Publications

Using diagnostic data from veterinary diagnostic laboratories to unravel macroepidemiological aspects of porcine circoviruses 2 and 3 in the United States from 2002–2023

Porcine circoviruses (PCVs), including porcine circovirus 2 (PCV2) and porcine circovirus 3 (PCV3), have been associated with clinical syndromes in swine, resulting in significant economic losses. To better understand the epidemiology and clinical relevance of PCV2 and PCV3, this study analyzed a dataset comprising diagnostic data from six veterinary diagnostic laboratories (VDLs) in the United States of America. The data comprised of polymerase chain reaction (PCR) test results, sample type, and age group for PCV2 and PCV3 submissions from 2002-2023. Findings indicated a decrease in the percentage of PCV2-positive submissions after introducing a commercial PCV2 vaccine in 2006 and a resurgence in positivity after 2018, particularly in breeding herds, associated with an increased number of submissions using processing fluid samples. After its first report in the U.S. in 2016, PCV3 detection had an upward trend in the percentage of positive cases, peaking in spring 2023. PCV3 detection was more frequent in adult/sow farms, while PCV2 was more frequently detected in the wean-to-market category. An additional analysis used results from tissue diagnostic data from 2019-2023 from one VDL to associate PCR cycle threshold (Ct) values with the probability of confirming a PCV2 or PCV3 disease diagnosis confirmation. An interpretative PCR Ct cutoff for PCV2 and PCV3 diagnoses was assessed based on the logistic regression model associating Ct values with the presence of tissue lesions. The analysis considered only cases tested for PCV2 and PCV3 by PCR with tissue evaluations by diagnosticians. An interpretative Ct cutoff of 22.4 for PCV2 was associated with a high probability of confirming a diagnosis of PCV2 clinical disease through histopathology. For PCV3, the interpretative cutoff with the highest performance was 26.7. These findings contribute to the ongoing efforts to monitor and understand the clinical relevance of PCV2 and PCV3 PCR results, identifying potential disease challenges.

Optimizing Tongue Fluid Sampling and Testing Protocols for Enhanced PRRSV Isolation from Perinatal Swine Mortalities

Porcine reproductive and respiratory syndrome virus (PRRSV) remains a major concern for swine health. Isolating PRRSV is essential for identifying infectious viruses and for vaccine formulation. This study evaluated the potential of using tongue fluid (TF) from perinatal piglet mortalities for PRRSV isolation. Four collection protocols were tested: extracting TF from fresh tissues using phosphate-buffered saline (PBS group), extracting TF from fresh tissues using virus transportation medium (VTM group), extracting TF from freeze-thawed tissue (freeze-thaw group), and using tissue homogenates (homogenate group). Two cell lines (ZMAC and MARC-145) and primary alveolar macrophages (PAM) were evaluated for their effect on successful PRRSV isolation. An eligible PRRSV-positive unstable breeding herd in Midwestern USA was chosen for the study. Tongues were collected in 20 batches (~30 mortalities per batch). Within each batch, each tongue tissue was cut into four quarters, with each quarter randomly assigned to one of the four collection protocols and RT-qPCR tested. Virus isolation (VI) was attempted on 10 batches. The mean RT-qPCR cycle threshold (Ct) values for the PBS, VTM, freeze-thaw, and homogenate groups were 21.9, 21.8, 22.6, and 24.8, respectively. The VI success rate was 22.6%, 12.1%, 2.8%, and 2.8% in the PBS, VTM, freeze-thaw, and homogenate groups, respectively. The probability of successful VI was 3.1% and 21.0% in the MARC-145 and ZMAC cell lines, respectively, and 4.8% in the PAM cells. TF from perinatal mortalities is an option for PRRS VI, aiding in PRRSV monitoring and control programs.

Evaluation of hepatitis E virus infection between different production systems of pigs in Brazil

PRRSV RNA Detection and Predictive Values Between Different Sow and Neonatal Litter Sample Types

Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), a major challenge for the swine industry, can be transmitted both vertically and horizontally. Common sample types, including processing fluid, serum, and family oral fluid, can neither determine whether PRRSV infection originates vertically or horizontally nor directly reflects the sow's PRRSV status. At around 90 days post live-virus inoculation (LVI), 555 sows were sampled by Tonsil-oral-scrubbing (TOSc) 2 weeks pre-farrowing and tested for PRRSV RNA. From these, 59 PRRSV-positive sows, and 88 PRRSV-negative sows matched by parity were conveniently selected. TOSc from sows, blood swabs from live piglet, and tongue fluid (TF) plus serum from dead piglet were collected individually from all study litters within 12 h post-farrowing. The pre-farrowing TOSc samples had significantly higher PRRSV positivity than TF, serum and blood swab pools, while dead piglet serum had significantly lower mean Ct values than all other sample types. TOSc samples had 25% positive predictive value (PPV) for "live litter" PRRSV status while the pre-farrow and post-farrow TOSc had 87.2% and 89.0% negative predictive value (NPV), respectively. In conclusion, we characterized PRRSV RNA detection among all sub-populations within a litter with easy-to-use TOSc samples and neonatal litter samples, suggesting the occurrence of vertical transmission 90 days post-LVI in sows. TOSc samples from sows had low PPV and high NPV for their respective litter's PRRSV status.

Polioencephalomyelitis in Domestic Swine Associated With Porcine Astrovirus Type 3

In the past decade, different members of the genus Mamastrovirus have been associated with outbreaks of neurologic disease in humans, cattle, sheep, mink, and, most recently, porcine astrovirus 3 (PoAstV3) in swine. We performed a retrospective analysis of 50 cases of porcine neurologic disease of undetermined cause but with microscopic lesions compatible with a viral encephalomyelitis to better understand the role and pathogenesis of PoAstV3 infection. Nucleic acid was extracted from formalin-fixed paraffin-embedded (FFPE) tissue for reverse transcription quantitative polymerase chain reaction (RT-qPCR) testing for PoAstV3. In addition, 3 cases with confirmed PoAstV3-associated disease were assayed by RT-qPCR to investigate PoAstV3 tissue distribution. PoAstV3 was detected in central nervous system (CNS) tissue via RT-qPCR and in situ hybridization in 13 of 50 (26%) FFPE cases assayed. PoAstV3 was rarely detected in any tissues outside the CNS. Positive cases from the retrospective study included pigs in various production categories beginning in 2010, the earliest year samples were available. Based on these results, PoAstV3 appears to be a recurring putative cause of viral encephalomyelitis in swine that is rarely detected outside of the CNS at the time of clinical neurologic disease, unlike other common viral causes of neurologic disease in swine.

Antigenic evolution of H3N2 influenza A viruses in swine in the United States from 2012 to 2016

Abstract Background Six amino acid positions (145, 155, 156, 158, 159, and 189, referred to as the antigenic motif; H3 numbering) in the globular head region of hemagglutinin ( HA 1 domain) play an important role in defining the antigenic phenotype of swine Clade IV (C‐ IV ) H3N2 IAV , containing an H3 from a late 1990s human‐to‐swine introduction. We hypothesized that antigenicity of a swine C‐ IV H3 virus could be inferred based upon the antigenic motif if it matched a previously characterized antigen with the same motif. An increasing number of C‐ IV H3 genes encoding antigenic motifs that had not been previously characterized were observed in the U.S. pig population between 2012 and 2016. Objectives A broad panel of contemporary H3 viruses with uncharacterized antigenic motifs was selected across multiple clades within C‐ IV to assess the impact of HA 1 genetic diversity on the antigenic phenotype. Methods Hemagglutination inhibition ( HI ) assays were performed with isolates selected based on antigenic motif, tested against a panel of swine antisera, and visualized by antigenic cartography. Results A previously uncharacterized motif with low but sustained circulation in the swine population demonstrated a distinct phenotype from those previously characterized. Antigenic variation increased for viruses with similar antigenic motifs, likely due to amino acid substitutions outside the motif. Conclusions Although antigenic motifs were largely associated with antigenic distances, substantial diversity among co‐circulating viruses poses a significant challenge for effective vaccine development. Continued surveillance and antigenic characterization of circulating strains is critical for improving vaccine efforts to control C‐ IV H3 IAV in U.S. swine.

Probability of Influenza A virus RNA detection at different pooling levels for commonly used sample types in swine breeding herds

Complete Genome Sequences of Two Novel Human-Like H3N2 Influenza A Viruses, A/swine/Oklahoma/65980/2017 (H3N2) and A/Swine/Oklahoma/65260/2017 (H3N2), Detected in Swine in the United States

Two novel human-like H3N2 influenza A virus strains, A/swine/Oklahoma/65980/2017 (H3N2) and A/swine/Oklahoma/65260/2017 (H3N2), were isolated from porcine samples submitted to the Iowa State University Veterinary Diagnostic Laboratory in the United States.

Detection and Characterization of Influenza A Virus Endemic Circulation in Suckling and Nursery Pigs Originating from Vaccinated Farms in the Same Production System

Inactivated influenza A virus (IAV) vaccines help reduce clinical disease in suckling piglets, although endemic infections still exist. The objective of this study was to evaluate the detection of IAV in suckling and nursery piglets from IAV-vaccinated sows from farms with endemic IAV infections. Eight nasal swab collections were obtained from 135 two-week-old suckling piglets from four farms every other week from March to September 2013. Oral fluid samples were collected from the same group of nursery piglets. IAV RNA was detected in 1.64% and 31.01% of individual nasal swabs and oral fluids, respectively. H1N2 was detected most often, with sporadic detection of H1N1 and H3N2. Whole-genome sequences of IAV isolated from suckling piglets revealed an H1 hemagglutinin (HA) from the 1B.2.2.2 clade and N2 neuraminidase (NA) from the 2002A clade. The internal gene constellation of the endemic H1N2 was TTTTPT with a pandemic lineage matrix. The HA gene had 97.59% and 97.52% nucleotide and amino acid identities, respectively, to the H1 1B.2.2.2 used in the farm-specific vaccine. A similar H1 1B.2.2.2 was detected in the downstream nursery. These data demonstrate the low frequency of IAV detection in suckling piglets and downstream nurseries from farms with endemic infections in spite of using farm-specific IAV vaccines in sows.

Comparison of tonsil-oral-scrubbing with serum, oral fluid, and tonsil scraping to detect PRRSV RNA in sows over time following live virus inoculation

Introduction Current common sample types for sows, such as serum and tonsil scraping, require snaring the animals, which can be labor-intensive and raise concerns regarding animal welfare. Obtaining oral fluids (OF) from individual sows in field conditions presents challenges, as not all sows readily respond to the rope method. The Tonsil-Oral-Scrubbing (TOSc) collector allows for the rapid retrieval of fluids from the sow’s oral and tonsillar areas without the need for snaring. Previous studies have reported comparable detection rates of porcine reproductive and respiratory syndrome virus (PRRSV) RNA between TOSc and tonsil scraping, with significantly higher positivity observed in TOSc compared to serum in acutely infected sows. Methods Given that PRRSV RNA detection rates can vary among different sample types and fluctuate over time, this field study aimed to compare PRRSV real-time reverse-transcription polymerase chain reaction (RT-rtPCR) positivity and cycle threshold (Ct) values between TOSc, serum, OF, and tonsil scraping at three time points following live-virus inoculation (LVI) in sows. This study was conducted within a breeding herd attempting to eliminate PRRSV following an outbreak. Four sample types were collected from each of the 61 conveniently selected sows at 30, 60, and 90 days post-LVI in the order of OF, TOSc, tonsil scraping, and serum, and subsequently tested for PRRSV RNA. Results The results indicated that TOSc and tonsil scraping exhibited decreased PRRSV RNA detection rates over time, whereas the detection rates for OF and serum remained relatively stable. Moreover, the median Ct values for TOSc and tonsil scraping were numerically lower than those for OF and serum at all sampling points. Specifically, tonsil scraping demonstrated significantly higher PRRSV RNA positivity than the other three sample types. TOSc also exhibited significantly higher PRRSV RNA positivity than OF and serum at both 30 and 60 days post-LVI. By 90 days post-LVI, there was a significant difference in the PRRSV RNA detection rates between TOSc and tonsil scraping. However, no significant difference was observed between TOSc and OF or between TOSc and serum. According to the RT-rtPCR results, most PRRSV RNA-positive sows detected via TOSc and tonsil scraping turned negative by 90 days post-LVI, although a small proportion remained positive. Conversely, a small number of previously negative sows tested positive at 60 and 90 days post-LVI, indicating an intermittent mode of PRRSV RNA detection for both sample types.

Discordant phylodynamic and spatiotemporal transmission patterns driving the long-term persistence and evolution of human coronaviruses

Four distinct species of human coronaviruses (HCoVs) circulate in humans. Despite the recent attention due to SARS-CoV-2, a comprehensive understanding of the molecular epidemiology and genomic evolution of HCoVs remains unclear. Here, we employed primary differentiated human nasal epithelial cells for the successful isolation and genome sequencing of HCoVs derived from two retrospective cohorts in Singapore and Tanzania. Phylodynamic inference shows that HCoV-229E and HCoV-OC43 were subject to stronger genetic drift and reduced purifying selection from the early 2000s onwards, primarily targeting spike Domain A and B. This resulted in increased lineage diversification, coinciding with a higher effective reproductive number (Re>1.0). However, HCoV-NL63 and HCoV-HKU1 experienced weaker genetic drift and selective pressure with prolonged regional persistence. Our findings suggest that HCoV-229E and HCoV-OC43 viruses are adept at generating new variants and achieving widespread intercontinental dissemination driven by continuous genetic drift, recombination, and complex migration patterns.

Machine-learning algorithms to identify key biosecurity practices and factors associated with breeding herds reporting PRRS outbreak

Concentration and Diversity in Collaboration Networks in Microelectronics: A Study on CNPq Productivity Fellows

In-silico characterization of the relationship between the Porcine reproductive and respiratory syndrome virus prevalence at the piglet and litter levels in a farrowing room

Abstract Background Family oral fluids (FOF) sampling has been described as a sampling technique where a rope is exposed to sows and respective suckling litters and thereafter wrung to obtain fluids. PCR-based testing of FOF reveals presence of PRRS virus RNA only at the litter level, as opposed to conventional individual-animal-based sampling methods that demonstrate PRRSV RNA at the piglet level. The relationship between the PRRSV prevalence at the individual piglet level and at the litter level in a farrowing room has not been previously characterized. Using Monte Carlo simulations and data from a previous study, the relationship between the proportion of PRRSV-positive (viremic) pigs in the farrowing room, the proportion of litters in the farrowing room with at least one viremic pig, and the likely proportion of litters to be positive by a FOF RT-rtPCR test in a farrowing room was characterized, taking into account the spatial distribution (homogeneity) of viremic pigs within farrowing rooms. Results There was a linear relationship between piglet-level- and litter-level prevalence, where the latter was always larger than the former. When the piglet-level prevalence was 1%, 5%, 10%, 20%, and 50%, the true-litter level prevalence was 5.36%, 8.93%, 14.29%, 23.21%, and 53.57%, respectively. The corresponding apparent-litter prevalence by FOF was 2.06%, 6.48%, 11.25%, 21.60%, and 51.56%, respectively. Conclusion This study provides matching prevalence estimates to help guide sample size calculations. It also provides a framework to estimate the likely proportion of viremic pigs, given the PRRSV RT-rtPCR positivity rate of FOF samples submitted from a farrowing room.

Forecasting Influenza A Pandemic lineage dominance in the United States using relative reproduction rates

Abstract The 2009 H1N1 pandemic (pdm09) lineage is a major component of the H1 influenza A virus (IAV) that causes seasonal outbreaks annually. Since its introduction in the 2009–10 season, this lineage has evolved into distinct, successive clades in humans. Predicting the fitness of influenza clades is essential to forecasting future prevalence, providing a critical opportunity to develop a response to mitigate infection. The relative fitness of pdm09 lineages was retrospectively inferred via relative reproduction rate (RRe) through RelRe, a programme that implements a renewal equation to estimate the relative difference in reproduction number between cocirculating clades. For this analysis, pdm09 lineage sequences from the USA, collected from 2017 to 2023 in both human and swine hosts, were downloaded from public databases. Clade designations were assigned using Nextclade. Human case count data were divided by each influenza season, and the RRe was estimated at 3-month intervals. The RRe was then used to forecast clade frequency 90 days into the future, and the predictions were compared to the historical data. The highest predicted frequency at 90 days corresponded to the most frequently detected lineage in 9 out of 13 predictions (69%). The pdm09 lineage plays an important role at the human–swine influenza interface. Bayesian inference using both human and swine data indicated unequal transmission rates of the pdm09 lineage, with 53–79 noted transmissions from human to swine and 0–2 in reverse using the available genetic data. Metadata analysis revealed that new clades of pdm09 in humans were typically detected in swine as early as ~8–20 months after clade emergence in humans. Understanding RRe and the fitness of contemporary IAV strains enables the identification of high-risk reverse-zoonotic strains and provides critical time for responding to emergent human clades.

Interpretable machine learning applied to on-farm biosecurity and porcine reproductive and respiratory syndrome virus

Effective biosecurity practices in swine production are key in preventing the introduction and dissemination of infectious pathogens. Ideally, biosecurity practices should be chosen by their impact on bio-containment and bio-exclusion, however quantitative supporting evidence is often unavailable. Therefore, the development of methodologies capable of quantifying and ranking biosecurity practices according to their efficacy in reducing risk have the potential to facilitate better informed choices. Using survey data on biosecurity practices, farm demographics, and previous outbreaks from 139 herds, a set of machine learning algorithms were trained to classify farms by porcine reproductive and respiratory syndrome virus status, depending on their biosecurity practices, to produce a predicted outbreak risk. A novel interpretable machine learning toolkit, MrIML-biosecurity, was developed to benchmark farms and production systems by predicted risk, and quantify the impact of biosecurity practices on disease risk at individual farms. Quantifying the variable impact on predicted risk 50% of 42 variables were associated with fomite spread while 31% were associated with local transmission. Results from machine learning interpretations identified similar results, finding substantial contribution to predicted outbreak risk from biosecurity practices relating to: the turnover and number of employees; the surrounding density of swine premises and pigs; the sharing of trailers; distance from the public road; and production type. In addition, the development of individualized biosecurity assessments provides the opportunity to guide biosecurity implementation on a case-by-case basis. Finally, the flexibility of the MrIML-biosecurity toolkit gives it potential to be applied to wider areas of biosecurity benchmarking, to address weaknesses in other livestock systems and industry relevant diseases.

Assessment of the Relationship Between Bioexclusion Practices Applied in Wean-to-Harvest Sites and PRRS Outbreaks

Porcine reproductive and respiratory syndrome virus (PRRSV) is a significant cause of economic loss in the swine industry, yet its control remains challenging in wean-to-harvest sites. This prospective observational study followed 95 wean-to-harvest sites across six U.S. states for one production cycle. Sites were required to be PRRSV-negative or vaccinated with a modified live virus (MLV) and free of major coronaviruses. Outbreaks were defined as RT-qPCR-positive in unvaccinated sites or detection of ORF5 sequences distinct from the MLV strain. Biosecurity data were collected through a survey, and oral fluids were tested every four weeks. PRRS outbreaks occurred in 14/42 nurseries (33.3%), 8/12 wean-to-finish (66.7%), and 35/41 finishers (82.4%), with lineage 1C.5 most frequently detected. In univariate models, higher odds of outbreaks were associated with transporting pigs of unknown status (OR 9.80, 1.73–55.37), rendering (OR 6.47, 1.62–25.84), and employee cohabitation (OR 6.15, 1.51–25.09). Protective factors included exclusive pumping equipment (OR 0.07, 0.01–0.43) and overnight downtime for multi-site workers (OR 0.15, 0.04–0.56). In multivariable models, finisher sites (OR 17.47, 2.44–125.19) and greater swine site density within one mile (OR 1.62, 1.09–2.41) significantly increased outbreak odds. These results support targeted biosecurity practices, helping farmers and the swine industry reduce PRRS outbreaks.

Effect of pooling family oral fluids on the probability of PRRSV RNA detection by RT-rtPCR

Risk Factors Associated with Piglet Pre-Weaning Mortality in a Midwestern U.S. Swine Production System from 2020–2022