Emily H. Waide

Evidence for a major QTL associated with host response to Porcine Reproductive and Respiratory Syndrome Virus challenge

Porcine reproductive and respiratory syndrome (PRRS) causes decreased reproductive performance in breeding animals and increased respiratory problems in growing animals, which result in significant economic losses in the swine industry. Vaccination has generally not been effective in the prevention of PRRS, partially because of the rapid mutation rate and evolution of the virus. The objective of the current study was to discover the genetic basis of host resistance or susceptibility to the PRRS virus through a genome-wide association study using data from the PRRS Host Genetics Consortium PRRS-CAP project. Three groups of approximately 190 commercial crossbred pigs from 1 breeding company were infected with PRRS virus between 18 and 28 d of age. Blood samples and BW were collected up to 42 d post infection (DPI). Pigs were genotyped with the Illumina Porcine 60k Beadchip. Whole-genome analysis focused on viremia at each day blood was collected and BW gains from 0 to 21 DPI (WG21) or 42 DPI (WG42). Viral load (VL) was quantified as area under the curve from 0 to 21 DPI. Heritabilities for WG42 and VL were moderate at 0.30 and litter accounted for an additional 14% of phenotypic variation. Genomic regions associated with VL were found on chromosomes 4 and X and on 1, 4, 7, and 17 for WG42. The 1-Mb region identified on chromosome 4 influenced both WG and VL, exhibited strong linkage disequilibrium, and explained 15.7% of the genetic variance for VL and 11.2% for WG42. Despite a genetic correlation of -0.46 between VL and WG42, genomic EBV for this region were favorably and nearly perfectly correlated. The favorable allele for the most significant SNP in this region had a frequency of 0.16 and estimated allele substitution effects were significant (P < 0.01) for each group when the SNP was fitted as a fixed covariate in a model that included random polygenic effects with overall estimates of -4.1 units for VL (phenotypic SD = 6.9) and 2.0 kg (phenotypic SD = 3 kg) for WG42. Candidate genes in this region on SSC4 include the interferon induced guanylate-binding protein gene family. In conclusion, host response to experimental PRRS virus challenge has a strong genetic component, and a QTL on chromosome 4 explains a substantial proportion of the genetic variance in the studied population. These results could have a major impact in the swine industry by enabling marker-assisted selection to reduce the impact of PRRS but need to be validated in additional populations.

Not All SCID Pigs Are Created Equally: Two Independent Mutations in the Artemis Gene Cause SCID in Pigs

Mutations in >30 genes are known to result in impairment of the adaptive immune system, causing a group of disorders collectively known as SCID. SCID disorders are split into groups based on their presence and/or functionality of B, T, and NK cells. Piglets from a line of Yorkshire pigs at Iowa State University were shown to be affected by T−B−NK+ SCID, representing, to our knowledge, the first example of naturally occurring SCID in pigs. In this study, we present evidence for two spontaneous mutations as the molecular basis for this SCID phenotype. Flow cytometry analysis of thymocytes showed an increased frequency of immature T cells in SCID pigs. Fibroblasts from these pigs were more sensitive to ionizing radiation than non-SCID piglets, eliminating the RAG1 and RAG2 genes. Genetic and molecular analyses showed that two mutations were present in the Artemis gene, which in the homozygous or compound heterozygous state cause the immunodeficient phenotype. Rescue of SCID fibroblast radiosensitivity by human Artemis protein demonstrated that the identified Artemis mutations are the direct cause of this cellular phenotype. The work presented in the present study reveals two mutations in the Artemis gene that cause T−B−NK+ SCID in pigs. The SCID pig can be an important biomedical model, but these mutations would be undesirable in commercial pig populations. The identified mutations and associated genetic tests can be used to address both of these issues.

Genomewide association of piglet responses to infection with one of two porcine reproductive and respiratory syndrome virus isolates.

Porcine reproductive and respiratory syndrome (PRRS) is a devastating disease in the swine industry. Identification of host genetic factors that enable selection for improved performance during PRRS virus (PRRSV) infection would reduce the impact of this disease on animal welfare and production efficiency. We conducted genomewide association study (GWAS) analyses of data from 13 trials of approximately 200 commercial crossbred nursery-age piglets that were experimentally infected with 1 of 2 type 2 isolates of PRRSV (NVSL 97-7985 [NVSL] and KS2006-72109 [KS06]). Phenotypes analyzed were viral load (VL) in blood during the first 21 d after infection (dpi) and weight gain (WG) from 0 to 42 dpi. We accounted for the previously identified QTL in the region on SSC4 in our models to increase power to identify additional regions. Many regions identified by single-SNP analyses were not identified using Bayes-B, but both analyses identified the same regions on SSC3 and SSC5 to be associated with VL in the KS06 trials and on SSC6 in the NVSL trials ( < 5 × 10); for WG, regions on SSC5 and SSC17 were associated in the NVSL trials ( < 3 × 10). No regions were identified with either method for WG in the KS06 trials. Except for the region on SSC4, which was associated with VL for both isolates (but only with WG for NVSL), identified regions did not overlap between the 2 PRRSV isolate data sets, despite high estimates of the genetic correlation between isolates for traits based on these data. We also identified genomic regions whose associations with VL or WG interacted with either PRRSV isolate or with genotype at the SSC4 QTL. Gene ontology (GO) annotation terms for genes located near moderately associated SNP ( < 0.003) were enriched for multiple immunologically (VL) and metabolism- (WG) related GO terms. The biological relevance of these regions suggests that, although it may increase the number of false positives, the use of single-SNP analyses and a relaxed threshold also increased the identification of true positives. In conclusion, although only the SSC4 QTL was associated with response to both PRRSV isolates, genes near associated SNP were enriched for the same GO terms across PRRSV isolates, suggesting that host responses to these 2 isolates are affected by the actions of many genes that function together in similar biological processes.

NK cells are intrinsically functional in pigs with Severe Combined Immunodeficiency (SCID) caused by spontaneous mutations in the Artemis gene

We have identified Severe Combined Immunodeficiency (SCID) in a line of Yorkshire pigs at Iowa State University. These SCID pigs lack B-cells and T-cells, but possess Natural Killer (NK) cells. This SCID phenotype is caused by recessive mutations in the Artemis gene. Interestingly, two human tumor cell lines, PANC-1 and A375-SM, survived after injection into these SCID pigs, but, as we demonstrate here, these cells, as well as K562 tumor cells, can be lysed in vitro by NK cells from SCID and non-SCID pigs. NK cells from both SCID and non-SCID pigs required activation in vitro with either recombinant human IL-2 or the combination of recombinant porcine IL-12 and IL-18 to kill tumor targets. We also showed that SCID NK cells could be activated to produce perforin, and perforin production was greatly enhanced in NK cells from both SCID and non-SCID pigs after IL-2 cytokine treatment. While CD16+, CD172- NK cells constituted an average of only 4% in non-SCID pigs, NK cells averaged 27% of the peripheral blood mononuclear cell population in SCID pigs. We found no significant differences in killing activity per NK cell between SCID and non-SCID pigs. We conclude that survival of human cancer cells in these SCID pigs is not due to an intrinsic defect in NK cell killing ability.

Pigs with Severe Combined Immunodeficiency Are Impaired in Controlling Influenza A Virus Infection

Influenza A viruses (IAV) infect many host species, including humans and pigs. Severe combined immunodeficiency (SCID) is a condition characterized by a deficiency of T, B, and/or natural killer (NK) cells. Animal models of SCID have great value for biomedical research. Here, we evaluated the pathogenesis and the innate immune response to the 2009 H1N1 pandemic IAV (H1N1pdm09) using a recently identified line of naturally occurring SCID pigs deficient in T and B lymphocytes that still have functional NK cells. SCID pigs challenged with H1N1pdm09 showed milder lung pathology compared to the non-SCID heterozygous carrier pigs. Viral titers in the lungs and nasal swabs of challenged SCID pigs were significantly higher than in carrier pigs 7 days postinfection, despite higher levels of IL-1β and IFN-α in the lungs of SCID pigs. The lower levels of pulmonary pathology were associated with the T and B cell absence in response to infection. The higher viral titers, prolonged shedding, and delayed viral clearance indicated that innate immunity was insufficient for controlling IAV in pigs. This recently identified line of SCID pigs provides a valuable model to understand the immune mechanisms associated with influenza protection and recovery in a natural host.

T Cell Lymphoma and Leukemia in Severe Combined Immunodeficiency Pigs following Bone Marrow Transplantation: A Case Report

After the discovery of naturally occurring severe combined immunodeficiency (SCID) within a selection line of pigs at Iowa State University, we found two causative mutations in the Artemis gene: haplotype 12 (ART12) and haplotype 16 (ART16). Bone marrow transplants (BMTs) were performed to create genetically SCID and phenotypically immunocompetent breeding animals to establish a SCID colony for further characterization and research utilization. Of nine original BMT transfer recipients, only four achieved successful engraftment. At approximately 11 months of age, both animals homozygous for the ART16 mutation were diagnosed with T cell lymphoma. One of these ART16/ART16 recipients was a male who received a transplant from a female sibling; the tumors in this recipient consist primarily of Y chromosome-positive cells. The other ART16/ART16 animal also presented with leukemia in addition to T cell lymphoma, while one of the ART12/ART16 compound heterozygote recipients presented with a nephroblastoma at a similar age. Human Artemis SCID patients have reported cases of lymphoma associated with a “leaky” Artemis phenotype. The naturally occurring Artemis SCID pig offers a large animal model more similar to human SCID patients and may offer a naturally occurring cancer model and provides a valuable platform for therapy development.

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

BackgroundGenomic 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.ResultsGenomic 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.ConclusionsResults 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.