First gene-edited calf with reduced susceptibility to a major viral pathogen.

Bovine viral diarrhea virus (BVDV) is one of the most important viruses affecting the health and well-being of bovine species throughout the world. Here, we used CRISPR-mediated homology-directed repair and somatic cell nuclear transfer to produce a live calf with a six amino acid substitution in the BVDV binding domain of bovine CD46. The result was a gene-edited calf with dramatically reduced susceptibility to infection as measured by reduced clinical signs and the lack of viral infection in white blood cells. The edited calf has no off-target edits and appears normal and healthy at 20 months of age without obvious adverse effects from the on-target edit. This precision bred, proof-of-concept animal provides the first evidence that intentional genome alterations in the CD46 gene may reduce the burden of BVDV-associated diseases in cattle and is consistent with our stepwise, in vitro and ex vivo experiments with cell lines and matched fetal clones.

Association of ARRDC3 and NFIA variants with bovine congestive heart failure in feedlot cattle.

Background: Bovine congestive heart failure (BCHF) has become increasingly prevalent among feedlot cattle in the Western Great Plains of North America with up to 7% mortality in affected herds. BCHF is an untreatable complex condition involving pulmonary hypertension that culminates in right ventricular failure and death. Genes associated with BCHF in feedlot cattle have not been previously identified. Our aim was to search for genomic regions associated with this disease. Methods: A retrospective, matched case-control design with 102 clinical BCHF cases and their unaffected pen mates was used in a genome-wide association study. Paired nominal data from approximately 560,000 filtered single nucleotide polymorphisms (SNPs) were analyzed with McNemar's test. Results: Two independent genomic regions were identified as having the most significant association with BCHF: the arrestin domain-containing protein 3 gene ( ARRDC3), and the nuclear factor IA gene ( NFIA, mid- p-values, 1x10 -8 and 2x10 -7, respectively). Animals with two copies of risk alleles at either gene were approximately eight-fold more likely to have BCHF than their matched pen mates with either one or zero risk alleles at both genes (CI 95 = 3-17). Further, animals with two copies of risk alleles at both genes were 28-fold more likely to have BCHF than all others ( p-value = 1×10 -7, CI 95 = 4-206). A missense variant in ARRDC3 (C182Y) represents a potential functional variant since the C182 codon is conserved among all other jawed vertebrate species observed. A two-SNP test with markers in both genes showed 29% of 273 BCHF cases had homozygous risk genotypes in both genes, compared to 2.5% in 198 similar unaffected feedlot cattle. This and other DNA tests may be useful for identifying feedlot animals with the highest risk for BCHF in the environments described here. Conclusions: Although pathogenic roles for variants in the ARRDC3 and NFIA genes are unknown, their discovery facilitates classifying animals by genetic risk and allows cattle producers to make informed decisions for selective breeding and animal health management.

Genome-wide association analysis for porcine reproductive and respiratory syndrome virus susceptibility traits in two genetic populations of pigs1.

Porcine reproductive and respiratory syndrome virus (PRRSV) is an economically important pathogen that continues to threaten swine industry sustainability. The complexity and high genetic diversity of PRRSV has prevented vaccines from conferring adequate protection against disease outbreaks. Genome-wide association analyses of PRRSV experimentally infected pigs representing two genetic lines (n = 174 to 176) revealed two major genomic regions accounting for ~1.2% of the genetic variation in PRRSV-specific antibody level in serum or lung. The major region for serum antibody was mapped to SSC7 near the SLAII complex, which has also been implicated in susceptibility to other swine viral pathogens. Haplotype substitution analysis uncovered potential DQB1 haplotypes associated with divergent effects. A novel major region for lung antibody was mapped to the proximal end of SSC17 with the top SNP overlapping two genes, PRAG1 and LONRF1. Sequencing LONRF1 uncovered polymorphisms within the coding region that may play a role in regulating PRRSV-specific antibody production in lung tissue following PRRSV infection. These data implicate novel host genomic regions (SSC17) that influence PRRSV-specific immune response as well as a common region (SSC7) potentially involved in susceptibility to multiple viral pathogens.

Variation in time and magnitude of immune response and viremia in experimental challenges with Porcine circovirus 2b.

BACKGROUND: Porcine circovirus 2 is the primary agent responsible for inducing a group of associated diseases known as Porcine Circovirus Associated Diseases (PCVAD), which can have detrimental effects on production efficiency as well as causing significant mortality. The objective of this study was to evaluate variation in viral replication, immune response and growth across pigs (n = 974) from different crossbred lines. The approach used in this study was experimental infection with a PCV2b strain of pigs at an average of 43 days of age. RESULTS: The sequence of the PCV2b isolate used in the challenge was similar with a cluster of PCV2b isolates known to induce PCVAD and increased mortality rates. The swine leukocyte antigen class II (SLAII) profile of the population was diverse, with nine DQB1 haplotypes being present. Individual viremia and antibody profiles during challenge demonstrate variation in magnitude and time of viral surge and immune response. The correlations between PCV2 specific antibodies and average daily gain (ADG) were relatively low and varied between - 0.14 to 0.08 for IgM and -0.02 and 0.11 for IgG. In contrast, PCV2 viremia was an important driver of ADG decline following infection; a moderate negative correlation was observed between viral load and overall ADG (r = - 0.35, P < 0.001). The pigs with the lowest 10% level of viral load maintained a steady increase in weekly ADG (P < 0.0001) compared to the pigs that had the 10% greatest viral load (P < 0.55). In addition, the highly viremic group expressed higher IgM and IgG starting with d 14 and d 21 respectively, and higher tumor necrosis factor - alpha (TNF-α) at d 21 (P < 0.005), compared to low viremic group. CONCLUSIONS: Molecular sources of the observed differences in viremia and immune response could provide a better understanding of the host factors that influence the development of PCVAD and lead to improved knowledge of swine immunity.