Production and characterization of anti-porcine CXCL10 monoclonal antibodies.

Research on C-X-C motif chemokine ligand 10 (CXCL10) has been widely reported for humans and select animal species, yet immune reagents are limited for pig chemokines. Our goal is to provide veterinary immunologists and the biomedical community with new commercial immune reagents and standardized assays. Recombinant porcine CXCL10 (rPoCXCL10) protein was produced by yeast expression and used to generate a panel of α CXCL10 monoclonal antibodies (mAbs). All mAbs were assessed for cross-inhibition and reactivity to orthologous yeast expressed CXCL10 proteins. Characterization of a panel of nine α PoCXCL10 mAbs identified six distinct antigenic determinants. A sensitive quantitative sandwich ELISA was developed with anti-PoCXCL10-1.6 and -1.9 mAb; reactivity was verified with both rPoCXCL10 and native PoCXCL10, detected in supernatants of peripheral blood mononuclear cells stimulated with rPoIFNγ or PMA/Ionomycin. Immunostaining of in vitro rPoIFNγ stimulated pig spleen and blood cells verified CXCL10 + cells as CD3-CD4-CD172+, with occasional CD3-CD4 + CD172 + subsets. Comparison studies determined that α PoCXCL10-1.4 mAb was the ideal mAb clone for intracellular staining, whereas with α PoCXCL10-1.1 and -1.2 mAbs were best for immunohistochemistry analyses. These techniques and tools will be useful for evaluating swine immune development, responses to infectious diseases and vaccines, as well as for improving utility of pigs as an important biomedical model.

Future of biomedical, agricultural, and biological systems research using domesticated animals.

Increased knowledge of reproduction and health of domesticated animals is integral to sustain and improve global competitiveness of U.S. animal agriculture, understand and resolve complex animal and human diseases, and advance fundamental research in sciences that are critical to understanding mechanisms of action and identifying future targets for interventions. Historically, federal and state budgets have dwindled and funding for the United States Department of Agriculture (USDA) National Institute of Food and Agriculture (NIFA) competitive grants programs remained relatively stagnant from 1985 through 2010. This shortage in critical financial support for basic and applied research, coupled with the underappreciated knowledge of the utility of non-rodent species for biomedical research, hindered funding opportunities for research involving livestock and limited improvements in both animal agriculture and animal and human health. In 2010, the National Institutes of Health and USDA NIFA established an interagency partnership to promote the use of agriculturally important animal species in basic and translational research relevant to both biomedicine and agriculture. This interagency program supported 61 grants totaling over $107 million with 23 awards to new or early-stage investigators. This article will review the success of the 9-year Dual Purpose effort and highlight opportunities for utilizing domesticated agricultural animals in research.

Gene expression in tonsils in swine following infection with porcine reproductive and respiratory syndrome virus.

BACKGROUND: Porcine reproductive and respiratory syndrome (PRRS) is a threat to pig production worldwide. Our objective was to understand mechanisms of persistence of PRRS virus (PRRSV) in tonsil. Transcriptome data from tonsil samples collected at 42 days post infection (dpi) were generated by RNA-seq and NanoString on 51 pigs that were selected to contrast the two PRRSV isolates used, NVSL and KS06, high and low tonsil viral level at 42 dpi, and the favorable and unfavorable genotypes at a genetic marker (WUR) for the putative PRRSV resistance gene GBP5. RESULTS: The number of differentially expressed genes (DEGs) differed markedly between models with and without accounting for cell-type enrichments (CE) in the samples that were predicted from the RNA-seq data. This indicates that differences in cell composition in tissues that consist of multiple cell types, such as tonsil, can have a large impact on observed differences in gene expression. Based on both the NanoString and the RNA-seq data, KS06-infected pigs showed greater activation, or less inhibition, of immune response in tonsils at 42 dpi than NVSL-infected pigs, with and without accounting for CE. This suggests that the NVSL virus may be better than the KS06 virus at evading host immune response and persists in tonsils by weakening, or preventing, host immune responses. Pigs with high viral levels showed larger CE of immune cells than low viral level pigs, potentially to trigger stronger immune responses. Presence of high tonsil virus was associated with a stronger immune response, especially innate immune response through interferon signaling, but these differences were not significant when accounting for CE. Genotype at WUR was associated with different effects on immune response in tonsils of pigs during the persistence stage, depending on viral isolate and tonsil viral level. CONCLUSIONS: Results of this study provide insights into the effects of PRRSV isolate, tonsil viral level, and WUR genotype on host immune response and into potential mechanisms of PRRSV persistence in tonsils that could be targeted to improve strategies to reduce viral rebreaks. Finally, to understand transcriptome responses in tissues that consist of multiple cell types, it is important to consider differences in cell composition.

Differential responses in placenta and fetal thymus at 12 days post infection elucidate mechanisms of viral level and fetal compromise following PRRSV2 infection.

BACKGROUND: A pregnant gilt infected with porcine reproductive and respiratory syndrome virus (PRRSV) can transmit the virus to her fetuses across the maternal-fetal-interface resulting in varying disease outcomes. However, the mechanisms leading to variation in fetal outcome in response to PRRSV infection are not fully understood. Our objective was to assess targeted immune-related gene expression patterns and pathways in the placenta and fetal thymus to elucidate the molecular mechanisms involved in the resistance/tolerance and susceptibility of fetuses to PRRSV2 infection. Fetuses were grouped by preservation status and PRRS viral load (VL): mock infected control (CTRL), no virus detected (UNINF), virus detected in the placenta only with viable (PLCO-VIA) or meconium-stained fetus (PLCO-MEC), low VL with viable (LVL-VIA) or meconium-stained fetus (LVL-MEC), and high VL with viable (HVL-VIA) or meconium-stained fetus (HVL-MEC). RESULTS: The host immune response was initiated only in fetuses with detectable levels of PRRSV. No differentially expressed genes (DEG) in either the placenta or thymus were identified in UNINF, PLCO-VIA, and PLCO-MEC when compared to CTRL fetuses. Upon fetal infection, a set of core responsive IFN-inducible genes (CXCL10, IFIH1, IFIT1, IFIT3, ISG15, and MX1) were strongly upregulated in both tissues. Gene expression in the thymus is a better differentiator of fetal VL; the strong downregulation of several innate and adaptive immune pathways (e.g., B Cell Development) are indicative of HVL. Gene expression in the placenta may be a better differentiator of fetal demise than the thymus, based-on principle component analysis clustering, gene expression patterns, and dysregulation of the Apoptosis and Ubiquitination pathways. CONCLUSION: Our data supports the concept that fetal outcome in response to PRRSV2 infection is determined by fetal, and more significantly placental response, which is initiated only after fetal infection. This conceptual model represents a significant step forward in understanding the mechanisms underpinning fetal susceptibility to the virus.

The effects of a globin blocker on the resolution of 3'mRNA sequencing data in porcine blood.

BACKGROUND: Gene expression profiling in blood is a potential source of biomarkers to evaluate or predict phenotypic differences between pigs but is expensive and inefficient because of the high abundance of globin mRNA in porcine blood. These limitations can be overcome by the use of QuantSeq 3'mRNA sequencing (QuantSeq) combined with a method to deplete or block the processing of globin mRNA prior to or during library construction. Here, we validated the effectiveness of QuantSeq using a novel specific globin blocker (GB) that is included in the library preparation step of QuantSeq. RESULTS: In data set 1, four concentrations of the GB were applied to RNA samples from two pigs. The GB significantly reduced the proportion of globin reads compared to non-GB (NGB) samples (P = 0.005) and increased the number of detectable non-globin genes. The highest evaluated concentration (C1) of the GB resulted in the largest reduction of globin reads compared to the NGB (from 56.4 to 10.1%). The second highest concentration C2, which showed very similar globin depletion rates (12%) as C1 but a better correlation of the expression of non-globin genes between NGB and GB (r = 0.98), allowed the expression of an additional 1295 non-globin genes to be detected, although 40 genes that were detected in the NGB sample (at a low level) were not present in the GB library. Concentration C2 was applied in the rest of the study. In data set 2, the distribution of the percentage of globin reads for NGB (n = 184) and GB (n = 189) samples clearly showed the effects of the GB on reducing globin reads, in particular for HBB, similar to results from data set 1. Data set 3 (n = 84) revealed that the proportion of globin reads that remained in GB samples was significantly and positively correlated with the reticulocyte count in the original blood sample (P < 0.001). CONCLUSIONS: The effect of the GB on reducing the proportion of globin reads in porcine blood QuantSeq was demonstrated in three data sets. In addition to increasing the efficiency of sequencing non-globin mRNA, the GB for QuantSeq has an advantage that it does not require an additional step prior to or during library creation. Therefore, the GB is a useful tool in the quantification of whole gene expression profiles in porcine blood.

Harnessing longitudinal information to identify genetic variation in tolerance of pigs to Porcine Reproductive and Respiratory Syndrome virus infection.

BACKGROUND: High resistance (the ability of the host to reduce pathogen load) and tolerance (the ability to maintain high performance at a given pathogen load) are two desirable host traits for producing animals that are resilient to infections. For Porcine Reproductive and Respiratory Syndrome (PRRS), one of the most devastating swine diseases worldwide, studies have identified substantial genetic variation in resistance of pigs, but evidence for genetic variation in tolerance has so far been inconclusive. Resistance and tolerance are usually considered as static traits. In this study, we used longitudinal viremia measurements of PRRS virus infected pigs to define discrete stages of infection based on viremia profile characteristics. These were used to investigate host genetic effects on viral load (VL) and growth at different stages of infection, to quantify genetic variation in tolerance at these stages and throughout the entire 42-day observation period, and to assess whether the single nucleotide polymorphism (SNP) WUR10000125 (WUR) with known large effects on resistance confers significant differences in tolerance. RESULTS: Genetic correlations between resistance and growth changed considerably over time. Individuals that expressed high genetic resistance early in infection tended to grow slower during that time-period, but were more likely to experience lower VL and recovery in growth by the later stage. The WUR genotype was most strongly associated with VL at early- to mid-stages of infection, and with growth at mid- to late-stages of infection. Both, single-stage and repeated measurements random regression models identified significant genetic variation in tolerance. The WUR SNP was significantly associated only with the overall tolerance slope fitted through all stages of infection, with the genetically more resistant AB pigs for the WUR SNP being also more tolerant to PRRS. CONCLUSIONS: The results suggest that genetic selection for improved tolerance of pigs to PRRS is possible in principle, but may be feasible only with genomic selection, requiring intense recording schemes that involve repeated measurements to reliably estimate genetic effects. In the absence of such records, consideration of the WUR genotype in current selection schemes appears to be a promising strategy to improve simultaneously resistance and tolerance of growing pigs to PRRS.

Genomic prediction of piglet response to infection with one of two porcine reproductive and respiratory syndrome virus isolates.

BACKGROUND: Genomic prediction of the pig's response to the porcine reproductive and respiratory syndrome (PRRS) virus (PRRSV) would be a useful tool in the swine industry. This study investigated the accuracy of genomic prediction based on porcine SNP60 Beadchip data using training and validation datasets from populations with different genetic backgrounds that were challenged with different PRRSV isolates. RESULTS: Genomic prediction accuracy averaged 0.34 for viral load (VL) and 0.23 for weight gain (WG) following experimental PRRSV challenge, which demonstrates that genomic selection could be used to improve response to PRRSV infection. Training on WG data during infection with a less virulent PRRSV, KS06, resulted in poor accuracy of prediction for WG during infection with a more virulent PRRSV, NVSL. Inclusion of single nucleotide polymorphisms (SNPs) that are in linkage disequilibrium with a major quantitative trait locus (QTL) on chromosome 4 was vital for accurate prediction of VL. Overall, SNPs that were significantly associated with either trait in single SNP genome-wide association analysis were unable to predict the phenotypes with an accuracy as high as that obtained by using all genotyped SNPs across the genome. Inclusion of data from close relatives into the training population increased whole genome prediction accuracy by 33% for VL and by 37% for WG but did not affect the accuracy of prediction when using only SNPs in the major QTL region. CONCLUSIONS: Results show that genomic prediction of response to PRRSV infection is moderately accurate and, when using all SNPs on the porcine SNP60 Beadchip, is not very sensitive to differences in virulence of the PRRSV in training and validation populations. Including close relatives in the training population increased prediction accuracy when using the whole genome or SNPs other than those near a major QTL.

Genomic regions associated with host response to porcine reproductive and respiratory syndrome vaccination and co-infection in nursery pigs.

BACKGROUND: The WUR1000125 (WUR) single nucleotide polymorphism (SNP) can be used as a genetic marker for host response to porcine reproductive and respiratory syndrome (PRRS), PRRS vaccination, and co-infection with porcine circovirus type 2b (PCV2b). Objectives of this study were to identify genomic regions other than WUR associated with host response to PRRS vaccination and PRRSV/PCV2b co-infection and regions with a different effect on host response to co-infection, depending on previous vaccination for PRRS. METHODS: Commercial crossbred nursery pigs were pre-selected for WUR genotype (n = 171 AA and 198 AB pigs) where B is the dominant and favorable allele. Half of the pigs were vaccinated for PRRS and 4 weeks later, all pigs were co-infected with PRRS virus and PCV2b. Average daily gain (ADG) and viral load (VL) were quantified post vaccination (Post Vx) and post co-infection (Post Co-X). Single-SNP genome-wide association analyses were then conducted to identify genomic regions associated with response to vaccination and co-infection. RESULTS: Multiple SNPs near the major histocompatibility complex were significantly associated with PCV2b VL (-log 10 P ≥ 5.5), regardless of prior vaccination for PRRS. Several SNPs were also significantly associated with ADG Post Vx and Post Co-X. SNPs with a different effect on ADG, depending on prior vaccination for PRRS, were identified Post Vx (-log 10 P = 5.6) and Post Co-X (-log 10 P = 5.5). No SNPs were significantly associated with vaccination VL (-log10 P ≤ 4.7) or PRRS VL (-log10 P ≤ 4.3). Genes near SNPs associated with vaccination VL, PRRS VL, and PCV2b VL were enriched (P ≤ 0.01) for immune-related pathways and genes near SNPs associated with ADG were enriched for metabolism pathways (P ≤ 0.04). SNPs associated with vaccination VL, PRRS VL, and PCV2b VL showed overrepresentation of health QTL identified in previous studies and SNPs associated with ADG Post Vx of Non-Vx pigs showed overrepresentation of growth QTL. CONCLUSIONS: Multiple genomic regions were associated with PCV2b VL and ADG Post Vx and Post Co-X. Different SNPs were associated with ADG, depending on previous vaccination for PRRS. Results of functional annotation analyses and novel approaches of using previously-reported QTL support the identified regions.

E-cadherin and ZEB2 modulate trophoblast cell differentiation during placental development in pigs.

It is one of the important events that trophoblast cells within the placental folds differentiate into two types that differ in cell shape during placental development in pigs. This study showed that all the trophoblast cells were of similar shape between Yorkshire and Chinese Meishan pigs on day 26 of gestation; thereafter, the trophoblast cells located at the top of the placental folds became high columnar, while those cells at the base of the placental folds were cuboidal on day 50 of gestation. Additionally, on day 95 of gestation, all the trophoblast cells in Meishan pigs became cuboidal, but the trophoblast cells located at the top of the placental folds in Yorkshire pigs still remained columnar. The membranous E-cadherin and ß-catenin were strongly co-expressed by the high columnar trophoblast cells but very weakly expressed by those cuboidal cells. Consistently, the expression pattern of ZEB2, the E-cadherin repressor, was inversely correlated with that of E-cadherin in the two types of trophoblast cells in the two breeds. Furthermore, electrophoretic mobility shift assays demonstrated the binding of ZEB2 to the E-cadherin promoter in nuclear extracts from porcine placental tissue. These findings suggest a ZEB2-dependent mechanism of trophoblast cell differentiation during placental development in pigs.

Use of multi-trait and random regression models to identify genetic variation in tolerance to porcine reproductive and respiratory syndrome virus.

BACKGROUND: A host can adopt two response strategies to infection: resistance (reduce pathogen load) and tolerance (minimize impact of infection on performance). Both strategies may be under genetic control and could thus be targeted for genetic improvement. Although there is evidence that supports a genetic basis for resistance to porcine reproductive and respiratory syndrome (PRRS), it is not known whether pigs also differ genetically in tolerance. We determined to what extent pigs that have been shown to vary genetically in resistance to PRRS also exhibit genetic variation in tolerance. Multi-trait linear mixed models and random regression sire models were fitted to PRRS Host Genetics Consortium data from 1320 weaned pigs (offspring of 54 sires) that were experimentally infected with a virulent strain of PRRS virus to obtain genetic parameter estimates for resistance and tolerance. Resistance was defined as the inverse of within-host viral load (VL) from 0 to 21 (VL21) or 0 to 42 (VL42) days post-infection and tolerance as the slope of the reaction-norm of average daily gain (ADG21, ADG42) on VL21 or VL42. RESULTS: Multi-trait analysis of ADG associated with either low or high VL was not indicative of genetic variation in tolerance. Similarly, random regression models for ADG21 and ADG42 with a tolerance slope fitted for each sire did not result in a better fit to the data than a model without genetic variation in tolerance. However, the distribution of data around average VL suggested possible confounding between level and slope estimates of the regression lines. Augmenting the data with simulated growth rates of non-infected half-sibs (ADG0) helped resolve this statistical confounding and indicated that genetic variation in tolerance to PRRS may exist if genetic correlations between ADG0 and ADG21 or ADG42 are low to moderate. CONCLUSIONS: Evidence for genetic variation in tolerance of pigs to PRRS was weak when based on data from infected piglets only. However, simulations indicated that genetic variance in tolerance may exist and could be detected if comparable data on uninfected relatives were available. In conclusion, of the two defense strategies, genetics of tolerance is more difficult to elucidate than genetics of resistance.

Genome-wide analysis of the transcriptional response to porcine reproductive and respiratory syndrome virus infection at the maternal/fetal interface and in the fetus.

BACKGROUND: Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) infection of pregnant pigs can result in congenital infection and ultimately fetal death. Little is known about immune responses to infection at the maternal-fetal interface and in the fetus itself, or the molecular events behind virus transmission and disease progression in the fetus. To investigate these processes, RNA-sequencing of two sites, uterine endothelium with adherent placental tissue and fetal thymus, was performed 21 days post-challenge on four groups of fetuses selected from a large PRRSV challenge experiment of pregnant gilts: control (CON), uninfected (UNINF), infected (INF), and meconium-stained (MEC) (n = 12/group). Transcriptional analyses consisted of multiple contrasts between groups using two approaches: differential gene expression analysis and weighted gene co-expression network analysis (WGCNA). Biological functions, pathways, and regulators enriched for differentially expressed genes or module members were identified through functional annotation analyses. Expression data were validated by reverse transcription quantitative polymerase chain reaction (RTqPCR) carried out for 16 genes of interest. RESULTS: The immune response to infection in endometrium was mainly adaptive in nature, with the most upregulated genes functioning in either humoral or cell-mediated immunity. In contrast, the expression profile of infected fetal thymus revealed a predominantly innate immune response to infection, featuring the upregulation of genes regulated by type I interferon and pro-inflammatory cytokines. Fetal infection was associated with an increase in viral load coupled with a reduction in T cell signaling in the endometrium that could be due to PRRSV-controlled apoptosis of uninfected bystander cells. There was also evidence for a reduction in TWIST1 activity, a transcription factor involved in placental implantation and maturation, which could facilitate virus transmission or fetal pathology through dysregulation of placental function. Finally, results suggested that events within the fetus could also drive fetal pathology. Thymus samples of meconium-stained fetuses exhibited an increase in the expression of pro-inflammatory cytokine and granulocyte genes previously implicated in swine infectious disease pathology. CONCLUSIONS: This study identified major differences in the response to PRRSV infection in the uterine endometrium and fetus at the gene expression level, and provides insight into the molecular basis of virus transmission and disease progression.

Bioinformatic analyses in early host response to Porcine Reproductive and Respiratory Syndrome virus (PRRSV) reveals pathway differences between pigs with alternate genotypes for a major host response QTL.

BACKGROUND: A region on Sus scrofa chromosome 4 (SSC4) surrounding single nucleotide polymorphism (SNP) marker WUR10000125 (WUR) has been reported to be strongly associated with both weight gain and serum viremia in pigs after infection with PRRS virus (PRRSV). A proposed causal mutation in the guanylate binding protein 5 gene (GBP5) is predicted to truncate the encoded protein. To investigate transcriptional differences between WUR genotypes in early host response to PRRSV infection, an RNA-seq experiment was performed on globin depleted whole blood RNA collected on 0, 4, 7, 10 and 14 days post-infection (dpi) from eight littermate pairs with one AB (favorable) and one AA (unfavorable) WUR genotype animal per litter. RESULTS: Gene Ontology (GO) enrichment analysis of transcripts that were differentially expressed (DE) between dpi across both genotypes revealed an inflammatory response for all dpi when compared to day 0. However, at the early time points of 4 and 7dpi, several GO terms had higher enrichment scores compared to later dpi, including inflammatory response (p < 10(-7)), specifically regulation of NFkappaB (p < 0.01), cytokine, and chemokine activity (p < 0.01). At 10 and 14dpi, GO term enrichment indicated a switch to DNA damage response, cell cycle checkpoints, and DNA replication. Few transcripts were DE between WUR genotypes on individual dpi or averaged over all dpi, and little enrichment of any GO term was found. However, there were differences in expression patterns over time between AA and AB animals, which was confirmed by genotype-specific expression patterns of several modules that were identified in weighted gene co-expression network analyses (WGCNA). Minor differences between AA and AB animals were observed in immune response and DNA damage response (p = 0.64 and p = 0.11, respectively), but a significant effect between genotypes pointed to a difference in ion transport/homeostasis and the participation of G-coupled protein receptors (p = 8e-4), which was reinforced by results from regulatory and phenotypic impact factor analyses between genotypes. CONCLUSION: We propose these pathway differences between WUR genotypes are the result of the inability of the truncated GBP5 of the AA genotyped pigs to inhibit viral entry and replication as quickly as the intact GBP5 protein of the AB genotyped pigs.

Comparison of host genetic factors influencing pig response to infection with two North American isolates of porcine reproductive and respiratory syndrome virus.

BACKGROUND: Porcine reproductive and respiratory syndrome (PRRS) is one of the most important swine diseases in the world and genetic selection of pigs for increased resistance to PRRS is an attractive method to improve the health status of the swine herd. This study compared phenotypic and genetic responses to infection with one of two genetically distinct type 2 PRRS virus (PRRSV) isolates: NVSL-97-7895 (NVSL) and KS-2006-72109 (KS06), and evaluated whether the single nucleotide polymorphism (SNP) WUR10000125 (WUR) on chromosome 4 that was associated with viral load and weight gain under infection with NVSL also has an effect on response to infection across North American PRRSV isolates. Wood's lactation curve was fitted to repeated viremia measurements to derive five curve characteristics that were evaluated. RESULTS: Infection with NVSL was characterized by reaching a 14 ± 2 % higher peak viremia (PV) 2.5 ± 0.6 days earlier (time to peak; TP) than KS06, followed by 36 ± 1 % faster virus clearance, which occurred 3.9 ± 0.7 days sooner. Weight gain from 0 to 42 days post-infection (WG) tended to be higher under infection with KS06 than NVSL (3.7 ± 1.5 kg). Estimates of heritability were moderate for both PRRSV isolates for viral load from 0 to 21 days post-infection (VL) (NVSL: 0.31 ± 0.06; KS06: 0.51 ± 0.09) and WG (NVSL: 0.33 ± 0.06; KS06: 0.31 ± 0.09). Strong negative genetic correlations were observed between VL and WG for both NVSL (-0.74 ± 0.10) and KS06 (-0.52 ± 0.17) infected pigs. Pigs with genotype AB at the WUR SNP had a more desirable phenotype than AA pigs for all traits under infection with NVSL, but only for VL and PV with KS06; effects on other traits were smaller and not significantly different from zero (P > 0.05). Genetic correlations of host response between isolates were strong for VL, WG and PV. Accounting for WUR genotype had little impact on these correlations, suggesting that response to PRRSV infection has a substantial polygenic component that is common between these two isolates. CONCLUSIONS: These results suggest that the KS06 PRRSV isolate is less virulent than NVSL but that genetic selection for increased resistance to either of these genetically distinct isolates is expected to increase resistance to the other isolate.

GO-FAANG meeting: a Gathering On Functional Annotation of Animal Genomes.

The Functional Annotation of Animal Genomes (FAANG) Consortium recently held a Gathering On FAANG (GO-FAANG) Workshop in Washington, DC on October 7-8, 2015. This consortium is a grass-roots organization formed to advance the annotation of newly assembled genomes of domesticated and non-model organisms (www.faang.org). The workshop gathered together from around the world a group of 100+ genome scientists, administrators, representatives of funding agencies and commodity groups to discuss the latest advancements of the consortium, new perspectives, next steps and implementation plans. The workshop was streamed live and recorded, and all talks, along with speaker slide presentations, are available at www.faang.org. In this report, we describe the major activities and outcomes of this meeting. We also provide updates on ongoing efforts to implement discussions and decisions taken at GO-FAANG to guide future FAANG activities. In summary, reference datasets are being established under pilot projects; plans for tissue sets, morphological classification and methods of sample collection for different tissues were organized; and core assays and data and meta-data analysis standards were established.

Whole blood microarray analysis of pigs showing extreme phenotypes after a porcine reproductive and respiratory syndrome virus infection.

BACKGROUND: The presence of variability in the response of pigs to Porcine Reproductive and Respiratory Syndrome virus (PRRSv) infection, and recent demonstration of significant genetic control of such responses, leads us to believe that selection towards more disease resistant pigs could be a valid strategy to reduce its economic impact on the swine industry. To find underlying molecular differences in PRRS susceptible versus more resistant pigs, 100 animals with extremely different growth rates and viremia levels after PRRSv infection were selected from a total of 600 infected pigs. A microarray experiment was conducted on whole blood RNA samples taken at 0, 4 and 7 days post infection (dpi) from these pigs. From these data, we examined associations of gene expression with weight gain and viral load phenotypes. The single nucleotide polymorphism (SNP) marker WUR10000125 (WUR) on the porcine 60 K SNP chip was shown to be associated with viral load and weight gain after PRRSv infection, and so the effect of the WUR10000125 (WUR) genotype on expression in whole blood was also examined. RESULTS: Limited information was obtained through linear modeling of blood gene differential expression (DE) that contrasted pigs with extreme phenotypes, for growth or viral load or between animals with different WUR genotype. However, using network-based approaches, molecular pathway differences between extreme phenotypic classes could be identified. Several gene clusters of interest were found when Weighted Gene Co-expression Network Analysis (WGCNA) was applied to 4 dpi contrasted with 0 dpi data. The expression pattern of one such cluster of genes correlated with weight gain and WUR genotype, contained numerous immune response genes such as cytokines, chemokines, interferon type I stimulated genes, apoptotic genes and genes regulating complement activation. In addition, Partial Correlation and Information Theory (PCIT) identified differentially hubbed (DH) genes between the phenotypically divergent groups. GO enrichment revealed that the target genes of these DH genes are enriched in adaptive immune pathways. CONCLUSION: There are molecular differences in blood RNA patterns between pigs with extreme phenotypes or with a different WUR genotype in early responses to PRRSv infection, though they can be quite subtle and more difficult to discover with conventional DE expression analyses. Co-expression analyses such as WGCNA and PCIT can be used to reveal network differences between such extreme response groups.

Identification of a putative quantitative trait nucleotide in guanylate binding protein 5 for host response to PRRS virus infection.

BACKGROUND: Previously, we identified a major quantitative trait locus (QTL) for host response to Porcine Respiratory and Reproductive Syndrome virus (PRRSV) infection in high linkage disequilibrium (LD) with SNP rs80800372 on Sus scrofa chromosome 4 (SSC4). RESULTS: Within this QTL, guanylate binding protein 5 (GBP5) was differentially expressed (DE) (p < 0.05) in blood from AA versus AB rs80800372 genotyped pigs at 7,11, and 14 days post PRRSV infection. All variants within the GBP5 transcript in LD with rs80800372 exhibited allele specific expression (ASE) in AB individuals (p < 0.0001). A transcript re-assembly revealed three alternatively spliced transcripts for GBP5. An intronic SNP in GBP5, rs340943904, introduces a splice acceptor site that inserts five nucleotides into the transcript. Individuals homozygous for the unfavorable AA genotype predominantly produced this transcript, with a shifted reading frame and early stop codon that truncates the 88 C-terminal amino acids of the protein. RNA-seq analysis confirmed this SNP was associated with differential splicing by QTL genotype (p < 0.0001) and this was validated by quantitative capillary electrophoresis (p < 0.0001). The wild-type transcript was expressed at a higher level in AB versus AA individuals, whereas the five-nucleotide insertion transcript was the dominant form in AA individuals. Splicing and ASE results are consistent with the observed dominant nature of the favorable QTL allele. The rs340943904 SNP was also 100 % concordant with rs80800372 in a validation population that possessed an alternate form of the favorable B QTL haplotype. CONCLUSIONS: GBP5 is known to play a role in inflammasome assembly during immune response. However, the role of GBP5 host genetic variation in viral immunity is novel. These findings demonstrate that rs340943904 is a strong candidate causal mutation for the SSC4 QTL that controls variation in host response to PRRSV.

Maternal and fetal predictors of fetal viral load and death in third trimester, type 2 porcine reproductive and respiratory syndrome virus infected pregnant gilts.

Minimal research has focused on understanding mechanisms underlying porcine reproductive and respiratory syndrome virus (PRRSV) induced reproductive failure. We have completed a large-scale project investigating phenotypic and genotypic predictors of reproductive PRRS severity in which numerous clinical, pathological, immunologic and viral responses were characterized in dams and fetuses. The goal was to determine which phenotypic responses were associated with fetal viral load and death after experimental infection of pregnant gilts with type 2 PRRSV, thereby elucidating mechanisms of reproductive PRRS in third trimester pregnant gilts. The presence of fetal infection and increasing RNA concentration at the maternal-fetal interface were strong predictors of the probability of fetal death, while PRRSV RNA concentration in dam sera and systemic tissues were not associated with the odds of fetal death. Fetal infection and death clustered, indicating that the status of adjacent fetuses is crucial for lateral transmission and fetal outcome. Several systemic immune responses of gilts were associated with fetal outcome and viral load: interferon-α contributed to the probability of fetal death, but absolute numbers of T helper cells in early infection, absolute numbers of myeloid cells over time and interleukin 12 levels appeared protective. These results suggest specific immune responses may either contribute to, or protect against, transplacental virus transmission. The WUR10000125 SNP on chromosome 4, associated with PRRS resilience in nursery pigs, was not associated with reproductive outcome. Whereas past research suggested that fetal death results from events occurring at the maternal-fetal interface, we conclude that viral replication within fetuses and spread of PRRSV to adjacent fetuses are pivotal events in the pathogenesis of reproductive PRRS.

Endometrial gene expression profiling in pregnant Meishan and Yorkshire pigs on day 12 of gestation.

BACKGROUND: Litter size in pigs is a major factor affecting the profitability in the pig industry. The peri-implantation window in pigs is characterized by the coordinated interactions between the maternal uterine endometrium and the rapidly elongating conceptuses and represents a period of time during which a large percentage of the developing conceptuses are lost. However, the gene expression and regulatory networks in the endometrium contributing to the establishment of the maternal: placental interface remain poorly understood. RESULTS: We characterized the endometrial gene expression profile during the peri-implantation stage of development by comparing two breeds that demonstrate very different reproductive efficiencies. We employed the porcine Affymetrix GeneChip® to assay the transcriptomic profiles of genes expressed in the uterine endometrium obtained from Meishan and Yorkshire gilts (n = 4 for each breed) on day 12 of gestation (M12 and Y12, respectively). Total of 17,076 probesets were identified as "present" in at least two arrays. A mixed model-based statistical analysis predicted a total of 2,656 (q < 0.1) transcripts as differentially expressed between Meishan and Yorkshire pigs. Eighteen differentially expressed transcripts of interest were validated by quantitative real-time PCR. Gene ontology (GO) annotation revealed that the known functions of the differentially expressed genes were involved in a series of important biological processes relevant to early pregnancy establishment in the pig. CONCLUSIONS: The results identified endometrial gene expression profiles of two breeds differing in litter size and identified candidate genes that are related to known physiological pathways related to reproductive prolificacy. These findings provide a deeper understanding of molecular pathways differing between two breeds at the critical peri-implantation stage of pregnancy, which can be utilized to better understand the events contributing to pregnancy establishment in the pig.

Increasing gene discovery and coverage using RNA-seq of globin RNA reduced porcine blood samples.

BACKGROUND: Transcriptome analysis of porcine whole blood has several applications, which include deciphering genetic mechanisms for host responses to viral infection and vaccination. The abundance of alpha- and beta-globin transcripts in blood, however, impedes the ability to cost-effectively detect transcripts of low abundance. Although protocols exist for reduction of globin transcripts from human and mouse/rat blood, preliminary work demonstrated these are not useful for porcine blood Globin Reduction (GR). Our objectives were to develop a porcine specific GR protocol and to evaluate the GR effects on gene discovery and sequence read coverage in RNA-sequencing (RNA-seq) experiments. RESULTS: A GR protocol for porcine blood samples was developed using RNase H with antisense oligonucleotides specifically targeting porcine hemoglobin alpha (HBA) and beta (HBB) mRNAs. Whole blood samples (n = 12) collected in Tempus tubes were used for evaluating the efficacy and effects of GR on RNA-seq. The HBA and HBB mRNA transcripts comprised an average of 46.1% of the mapped reads in pre-GR samples, but those reads reduced to an average of 8.9% in post-GR samples. Differential gene expression analysis showed that the expression level of 11,046 genes were increased, whereas 34 genes, excluding HBA and HBB, showed decreased expression after GR (FDR <0.05). An additional 815 genes were detected only in post-GR samples. CONCLUSIONS: Our porcine specific GR primers and protocol minimize the number of reads of globin transcripts in whole blood samples and provides increased coverage as well as accuracy and reproducibility of transcriptome analysis. Increased detection of low abundance mRNAs will ensure that studies relying on transcriptome analyses do not miss information that may be vital to the success of the study.

Changes in leukocyte subsets of pregnant gilts experimentally infected with porcine reproductive and respiratory syndrome virus and relationships with viral load and fetal outcome.

In spite of more than two decades of extensive research, the understanding of porcine reproductive and respiratory syndrome virus (PRRSv) immunity is still incomplete. A PRRSv infection of the late term pregnant female can result in abortions, early farrowings, fetal death, and the birth of weak, congenitally infected piglets. The objectives of the present study were to investigate changes in peripheral blood mononuclear cell populations in third trimester pregnant females infected with type 2 PRRSv (NVSL 97-7895) and to analyze potential relationships with viral load and fetal mortality rate. PRRSv infection caused a massive, acute drop in total leukocyte counts affecting all PBMC populations by two days post infection. Except for B cells, cell counts started to rebound by day six post infection. Our data also show a greater decrease of naïve B cells, T-helper cells and cytolytic T cells than their respective effector or memory counterparts. Absolute numbers of T cells and γδ T cells were negatively associated with PRRSv RNA concentration in gilt serum over time. Additionally, absolute numbers of T helper cells may be predictive of fetal mortality rate. The preceding three leukocyte populations may therefore be predictive of PRRSv-related pathological outcomes in pregnant gilts. Although many questions regarding the immune responses remain unanswered, these findings provide insight and clues that may help reduce the impact of PRRSv in pregnant gilts.