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.

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.

Infectivity of GII.4 human norovirus does not differ between T-B-NK+ severe combined immunodeficiency (SCID) and non-SCID gnotobiotic pigs, implicating the role of NK cells in mediation of human norovirus infection.

Human noroviruses (HuNoVs) are a leading cause of acute gastroenteritis worldwide. It is unclear which arm of the immune system regulates resistance to HuNoV infection. Thus, we studied the pathogenesis of human norovirus (HuNoV) in T-B-NK+ Severe Combined Immunodeficiency (SCID) gnotobiotic pigs to investigate the role of innate (especially, natural killer (NK) cells) immunity in HuNoV infection. Forty SCID and non-SCID pigs were randomly grouped: 1) SCID+HuNoV (n = 12); 2) non-SCID+HuNoV (n = 14); 3) SCID mock-inoculated (n = 6); and 4) non-SCID mock-inoculated (n = 8). Pigs (8-14-day-old) were inoculated orally with GII.4 HuNoV strain HS292 (mean 9.1 log10 genomic equivalents/pig) or mock. Daily fecal consistency and fecal viral RNA shedding, and histopathology (at euthanasia) were evaluated. Frequencies of blood and ileal T, B, and NK cells were analyzed by flow cytometry, and a NK cell cytotoxicity assay was performed at post-inoculation day (PID) 8. Unlike the increased infectivity of HuNoV observed previously in T-B-NK- SCID pigs (Lei et al., 2016. Sci. Rep. 6, 25,222), there was no significant difference in frequency of pigs with diarrhea and diarrhea days between T-B-NK+ SCID+HuNoV and non-SCID+HuNoV groups. Cumulative fecal HuNoV RNA shedding at PIDs 1-8, PIDs 9-27, and PIDs 1-27 also did not differ statistically. These observations coincided with the presence of NK cells and NK cell cytotoxicity in the ileum and blood of the SCID pigs. Based on our observations, innate immunity, including NK cell activity, may be critical to mediate or reduce HuNoV infection in T-B-NK+ SCID pigs, and potentially in immunocompetent patients.

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-1ß 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.

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.