIANA; ODOGWU, NKECHI M.; KIEHNE, ROSS; VANDERWAAL, KIMBERLY, SCHROEDER, DECLAN; CORZO, CESAR A. Porcine Reproductive and Respiratory Syndrome (PRRSV2) Viral Diversity within a Farrow-to-Wean Farm Cohort Study. VIRUSES, DOI: 10.3390/v15091837, 2023.
• Type: Journal Articles Status: Published Year Published: 2023 Citation: PAMORNCHAINAVAKUL, NAKARIN; PAPLOSKI, IGOR A.D.; MAKAU, DENNIS N.; KIKUTI, MARIANA; ROVIRA, ALBERT; LYCETT SAMANTHA; CORZO, CESAR A.; VANDERWAAL, KIMBERLY. Mapping the Dynamics of Contemporary PRRSV-2 Evolution and Its Emergence and Spreading Hotspots in the U.S. Using Phylogeography. PATHOGENS, DOI: 10.3390/pathogens12050740, 2023.
• Type: Journal Articles Status: Published Year Published: 2023 Citation: PAPLOSKI, IGOR A.D.; ARRUDA, ANDREIA G.; VANDERWAAL, KIMBERLY. Editorial: Rising stars in veterinary epidemiology and economics 2022: Porcine Reproductive and Respiratory Syndrome Virus: Epidemiology, immunology and virology. FRONTIERS IN VETERINARY SCIENCE, DOI: 10.3389/fvets.2023.1111668, 2023.
• Type: Journal Articles Status: Published Year Published: 2022 Citation: PAPLOSKI, IGOR A.D.; MAKAU, DENNIS N.; PAMORNCHAINAVAKUL, NAKARIN; BAKER, JULIA P.; SCHROEDER, DECLAN; ROVIRA, ALBERT; VANDERWAAL, KIMBERLY. Potential Novel N-Glycosylation Patterns Associated with the Emergence of New Genetic Variants of PRRSV-2 in the U.S.. VACCINES, DOI: 10.3390/vaccines10122021 , 2022.
• Type: Journal Articles Status: Published Year Published: 2022 Citation: MAKAU, DENNIS N.; PRIETO, CINTA; MARTINEZ-LOBO, FRANCISCO; PAPLOSKI, IGOR A.D.; VANDERWAAL, KIMBERLY. Predicting Antigenic Distance from Genetic Data for PRRSV-Type 1: Applications of Machine Learning. MICROBIOLOGY SPECTRUM, DOI: 10.1128/spectrum.04085-22 , 2022.

Progress 08/15/21 to 08/14/22

Outputs
Target Audience: Scientists and epidemiologists Government and policy making agencies Project participants from study regions for the longitudinal sampling study Swine veterinarians Industry players (swine production companies, pharmaceutical companies biosecurity companies etc.) Changes/Problems:Changes NA Problems While working with samples from farms recruited for objective 2, we realized that processing fluid samples generated poor quality reads and it was difficult to obtain whole genome reads from them. We changed our analytical approach to allow us answer objective 2 questions using both full genome and short genome segments where necessary to avoid further delays in this analysis. What opportunities for training and professional development has the project provided? The project has enhanced four postdoctoral research associates who are learning different aspects of phylodynamic and phylogeographic analysis. Three PhD students have the opportunity to participate in experiments and use data generated from the project to learn and enhance their skills on immunology, sequence assembly and analysis. How have the results been disseminated to communities of interest?We continue to enhance our partnerships with the industry and other diagnostic institutions. In the past year we have worked with a vaccine company that specializes in protein-based classification of PRRSV to develop strain specific vaccines. Through data sharing we have been able to assist with the classification of PRRSV in different lineages and map temporal changes in PRRSV epitopes over time. We have also held several meetings with interested swine industry players including company veterinarians and vaccine manufacturing companies. These meetings have been good forums for sharing observed trends of PRRSV spread and evolution over the last 10 years. Moreover, vaccine companies have been keen to understand the importance of observed viral phylogenetic divergence in relation to market vaccines and their effectiveness in PRRS management. From these discussions, both swine production companies and pharmaceutical companies have been discussing how the current knowledge would modify vaccine development in the future and employment of different immunization regimes in swine farms. Findings of this project have so far been presented in industry-oriented meetings largely attended by the target audience, the 2019- 2021 Allen D. Leman Swine Conference, the 2019-2021 Swine Disease Eradication Center meetings and the 26th International pig veterinary society congress, 2022. Additionally, some of the research findings have been disseminated through scientific manuscripts published in peer reviewed journals and other media outlets. Below is a detailed list of these outreach efforts: Academic conference attended by industry: Dennis Makau, Moh Alkhamis, Igor Paploski, Cesar Corzo, Samantha Lycett, Kimberly VanderWaal. Using animal movement data to understand the spread and evolution of PRRSV, (June 21st to 24th , 2022). In 26th international pig veterinary society congress, Rio de Janeiro, Brazil. Igor Paploski, Nakarin Pamornchainavakul, Dennis Makau, Albert Rovira, Cesar Corzo, Mariana Kikuti, Declan Schroeder, Maxim Cheeran, Andrea Doeschl-Wilson, Rowland Kao, Samantha Lycett, Kimberly VanderWaal. N-glycosylation patterns of epidemic PRRSV2 sub-lineages in the U.S., (June 21st to 24th , 2022). In 26th international pig veterinary society congress, Rio de Janeiro, Brazil. Julia P. Baker, Igor Paploski, Albert Rovira, Dwain Guggenbiller, Ronald Kaptur, Kim VanderWaal. Moving targets: how porcine reproductive and respiratory syndrome epitopes shift over time. In 2022 CVM Points of Pride Research Day, University of Minnesota, MN, USA. Pamornchainavakul N, Paploski IAD, Makau DN, Kikuti M, Rovira A, Lycett S, Corzo C, and VanderWaal K. Tempo and pattern of porcine reproductive and respiratory syndrome virus 2 (PRRSV-2) in USA. In 2022 CVM Points of Pride Research Day, University of Minnesota, MN, USA. C. A. Corzo, M. Kikuti, I. A. D. Paploski, N. Pamornchainavakul, C. Picasso-Risso, D. Makau, A. Rovira, K. VanderWaal. Understanding the emergence of a new PRRSV variant (L1C 1-4-4) through the implementation of epidemiological tools. (26th Feb - 1st March 2022). In American Association of Swine Veterinarians (AASV) Annual meeting, Indianapolis, Indiana, USA. Pamornchainavakul, N.; Paploski, I.; Makau, D.; Corzo, C.; VanderWaal, K. Revealing invisible links and modes of between-farm PRRSV transmission using genetic-based network analysis (3rd - 7th December 2021). In Conference of Research Workers in Animal Diseases (CRWAD) , Chicago, IL, USA (virtual format). Paploski, I.; Pamornchainavakul, N.; Makau, D.; Rovira, A.; Corzo, C.; Schroeder, D.; Cheeran, M.; Doeschl-Wilson, A.; Kao, R.; Lycett, S.; VanderWaal, K. Multi-strain dynamics of PRRSV type-2 in U.S. pig populations (3rd - 7th December 2021). In Conference of Research Workers in Animal Diseases (CRWAD) , Chicago, IL, USA (virtual format). Daniel Balaz, Andrea Doeschl-Wilson, Rowland Kao, Kimberly VanderWaal, Samantha Lycett, A simulation model for vaccine pressure and immune escape of RNA viruses. (30th November - 3rd December 2021). In Epidemics8, International Conference on Infectious Disease Dynamics (virtual format). Dennis N. Makau, Moh A. Alkhamis, Igor A. D. Paploski, Cesar A. Corzo, Samantha Lycett, Kimberly VanderWaal. Integrating animal movements with phylogeography to model the spread of PRRS virus in the U.S. (3rd - 7th December 2021). In Conference of Research Workers in Animal Diseases (CRWAD) , Chicago, IL, USA (virtual format). Dennis N. Makau, Moh A. Alkhamis, Igor A. D. Paploski, Cesar A. Corzo, Samantha Lycett, Kimberly VanderWaal. Integrating animal movements with phylogeography to model the spread of PRRS virus in the U.S. (30th November - 3rd December 2021). In Epidemics8, International Conference on Infectious Disease Dynamics (virtual format). D. Makau, K. VanderWaal, M. Kikuti, C. Picasso-Risso, E. Geary, C. Corzo. Post-epidemic dynamics of PEDV in the United States: Current trends and patterns (18th - 21st September 2021). Allen D Leman Conference, St. Paul, MN, USA. Kikuti, M.; Sanhueza, J.; Vilalta, C.; Paploski, I.; VanderWaal, K.; Corzo, C. Porcine Reproductive and Respiratory Syndrome Virus 2 (PRRSV-2) genetic diversity and occurrence of wild type and vaccine-like strains in the United States swine industry. Poster. North American PRRS Symposium (NAPRRS), Chicago, 2021. Kikuti, M.; Paploski, I.; Pamornchainavakul, N.; Picasso-Risso, C.; Schwartz, M.; Yeske, P.; Leuwerke, B.; Bruner, L.; Murray, D.; Roggow, B.; Thomas, P.; Feldmann, L.; Allerson, M.; Hensch, M.; Bauman, T.; Sexton, B.; Rovira, A.; VanderWaal, K.; Corzo, C. Emergence of a new lineage 1C variant of Porcine Reproductive and Respiratory Syndrome Virus 2 in the United States. Poster. North American PRRS Symposium (NAPRRS), Chicago, 2021. Paploski, I.; Makau, D.; Pamornchainavakul, N.; Kikuti, M.; Cheeran, M.; Corzo, C.; Doeschl-Wilson, A.; Kao, R.; Lycett, S.; Schroeder, D.; VanderWaal, K. Emergence and spread of PRRS virus sub-lineages. Oral presentation. James D. McKean Swine Disease Conference, Ames, 2021. Technical seminars and presentations for industry audience: Kimberly VanderWaal & Dennis Makau. Predicting antigenic distance for PRRSV, : Invited speakers University of Minnesota - Zoetis Swine Pathogen Bio Surveillance Meeting (Pharmaceuticals - PRRS vaccine producer. May 24th, 2022. Dennis Makau: "Predicting antigenic distance for PRRSV". Swine Seminar, Swine Health Information Center, April 29th, 2022. Nakarin Pamornchainavakul: "Measuring how recombination re-shapes the evolutionary history of PRRSV-2". Swine Seminar, Swine Health Information Center, April 1st, 2022. Media 2022 Tracking lineage of PRRS viariants across the US. Podcast in Swine Health backbelt#8 . https://www.linkedin.com/pulse/swine-health-blackbelt-8-tracking-lineage-prrs-variants-/?trackingId=uTTxkWzKQBSJPHXUzGAV6g%3D%3D 2022 Tracing the origin of the novel PRRSV-2 L1C-1-4-4 variant. By: Tracing the origin of the novel PRRSV-2 L1C-1-4-4 variant https://www.nationalhogfarmer.com/animal-health/tracing-origin-novel-prrsv-2-l1c-1-4-4-variant 2022 Article in National Hog Farmer: What are porcine reproductive and respiratory syndrome RFLPs? By Igor Paploski and Kimberly VanderWaal https://www.nationalhogfarmer.com/animal-health/what-are-porcine-reproductive-and-respiratory-syndrome-rflps 2021 Article in National Hog Farmer: Emergent Lineage 1C RFLP 1-4-4 PRRS virus in the U.S. By: Mariana Kikuti, Igor A. D. Paploski, Nakarin Pamornchainavakul, Catalina Picasso-Risso, Albert Rovira, Kimberly VanderWaal, Cesar A. Corzo https://www.nationalhogfarmer.com/news/emergent-lineage-1c-rflp-1-4-4-prrs-virus-us 2021 Article in National Hog Farmer: Feeder pig movements to blame for spread of PRRSV? By: D Makau, M Alkhamis, I Paploski, C Corzo, S Lycett, K VanderWaal https://www.nationalhogfarmer.com/news/feeder-pig-movement-blame-spread-prrsv What do you plan to do during the next reporting period to accomplish the goals?Challenges and delays due to covid notwithstanding, we are doing our best to catch up on the research timelines. We hope to continue undertaking the different project activities with minimal disruptions. Details of how we intend to proceed are: Objective 1 - We plan to sequence samples from the infection chain experiment (objective 1d) by end of August. Subsequently, data obtained from these sequences will be used in the analysis to understand the relationship between host immunity, viral evolution the occurrence of quasi species. Soon after completing the infection chain live animal experiment, we plan to set up the live animal experiments to generate hyperimmune sera to be used for invitro cross-neutralization assays and other immunological assays in objective 1a & b. Objective 2 - Since RNA extraction has been completed for all samples received from the farms and the sequencing pipeline has been optimized, we plan to continue generating sequence data from these samples. We will also share the results of viral sequencing with the participating farms. Objective 3c - Since this objective relies on the optimized models from the analysis time-varying effect of the immune profile and phylodynamic models, we will use the models from the analysis in objective 3 a & b to analyze data obtained from the longitudinal study. Subsequently these findings shall also be published. Objective 4 - We intend to continue optimizing the phylodynamic models generated using retrospective PRRS ORF5 sequences, and to add new whole genome sequences data and spatial data as they become available from objective 2. Furthermore, we intend to present our research findings in the upcoming conferences (International symposium for veterinary epidemiology and economics (ISVEE) 2022 and Allen D. Leman Swine Conference 2022).

Impacts
What was accomplished under these goals? Objective 1a - Completion status = 80% Several steps have been undertaken towards the achievement of this objective. An experiment to generate hyperimmune serum to one of the target viral lineages has been successfully completed. Protocols for immunological assays and detection of antibody producing cells have also been standardized and can be applied to other viral lineages of interest. We also expanded adequate volumes and titers for all 9 target isolates to be used for both hyper immune sera generation and the infection chain experiment (objective 1d). This will subsequently be followed by in vitro assays. In addition, we jsut completed a 7-month in vivo "infection chian" experiement wherein we following the evoltuion of PRRSV across an infection chain consisting of six sequentially infected batches of pigs. Treatments included non-vaccinated, vaccianted with a homologous vaccine, and vaccinated with a non-homology vaccine. Samples collected from this experiment will be used to investigate antibody avidity to epitopes (part of aim 1C), within-host evolution (aim 1a), and parameterize the evoltuionary models (aim 1d). This experiment concluded in August 2022, and samples have yet to be sequenced. Objective 1c/d - Completion status = 50% To develop within/between host model to forward-simulate evolutionary processes, we have conducted 90% of live animal experiments and collected necessary serum samples for PCR and NGS sequencing for quasi-species assessment. We have also developed cellular assays and protocols to examine viral kinetics and variation of immune response. Objective 2a - Completion status = 40% We have completed sample collection from all farms recruited for this phase of the project (November 2019-January 2021). All farms submitted samples for up to12 months on a majority frequency. All samples have been catalogued and RNA extraction completed. Sequencing pipelines have also been developed and parameterized and some sequencing results have already been generated and assembled. Objective 2b & 2c - Completion status = 15% Sequencing pipelines have been optimized and some sequencing results from one of the farms have already been generated and assembled. We hope to complete these procedures swiftly in the next few months. Objective 3a - Completion status = 100% Using virus isolate data and animal movement data from farms in two production systems in the US, we have conducted temporal correlation analyses to evaluate the effect of previous exposures on PRRS occurrence on farms. This manuscript has been published. From these analyses, several time-varying exposures were observed to influence PRRS occurrence on farms. These included prior exposure to PRRS 6 months before, frequency of animal shipments, and direct and indirect farm contacts via animal movements. The methods developed under this objective can also be applied to other studies, while the findings of the study will help guide decisions on disease prevention and management. Objective 3b - Completion status= 100% Phylogenetic analysis of drivers of spatial transition of PRRS have been developed using previously collected data. This analysis has been completed and published. From the analysis we observed that movement of feeder pigs shaped the spatial spread of PRRSV L1A more than weaned and breeding pig movements and spatial proximity and farm density were also factors that enhanced the spatial spread of the PRRSV. This work has been published (peer reviewed journal article, lay report, magazine article) and presented in several conferences Objective 3c - Completion status = 30% The challenges mentioned above in the longitudinal phase (objective 2) have occasioned a delay in generating the sequence data hence slowing down model parameterization. Using sequence data generated by the UMN VDL, previously collected spatial data, we have developed model parameters for simulations. Additionally, by inferring from other studies where antigenic data has been generated, we have been able to develop immunity related model parameters. The model framework has already been tested using thousands of SAR-CoV2 sequences available to us has worked well. Using PRRSV data, some simulation has been done though the model needs more tuning and parameterization. Part of this work was presented in the Epidemics 8 conference of 2021. Once data from objectives 1 and 2 is generated, we hope to develop a robust model, tuned using more current and well annotated data. Objective 4a - Completion status = 90% Analysis of temporal dynamics of PRRS populations in the US has been done using multiple datasets and published. These data have included sequence data from Morison Swine Health Monitoring Project, the UMN Veterinary Diagnostic Laboratory and other collaborating VDLs. From the analysis it is evident that a new PRRSV lineage emerge every 3-4 years and over the past decade there most of the dominant viruses have belonged to lineage 1. Changes in specific motifs have also been shown to correlate with lineage turnover and these motifs appear to be associated with gain or loss of N-glycosylation sites associated with immune response. Changes in specific epitopes in GP5 is also suspected to occur in PRRSV as different strains emerge. This area of research is ongoingbut key findings so farsuggest that GP5 epitope turnover occurs over time and these changes in epitopes also cluster with specific lineages and sub lineages. In addition, we have had the opportunity to document the emergence of a new strain of PRRSV-2 that was first detected in 2020. This new strain appears to be the product of recombination, and is more virulent than other strians of PRRSV-2. We have contributed to several epidemiolgoical and evolutionary studies on the origin and spread of this new strain within the context of multi-strain dynamics. Objective 4b & 4c - Completion status = 25% From the knowledge obtained in objectives 1a, 3a-c and 4a, we are developing multi-strain models using old and new data to answer questions on cross-immunity, dominance and co-circulation of old and emerging PRRSV. Specifically using an individual-based stochastic metapopulation model ("DSPS:Discrete Spatial Phylo Simulator" development lead at Roslin, UK), to model PRRSV transmission in pigs, we have implemented tracking mutations to the viral sequence and their impact on reinfection of hosts by novel strains. Thus, we can model response to vaccination and immune escape of the virus. Additionally we can modify model parameters at specific points of the simulation to reflect the implementation of time dependent control measures. Currently, temporally variable networks between farms are being implemented in order to capture the changing nature of contacts between farms.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: VanderWaal, K. (presented by I. Paploski) Evolution of PRRS and lineages. McKean Swine Disease Conference. Ames, IA. Nov 3  5, 2021.
• Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: VanderWaal, K. Emergence and spread of PRRS sub-lineages. Allen D. Leman Swine Conference. St. Paul, MN. September 18-21, 2021.
• Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Kikuti, M., [&], VanderWaal, K. Characterization of the current PRRS Lineage 1C 1-4-4 outbreak. Allen D. Leman Swine Conference. St. Paul, MN. September 18-21, 2021.
• Type: Journal Articles Status: Published Year Published: 2022 Citation: Nakarin Pamornchainavakul, Mariana Kikuti, Igor A. D. Paploski, Dennis N. Makau, Albert Rovira, Cesar A. Corzo, Kimberly VanderWaal. 2022. 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 (Frontiers in Veterinary) https://doi.org/10.3389/fvets.2022.846904
• Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: C. A. Corzo, M. Kikuti, I. A. D. Paploski, N. Pamornchainavakul, C. Picasso-Risso, D. Makau, A. Rovira, K. VanderWaal. Understanding the emergence of a new PRRSV variant (L1C 1-4-4) through the implementation of epidemiological tools. American Association of Swine Veterinarians (AASV) Annual meeting, Indianapolis, Indiana, USA https://doi.org/10.54846/am2022/164
• Type: Journal Articles Status: Published Year Published: 2021 Citation: Mariana Kikuti, Juan Sanhueza, Carles Vilalta, Igor Adolfo Dexheimer Paploski,Kimberly VanderWaal, Cesar A. Corzo. Porcine reproductive and respiratorysyndrome virus 2 (PRRSV-2) genetic diversityand occurrence of wild type and vaccine-likestrains in the United States swine industry PLoS ONE 16(11): e0259531. https://doi.org/10.1371/journal.pone.0259531 Emergence of a New Lineage 1C Variant of Porcine Reproductive and Respiratory Syndrome Virus 2 in the United States.
• Type: Journal Articles Status: Published Year Published: 2021 Citation: Kikuti M, Paploski IAD, Pamornchainavakul N, Picasso-Risso C, Schwartz M, Yeske P, Leuwerke B, Bruner L, Murray D, Roggow BD, Thomas P, Feldmann L, Allerson M, Hensch M, Bauman T, Sexton B, Rovira A, VanderWaal K and Corzo CA (2021) Emergence of a New Lineage 1C Variant of Porcine Reproductive and Respiratory Syndrome Virus 2 in the United States. Front. Vet. Sci. 8:752938. https://doi.org/10.3389/fvets.2021.752938
• Type: Journal Articles Status: Accepted Year Published: 2022 Citation: Dennis N. Makau, Samantha Lycett, Matthew Michalska-Smith, Igor Paploski, Maxim Cheeran, Meggan Craft, Rowland Kao, Declan Schroeder, Andrea Wilson. Ecological and evolutionary dynamics of multi-strain viruses (Nature Ecology and Evolution).
• Type: Journal Articles Status: Published Year Published: 2022 Citation: Michalska-Smith, M., K. VanderWaal, M. Torremorell, C. Corzo, M.E Craft. 2022. Asymmetric host movement reshapes local disease dynamics in metapopulations. Nature Scientific Reports 12: 9365
• Type: Journal Articles Status: Accepted Year Published: 2021 Citation: Paploski IAD, Bhojwani RK, Sanhueza JM, Corzo CA, VanderWaal K. Forecasting viral disease outbreaks at the farm-level for commercial sow farms in the U.S. Prev Vet Med. 2021:105449.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2021 Citation: Makau, D, Moh A. Alkhamis, Igor A. D. Paploski, Cesar A. Corzo, Samantha Lycett, Kimberly VanderWaal. Integrating animal movements with phylogeography to model the spread of PRRS virus in the U.S. Conference of Research Workers in Animal Diseases (CRWAD) Chicago, IL. December 3-7, 2021.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2021 Citation: Pamornchainvakul, N., [&], K. VanderWaal. Revealing invisible links and modes of between-farm PRRSV transmission using genetic-based network analysis. Conference of Research Workers in Animal Diseases (CRWAD) Chicago, IL. December 3-7, 2021.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2021 Citation: Paploski, I, [&], K. VanderWaal. Multi-strain dynamics of PRRSV type-2 in U.S. pig populations. Conference of Research Workers in Animal Diseases (CRWAD) Chicago, IL. December 3-7, 2021
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2021 Citation: Makau, D, Moh A. Alkhamis, Igor A. D. Paploski, Cesar A. Corzo, Samantha Lycett, Kimberly VanderWaal. Integrating animal movements with phylogeography to model the spread of PRRS virus in the U.S. Epidemics8, International Conference on Infectious Disease Dynamics (virtual format). Nov 30  Dec 3, 2021

Progress 08/15/20 to 08/14/21

Outputs
Target Audience: Scientists and epidemiologists Government and policy making agencies Project participants from study regions for the longitudinal sampling study Swine veterinarians Industry players (swine production companies, pharmaceutical companies biosecurity companies etc.) Changes/Problems: Due for supply chain disruptions by COVID-19 , we have had some challenges in procuring certain consumables for our sample processing, immunological assays and sequencing. However, we Identified alternative ways of procuring the materials and are now able to proceed with our wet lab activities. Some delays with sequencing have also been due to hacking of our sequencing server but we have since migrated our work to more secure platforms and hope that these challenges will not recur. What opportunities for training and professional development has the project provided? The project has enhanced four postdoctoral research associates who are learning different aspects of phylodynamic and phylogeographic analysis. Two graduate student undertaking his PhD have the opportunity to participate in experiments and use data generated from the project to learn and enhance their skills on immunology, sequence assembly and analysis. How have the results been disseminated to communities of interest?We have held several meetings with interested swine industry players including company veterinarians and vaccine manufacturing companies. These meetings have been good forums for sharing observed trends of PRRSV spread and evolution over the last 10 years. Moreover, vaccine companies have been keen to understand the importance of observed viral phylogenetic divergence in relation to market vaccines and their effectiveness in PRRS management. From these discussions, both swine production companies and pharmaceutical companies have been discussing how the current knowledge would modify vaccine development in the future and employment of different immunization regimes in swine farms. Findings of this project have so far been presented in industry-oriented meetings largely attended by the target audience, the 2091 & 2020 Allen D. Leman Swine Conference and the 2019-2021 Swine Disease Eradication Center meetings. Additionally, some of the research findings have been disseminated through scientific manuscripts published in peer reviewed journals and other media outlets. Below is a detailed list of these outreach efforts: Academic conference attended by industry: Dennis Makau, Mohammad Alkhamis, Igor Paploski, Cesar Corzo, Kimberly VanderWaal Feeder pig movements as a potential driver of the spread of PRRS lineage 1 viruses in the US (27th Feb-2nd March 2021). In American Association of Swine Veterinarians (AASV) Annual meeting (virtual format). N. Pamornchainavakul, I. Paploski, D. Makau, C. Corzo, K. VanderWaal. Estimating farm-level reproduction numbers for PRRSV using sequence-based transmission tree analysis (27th Feb-2nd March 2021). In American Association of Swine Veterinarians (AASV) Annual meeting (virtual format). Paploski, D. Makau, N. Pamornchainavakul, D. Schroeder, A. Rovira, K. VanderWaal Estimating viral population size of PRRSV sublineages based on genetic diversity (27th Feb-2nd March 2021). In American Association of Swine Veterinarians (AASV) Annual meeting (virtual format). Kimberly VanderWaal, Igor Paploski, Rahul Bhojwani, Andres Perez, Cesar Corzo: Evolution of PRRSV: lessons from the last 10 years and implications for the future. AASV Annual Meeting. March 7-10, 2020, Atlanta, GA. Paploski, I., C. Corzo, A. Rovira, M. Murtaugh, J. Sanhueza, C. Vilalta, D.C. Schroeder, K. VanderWaal: Temporal dynamics of co-circulating lineages of porcine reproduction and respiratory syndrome virus. Epidemics 7. Dec 03 - 06 2019, Charlston, SC. Igor Paploski, Mariana Kikuti, C. Corzo, K. VanderWaal: Dynamics of PRRSV in the US over the past decade: A molecular epidemiology perspective. Allen D. Leman Swine Conference, St. Paul, MN Sept 14-17, 2019. Technical seminars and presentations for industry audience: Kimberly VanderWaal : Invited speaker. Veterinary Advisory Board for Elanco Animal Health (Pharmaceuticals - PRRS vaccine producer. Emergence and spread of PRRS sub-lineages. August 31, 2021. Kimberly VanderWaal : Invited speaker. Veterinary Advisory Council for Phibro Animal Health (Pharmaceuticals - PRRS vaccine producer). Understanding emergence, spread, and classification of PRRSV variants. July 28, 2021. Colorado Springs, CO. Kimberly VanderWaal : Invited speaker. Morrison Forum for Advancing Swine Production Medicine. Network analysis of livestock movement data. April 2, 2021. Kimberly VanderWaal : Speaker. UMN/Zoetis Biosurveillance Review. Research updates on PRRS Lineages. May 3, 2021. Kimberly VanderWaal : Speaker at the annual meeting of the board of directors for the Swine Disease Eradication Center. Risks associated with feeder pig movements.(2020) Kimberly VanderWaal : Speaker at the annual meeting of the board of directors for the Swine Disease Eradication Center. Genetic diversity and evolution of PRRSV. St. Paul, MN. (2020) Dennis Makau: "Network connectivity and phylogeographic diffusion of PRRSV sub-lineage 1A". Swine Seminar, Swine Health Information Center, October 23rd, 2020. Nakarin Pamornchainavakul: "How can Time-based phylogeny tell us about the disease transmission: The preliminary study of PRRSV transmission tree inference". Graduate Seminar Series, University of Minnesota, Jan 30th, 2020. Kimberly VanderWaal : Featured speaker in National Hog Farmer's "Science talks" webinar series. A review of PRRS nomenclature and currently circulating PRRSV strains in the U.S. https://www.nationalhogfarmer.com/animal-health/webinar-examines-current-prrsv-strains-us. Jul 18, 2019 Kimberly VanderWaal : "New developments in PRRS epidemiology and lineages". Elanco technical seminar at the American Association of Swine Veterinarians annual meeting. March 9th, 2019. Orlando, FL. Igor Paploski: "Temporal dynamics of PRRSv in US swine populations". Swine Seminar, Swine Health Information Center, August 16th, 2019. Outreach articles: 2021 Morison Swine Health Monitoring Program: Phylogenetic Structure and Sequential Dominance of Sub?Lineages of PRRSV Type?2 Lineage 1 in the United States. By I. Paploski, N Pamornchainavakul, DN Makau, A Rovira, CA Corzo, DC Schroeder, MC-J Cheeran, A Doeschl-Wilson, RR Kao, S Lycett, K VanderWaal. https://umnswinenews.com/2021/08/27/phylogenetic-structure-and-sequential-dominance-of-sub-lineages-of-prrsv-type-2-lineage-1-in-the-united-states-2/ 2021 Article in National Hog Farmer:" Understanding stability of movement networks through time can inform decisions for designing, implementing disease management." By: D. Makau, I. Paploski, K. VanderWaal. https://www.nationalhogfarmer.com/news/practical-use-pig-movement-data-disease-interventions 2021 Morison Swine Health Monitoring Program: "Temporal stability of swine movement networks in the U.S." By : D. Makau, I. Paploski and K. VanderWaal https://umnswinenews.com/2021/06/18/temporal-stability-of-swine-movement-networks-in-the-u-s-2/ 2021 UMN swine news: Dynamic network connectivity influences the spread of a sub-lineage of PRRS virus. By: D. Makau, I. Paploski, C. Corzo and K. VanderWaal. https://umnswinenews.com/2021/03/09/dynamic-network-connectivity-influences-the-spread-of-a-sub-lineage-of-prrs-virus/ 2020 Article in National Hog Farmer: "Animal movements: The Achilles heel for PRRS management" By: Dennis Makau, Igor Paploski, Cesar Corzo and Kimberly VanderWaal. https://www.nationalhogfarmer.com/animal-health/animal-movements-achilles-heel-prrs-management 2020 Morison Swine Health Monitoring Program: "How much is the spread of 1?7?4?related PRRS viruses due to animal movement?" By: Dennis Makau, Igor Paploski, Cesar Corzo and Kimberly VanderWaal. https://umnswinenews.com/2020/05/22/how-much-is-the-spread-of-1-7-4-related-prrs-viruses-due-to-animal-movement/ 2020 Article in Pig Health Today: "Outmaneuvering PRRSV requires a better understanding of genetic diversity" By: Kimberly VanderWaal. https://www.swineweb.com/outmaneuvering-prrsv-requires-a-better-understanding-of-genetic-diversity/ 2019 Article in National Hog Farmer: "Why is PRRS virus so genetically diverse?" By: Igor Paploski, Cesar Corzo, Albert Rovira, Juan Sanhueza, Carles Vilalta and Kimberly VanderWaal. https://www.nationalhogfarmer.com/animal-health/why-prrs-virus-so-genetically-diverse Other submitted conference abstracts: These conferences are yet to be held although calls for abstracts have been honored. 2021 Emergence and spread of PRRS virus sub-lineages 2021 Revealing the invisible links and modes of between-farm PRRSV transmission using genetic-based network analysis 2021 PRRSV type-2 Lineage 1 diversity and sequential dominance in the U.S. 2021 Integrating animal movements with phylogeography to model the spread of PRRS virus in the U.S. What do you plan to do during the next reporting period to accomplish the goals?Having resumed near normal operations at the university, we have now restarted all halted experiments and we hope to continue undertaking the different project activities with minimal disruptions. Details of how we intend to proceed are: Objective 1 - We hope to finalize the viral expansion phase in live animals for the remaining 6 lineages. We will then inoculate other pigs to generate hyperimmune sera as initially planned and processed with the invitro cross-neutralization assays and other immunological assays. Objective 2 - Since all samples have been received and cataloged, we plan to complete RNA extraction using the optimized protocols and sequence the isolated viruses. We will also share the results of viral sequencing with the participating farms. Objective 3c - Since this objective relies on the optimized models from the analysis time-varying effect of the immune profile and phylodynamic models, we will use the models from the analysis in objective 3 a & b to analyze data obtained from the longitudinal study. Subsequently these findings shall also be published. Objective 4 - We intend to continue optimizing the phylodynamic models generated using retrospective PRRS ORF5 sequences, add new whole genome sequences data and spatial data as they become available form objective 2 to optimize it. Furthermore, we intend to present our research findings in the upcoming conferences (CRWAD 2021, Epidemics 2021, and Allen D. Leman Swine Conference 2021).

Impacts
What was accomplished under these goals? Objective 1a - Completion status = 35% Several steps have been undertaken towards the achievement of this objective. An experiment to generate hyperimmune serum to one of the target viral lineages has been successfully completed. Protocols for immunological assays and detection antibody producing cells have also been standardized and can be applied to other viral lineages of interest. We also expanded adequate volumes and titers for two of the target isolates to be used for both hyper immune sera generation and the infection chain experiment (objective 1d). Plans to expand the remaining isolates and complete the hyperimmune sera generation are currently underway. Objective 2a - Completion status = 50% We have completed sample collection from all farms initially recruited for this phase of the project (November 2019-January 2021). All farms submitted samples for up to12 months on a majority frequency. All samples have been catalogued and RNA extraction is currently underway. Sequencing pipelines have also been developed in readiness for sequencing and assembly of the data. Objective 2b & 2c - Completion status = 5% Due to supply chain disruptions for consumable materials due to COVID-19, a hacking event of our sequencing server, the scheduled lab work to evaluate genetic distance of viruses isolated from farms participating in the longitudinal study was delayed. Although initial steps of sample processing, virus isolation protocols and sequence assembly have so far been optimized, we are yet to commence full profiling of the field viruses, but we hope to undertake these procedures swiftly in the next few months. Objective 3a - Completion status = 80% Using virus isolate data and animal movement data from farms in two production systems in the US, we have conducted temporal correlation analyses to evaluate the effect of previous exposures on PRRS occurrence on farms. This manuscript has been published. From these analyses, several time-varying exposures were observed to influence PRRS occurrence on farms. These included prior exposure to PRRS 6 months before, frequency of animal shipments, and direct and indirect farm contacts via animal movements. The methods developed under this objective can also be applied to other studies, while the findings of the study will help guide decisions on disease prevention and management. Objective 3b - Completion status= 80% Phylogenetic analysis of drivers of spatial transition of PRRS have been developed using previously collected data. This analysis has been completed and published. From the analysis we observed that movement of feeder pigs shaped the spatial spread of PRRSV L1A more than weaned and breeding pig movements and spatial proximity and farm density were also factors that enhanced the spatial spread of the PRRSV. Objective 3c - Completion status = 25% Delays in generating the sequence data from the longitudinal phase (objective 2) due to the challenges mentioned above have slowed down model parameterization process. However, we have been able to use previously collected PRRS data and add some spatial data to develop and enhance phylodynamic models to be used in this project. Current efforts are aimed and optimizing immunity related model parameters for PRRSV before commencing simulations model tuning. The model framework has already been tested using thousands of SAR-CoV2 sequences available to us which helps us quicken a model development process for PRRSV. Objective 4a - Completion status = 80% Analysis of temporal dynamics of PRRS populations in the US has been done using multiple datasets and published. These data have included sequence data from Morison Swine Health Monitoring Project, the UMN Veterinary Diagnostic Laboratory and other collaborating VDLs. From the analysis it is evident that a new PRRSV lineage emerge every 3-4 years and over the past decade there most of the dominant viruses have belonged to lineage 1. Certain mutation shave also been identifying to occur parallel to the lineage turnover and these are being investigated further. Objective 4a & 4b - Completion status = 30% From the knowledge obtained in objectives 1a, 3a-c and 4a, we are developing multi-strain models using old and new data to answer questions on cross-immunity, dominance and co-circulation of old and emerging PRRSV.

Publications

• Type: Journal Articles Status: Published Year Published: 2021 Citation: Schroeder DC, Odogwu NM, Kevill J, Yang M, Krishna VD, Kikuti M, Pamornchainavakul N, Vilalta C, Sanhueza J, Corzo CA, Rovira A, Dee S, Nelson E, Singrey A, Zhitnitskiy P, Balestreri C, Makau DN, Paploski IAD, Cheeran MC, VanderWaal K, Torremorell M. 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. Microbiol Resour Announc. 2021 Aug 19;10(33):e0026021. https://journals.asm.org/doi/10.1128/MRA.00260-21
• Type: Journal Articles Status: Published Year Published: 2021 Citation: Dennis N. Makau, Moh A. Alkhamis, Igor A. D. Paploski, Cesar A. Corzo, Samantha Lycett, Kimberly VanderWaal, 2021. Integrating animal movements with phylogeography to model the spread of PRRS virus in the U.S. Virus Evolution. https://doi.org/10.1093/ve/veab060
• Type: Journal Articles Status: Published Year Published: 2021 Citation: Paploski IAD, Pamornchainavakul N, Makau DN, Rovira A, Corzo CA, Schroeder DC, Cheeran MC-J, Doeschl-Wilson A, Kao RR, Lycett S, VanderWaal K, 2021. Phylogenetic structure and sequential dominance of sub-lineages of PRRSV Type 2 Lineage 1 in the United States; Vaccines. https://doi.org/10.3390/vaccines9060608
• Type: Journal Articles Status: Published Year Published: 2021 Citation: U.S.; Preventive Veterinary Medicine. https://doi.org/10.1016/j.prevetmed.2021.105369 Dennis N. Makau, Igor A. D. Paploski, Cesar A. Corzo, Kimberly VanderWaal, 2021. Dynamic network connectivity influences the spread of a sub-lineage of porcine reproductive and respiratory syndrome virus; Transboundary and Emerging Diseases. https://doi.org/10.1111/tbed.14016

Progress 08/15/19 to 08/14/20

Outputs
Target Audience: Scientists and epidemiologists Government and policy making agencies Project participants from study regions for the longitudinal sampling study Swine veterinarians and producers Industry players (swine production companies, pharmaceutical companies, biosecurity companies etc.) Changes/Problems: We made some modifications to objective 1c to enable us to answer our research question more clearly. Instead of analyzing the immunological important epitopes, this analysis was replaced with a cross neutralization test for the nine different viral lineages. This is a more functional approach will help us understand the relationship between host immune response and epidemiology of the infective virus. Due to workplace disruptions occasioned by COVID-19, we have had to adjust our timelines for different aspects of the project. These delays have been experienced most in objectives that involve wet lab activities in objectives 1 and 2. But with the phased reopening of the university, we hope to resume these activities very soon. We have also had some challenges in culturing some of the virus isolates in primary porcine alveolar macrophages to achieve the adequate infective dose for the live animal inoculation experiments. In addition, some virus isolates do not show the expected CPE on MARC-145 cultures and thus it is difficult to quantify them successfully. However, we have adjusted and made modifications to the study protocols to accommodate alternative ways to increase the virus stocks, such as inoculating viral isolates in live animals to allow for viral replication then isolating the virus for the next steps in the experiment. What opportunities for training and professional development has the project provided? The project has enhanced four postdoctoral research associates who are learning different aspects of phylodynamic and phylogeographic analysis. One graduate student undertaking his PhD has the opportunity to use data generated from the project to learn and enhance their skills on sequence assembly and analysis. How have the results been disseminated to communities of interest?We have held several meetings with interested swine industry players including company veterinarians and vaccine manufacturing companies. These meetings have been good forums for sharing observed trends of PRRSV spread and evolution over the last 10 years. Moreover, vaccine companies have been keen to understand the importance of observed viral phylogenetic divergence in relation to market vaccines and their effectiveness in PRRS management. From these discussions, both swine production companies and pharmaceutical companies have been discussing how the current knowledge would modify vaccine development in the future and employment of different immunization regimes in swine farms. Findings of this project have so far been presented in industry-oriented meetings largely attended by the target audience, the 2019 Allen D. Leman Swine Conference and the 2019-2020 Swine Disease Eradication Center meetings. Additionally, some of the research findings have been disseminated through scientific manuscripts published in peer reviewed journals and other media outlets. Below is a detailed list of these outreach efforts: Academic conference attended by industry: Kimberly VanderWaal, Igor Paploski, Rahul Bhojwani, Andres Perez, Cesar Corzo: Evolution of PRRSV: lessons from the last 10 years and implications for the future. AASV Annual Meeting. March 7-10, 2020, Atlanta, GA. Paploski, I., C. Corzo, A. Rovira, M. Murtaugh, J. Sanhueza, C. Vilalta, D.C. Schroeder, K. VanderWaal: Temporal dynamics of co-circulating lineages of porcine reproduction and respiratory syndrome virus. Epidemics 7. Dec 03 - 06 2019, Charlston, SC. Igor Paploski, Mariana Kikuti: Dynamics of PRRSV in the US over the past decade: A molecular epidemiology perspective. Allen D. Leman Swine Conference, St. Paul, MN Sept 14-17, 2019. Technical seminars for industry audience: Kimberly VanderWaal : "New developments in PRRS epidemiology and lineages". Elanco technical seminar at the American Association of Swine Veterinarians annual meeting. March 9th, 2019. Orlando, FL. Presentations to industry groups: Nakarin Pamornchainavakul: "How can Time-based phylogeny tell us about the disease transmission: The preliminary study of PRRSV transmission tree inference". Graduate Seminar Series, University of Minnesota, January 30th, 2020. Igor Paploski: "Temporal dynamics of PRRSv in US swine populations". Swine Seminar, Swine Health Information Center, August 16th, 2019. Media: 2020 Article in National Hog Farmer: "Animal movements: The Achilles heel for PRRS management" By: Dennis Makau, Igor Paploski, Cesar Corzo and Kimberly VanderWaal. https://www.nationalhogfarmer.com/animal-health/animal-movements-achilles-heel-prrs-management 2020 Morison Swine Health Monitoring Program: "How much is the spread of 1?7?4?related PRRS viruses due to animal movement?" By: Dennis Makau, Igor Paploski, Cesar Corzo and Kimberly VanderWaal. https://umnswinenews.com/2020/05/22/how-much-is-the-spread-of-1-7-4-related-prrs-viruses-due-to-animal-movement/ 2020 Article in Pig Health Today: "Outmaneuvering PRRSV requires a better understanding of genetic diversity" By: Kimberly VanderWaal. https://www.swineweb.com/outmaneuvering-prrsv-requires-a-better-understanding-of-genetic-diversity/ 2019 Article in National Hog Farmer: "Why is PRRS virus so genetically diverse?" By: Igor Paploski, Cesar Corzo, Albert Rovira, Juan Sanhueza, Carles Vilalta and Kimberly VanderWaal. https://www.nationalhogfarmer.com/animal-health/why-prrs-virus-so-genetically-diverse Other submitted conference abstracts: These conferences are yet to be held although calls for abstracts have been honored. 2020 Quantifying sequential dominance of PRRSV strains through classification of sub-lineages 2020 Estimating farm-level reproductive numbers for porcine reproductive and respiratory syndrome virus using sequence-based transmission tree analysis 2020 Role of animal movements in PRRS spread in the U.S. swine industry 2020 Spatiotemporal diffusion of PRRS virus L1A in US swine herds, 2014-2017 What do you plan to do during the next reporting period to accomplish the goals?When the COVID-19 pandemic spread in the US, the University of Minnesota shut its on-site activities for non-essential tasks, which included animal experiments not related to COVID-19 that had not begun yet. As such, we developed a hibernation plan that included provisions on how to deal with activities of this project. The prospective collection of samples from farms throughout the country to accomplish the long-term PRRSv surveillance goal was maintained. These samples are still being collected and sent routinely to us. We developed a plan to ensure that samples are received, cataloged and stored upon arrival. So far we have received samples from participating farms of different PRRSv status from different regions of the country every month since November 2019. Other project goals were temporarily put on hold, such as the animal experiment aimed at generating lineage-specific antibodies to better understand cross-reactivity and humoral immune response between these antibodies. Although these activities have now been put on hold, we have initiated the process to resume laboratory operations through the phased reopening protocols of the university. Details of how we intend to proceed are: Objective 1 - We hope to expand and purify the 9 viral isolates we intent to use for the cross-neutralization experiments. We will then inoculate these isolates in live piglets, extract antisera which will subsequently be used for the invitro cross-neutralization tests. Once genetic and antigenic distances have been established between different viral lineages, chain infection experiment in live animals will then be performed to answer the question in Objective 1. Objective 2 - Since we are still routinely receiving samples of processing fluids and serum from farms participating in the longitudinal study, this engagement with the study farms will continue. As for the samples already received, we plan to optimize virus isolation protocols from processing fluids and proceed with sequencing and typing of the different virus isolated from these farms overtime. We will also share the results of viral sequencing with the participating farms. This engagement with swine production systems will continue until the end of our study period as we plan to share our findings with them through meetings. Objective 3a and 3b - We plan to finalize the manuscripts addressing these objectives and publish the findings in peer reviewed journals. The manuscripts will be submitted to Frontiers in Microbiology, Journal of Transboundary and Emerging Diseases. Objective 3c - Since this objective relies on the optimized models from the analysis time-varying effect of the immune profile and phylodynamic models, we will use the models from the analysis in objective 3 a & b to analyze data obtained from the longitudinal study. Subsequently these findings shall also be published. Objective 4 - We intend to continue optimizing the phylodynamic models generated using retrospective PRRS ORF5 sequence data using the thousands of sequences already available to us to model viral evolution. The methods of model generation and fitting have been improved and inspired by separate work on thousands of SARS-CoV-2 sequences on other projects. Ultimately these models will be used to analyze the data obtained from the longitudinal study as well as objective 1 live animal infection chain experiments. Furthermore, we intend to present our research findings in the upcoming conferences (CRWAD 2020 and Allen D. Leman Swine Conference 2020).

Impacts
What was accomplished under these goals? Objective 1a - Completion status = 23% Several steps have been undertaken towards the achievement of this objective. Firstly, all ethical protocols (IACUC and IBC) have been approved enabling commencement of all relevant in vivo and in vitro experiments. We have identified 9 relevant wild type PRRS virus isolates that fall under nine different lineages and have conducted initial tests to assess their biological properties, such viral viability, cytopathic effects, and replication characteristics in primary porcine alveolar macrophages and MARC-145 cells. Based on the replication characteristics, viral isolates will be expanded in cell culture for animal experiments. Preparations for live animal virus inoculations are now underway and adjustments in light of COVID-19 disruptions have been instituted to facilitate these experiments. Objective 2a - Completion status = 36% We have recruited 20 farms with different immunological backgrounds (naïve, vaccinated or previous exposure to field virus) to the study, from which we have continue to receive samples (processing fluids and/serum) for laboratory analysis. Majority of these have consistently sent samples on a monthly basis since November 2019 and continue to participate in the study. We anticipate that these farms will continue to participate in the study till the end of our sampling period. Due to COVID-19, there have been delays in the isolation and sequencing of viruses from the processing fluids and serum samples received so far. However, we hope to resume wet lab activities as soon as university operations resume. Objective 2b, 2c & 2d - Completion status = 5% Due to COVID-19 disruptions, the scheduled lab work to evaluate genetic distance of viruses isolated from farms participating in the longitudinal study was delayed. Although initial steps of sample processing, virus isolation protocols and sequence assembly have so far been optimized, we are yet to commence actual profiling of the field viruses. Given the progress made on setting up protocols and a robust assembly pipeline, we hope to move swiftly with these analyses once university operations resume. Objective 3a - Completion status = 75% Using virus isolate data and animal movement data from farms in two production systems in the US, we have conducted temporal correlation analyses to evaluate the effect of previous exposures on PRRS occurrence on farms. These analysis have been completed and a draft manuscript has been prepared. From these analyses, several time-varying exposures were observed to influence PRRS occurrence on farms. These included prior exposure to PRRS 6 months before, frequency of animal shipments, and direct and indirect farm contacts via animal movements. The methods developed under this objective can also be applied to other studies, while the findings of the study will help guide decisions on disease prevention and management. Objective 3b - Completion status= 30% Phylogenetic analysis of drivers of spatial transition of PRRS have been developed using previously collected data. These models have been optimized and are now being tested on the previously collected data to identify factors that enhance spread of certain PRRS viral strains. Objective 3c - Completion status = 5% COVID-19 disruptions notwithstanding, we have been able to use previously collected PRRS data to develop and enhance phylodynamic models to be used in this project. Furthermore, these models are also being optimized using thousands of SAR-CoV2 sequences available to us to model viral evolution. Ultimately these optimized computational tools will be used to understand viral evolutionary dynamics of PRRS reducing the execution time for this project. Objective 4a & 4b - Completion status = 30% Analysis of temporal dynamics of PRRS is underway using different ORF5 PRRS sequence datasets available to us from different diagnostic laboratories. We have so far been able to develop and publish the outcome of an initial analysis of the genetic diversity and dynamics of co-circulating PRRS viruses for the last 10 years in the US. These methods are now being applied to other datasets to enable the development of multi-strain network models.

Publications

• Type: Journal Articles Status: Published Year Published: 2020 Citation: Kimberly VanderWaal, Igor A.D. Paploski, Dennis N. Makau, Cesar A. Corzo 2020. Contrasting animal movement and spatial connectivity networks in shaping transmission pathways of a genetically diverse virus; Preventive Veterinary Medicine. https://doi.org/10.1016/j.prevetmed.2020.104977
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Paploski, I. A. D., Corzo, C., Rovira, A., Murtaugh, M. P., Sanhueza, J. M., Vilalta, C., Schroeder, D.C., VanderWaal, K. (2019). Temporal Dynamics of Co-circulating Lineages of Porcine Reproductive and Respiratory Syndrome Virus. Frontiers in Microbiology, 10, 123. https://doi.org/10.3389/fmicb.2019.02486
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Kimberly VanderWaal, Igor Paploski, Rahul Bhojwani, Andres Perez, Cesar Corzo. "Evolution of PRRSV: lessons from the last 10 years and implications for the future" Poster presentation at the American Association of Swine Veterinarians Annual Meeting. March 7-10, 2020, Atlanta, GA.
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Paploski, I., C. Corzo, A. Rovira, M. Murtaugh, J. Sanhueza, C. Vilalta, D.C. Schroeder, K. VanderWaal. Temporal dynamics of co-circulating lineages of porcine reproduction and respiratory syndrome virus. Poster presentation at Epidemics 7. Dec 03 - 06 2019, Charleston, SC.
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Kikuti M, Sanhueza J, Vilalta C, VanderWaal K and Corzo C. Genetic and spatio-temporal patterns of porcine reproductive and respiratory syndrome virus in U.S. swine populations. Front. Vet. Sci. Conference Abstract: GeoVet 2019. Novel spatio-temporal approaches in the era of Big Data. Davis, CA. Oct 8-10, 2019.
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Makau, D.N., I.A.D Paploski, C. Corzo, K. VanderWaal. Farm contacts and PRRSV occurrence in the U.S. swine industry: the time factor. Poster presentation at University of Minnesota College of Veterinary Medicine Points of Pride Research Day. October 4, 2019, St. Paul, MN.
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Paploski, I., C. Corzo, A. Rovira, M. Murtaugh, J. Sanhueza, C. Vilalta, D.C. Schroeder, K. VanderWaal. Temporal dynamics of co-circulating lineages of porcine reproduction and respiratory syndrome virus. Poster presentation at University of Minnesota College of Veterinary Medicine Points of Pride Research Day. October 4, 2019, St. Paul, MN.
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Kikuti, M. J. Sanhueza, C. Vilalta, K. VanderWaal, C. Corzo. Spatio-temporal dynamics of porcine reproductive and respiratory syndrome virus (PRRSv). Poster presentation at University of Minnesota College of Veterinary Medicine Points of Pride Research Day. October 4, 2018, St. Paul, MN.