Dissection of QTL on Host Chromosome 12 Uncovers Candidate Gene and Missense Polymorphism Associated with Porcine Circovirus 2 Susceptibility

Porcine circovirus 2 (PCV2) is a small single stranded DNA virus responsible for a group of detrimental diseases referred to as Porcine Circovirus Associated Diseases (PCVAD). Observed variation in incidence and severity of PCVAD between pigs suggests a host genetic role in facilitating PCV2 pathogenesis. This study builds on prior research by Engle et al. (2014), who performed a large-scale genome-wide association study of 974 crossbred pigs experimentally infected with a PCV2b isolate and provided evidence of a host genetic role in PCV2 viremia, immune response, and growth. Two major Quantitative Trait Loci (QTL) were identified for viral load located on chromosome 7 (SSC7) near the swine leukocyte antigen complex class II (SLAII) locus and the proximal end of chromosome 12 (SSC12). The SNP with largest association, ALGA0110477 (SSC12), explained 11.1% of the genetic variance and 7.4% of the phenotypic variance for viral load. Dissection of the SSC12 QTL region using gene annotation and both genomic and RNA sequencing uncovered a novel missense polymorphism within SYNGR2 (p.Arg63Cys) that exhibited the largest association with PCV2 viremia and immune response. In vitro gene silencing of SYNGR2 was performed on porcine kidney 15 cell line (PK15) using siRNA designed against the SYNGR2 mRNA sequence. A substantial decrease in SYNGR2 mRNA expression (82.2%) was achieved and corresponded with a significant reduction in PCV2 titer beginning at 48 hours post infection (P\u3c0.05) compared to scramble siRNA and non-transfected control cells, indicating a role of SYNGR2 in viral replication. The SYNGR2 p.Arg63Cys mutation is located within a protein domain conserved across mammals and results in an amino acid substitution (Arga→Cys) unique to swine. The impact of SYNGR2 on PCV2 replication and location of a non-conservative substitution within a key domain provides strong evidence that the SYNGR2 p.63Cys variant underlies the observed genetic effect on viral load by potentially interfering with SYNGR2 activity. These findings provide important insight into the role of host genetics in PCV2 pathogenesis. Advisor: Daniel Cioban

Functional and evolutionary analysis of host Synaptogyrin-2 in porcine circovirus type 2 susceptibility

Mammalian evolution has been influenced by viruses for millions of years, leaving signatures of adaptive evolution within genes encoding for viral interacting proteins. Synaptogyrin- 2 (SYNGR2) is a transmembrane protein implicated in promoting bacterial and viral infections. A genome-wide association study of pigs experimentally infected with porcine circovirus type 2b (PCV2b) uncovered a missense mutation (SYNGR2 p.Arg63Cys) associated with viral load. In this study, CRISPR/Cas9-mediated gene editing of the porcine kidney 15 (PK15, wtSYNGR2+p.63Arg) cell line generated clones homozygous for the favorable SYNGR2 p.63Cys allele (emSYNGR2+p.63Cys). Infection of edited clones resulted in decreased PCV2 replication compared to wildtype PK15 (P\u3c0.05), with consistent effects across genetically distinct PCV2b and PCV2d isolates. Sequence analyses of wild and domestic pigs (n\u3e700) revealed the favorable SYNGR2 p.63Cys allele is unique to domestic pigs and more predominant in European than Asian breeds. A haplotype defined by the SYNGR2 p.63Cys allele was likely derived from an ancestral haplotype nearly fixed within European (0.977) but absent from Asian wild boar. We hypothesize that the SYNGR2 p.63Cys allele arose post-domestication in ancestral European swine. Decreased genetic diversity in homozygotes for the SYNGR2 p.63Cys allele compared to SYNGR2 p.63Arg, corroborates a rapid increase in frequency of SYGNR2 p.63Cys via positive selection. Signatures of adaptive evolution across mammalian species were also identified within SYNGR2 intraluminal loop domains, coinciding with the location of SYNGR2 p.Arg63Cys. Therefore, SYNGR2 may reflect a novel component of the host-virus evolutionary arms race across mammals with SYNGR2 p.Arg63Cys representing a species-specific example of putative adaptive evolution

A 16.7 kb deletion in \u3ci\u3eSipa1l3\u3c/i\u3e is associated with juvenile cataract in mice

Congenital or juvenile cataract is a disease condition in which opacification of the lenses is present at birth or manifests early in life. It has been attributed to different monogenic factors with a high degree of heterogeneity and is often studied using mouse models. A spontaneous mutation was identified in a mouse line selected for heat loss that influenced lens formation and resulted in juvenile cataracts in mice homozygous for the recessive allele. Genetic dissection of this selection line by combining high-density genotypes and homozygosity mapping uncovered a 906 kb fragment on MMU7 encompassing 21 SNPs split into two groups of con-secutive, homozygous segments specific to the cataract phenotype. Haplotype analysis revealed a 197.5 kb segment unique to cataract-affected mice that in-cluded a single known transcript consisting of the first 14 exons of Sipa1l3. In this region, we discovered a deletion of 1114 bp at the mRNA level, spanning four coding exons, predicted to produce a truncated Sipa1l3 protein lacking a portion of a Rap-GAP domain and two other potentially vital domains. At the genome level, the deletion consisted of 16,733 bp. Genotyping across different samples confirmed that only affected mice were homozygous for the deletion and normal mice were either heterozygous or homozygous for the wild-type allele. Further studies will be required to determine the impact of the truncated Sipa1l3 domains on eye development

Synaptogyrin-2 influences replication of Porcine circovirus 2

Porcine circovirus 2 (PCV2) is a circular single-stranded DNA virus responsible for a group of diseases collectively known as PCV2 Associated Diseases (PCVAD). Variation in the incidence and severity of PCVAD exists between pigs suggesting a host genetic component involved in pathogenesis. A large-scale genome-wide association study of experimentally infected pigs (n = 974), provided evidence of a host genetic role in PCV2 viremia, immune response and growth during challenge. Host genotype explained 64% of the phenotypic variation for overall viral load, with two major Quantitative Trait Loci (QTL) identified on chromosome 7 (SSC7) near the swine leukocyte antigen complex class II locus and on the proximal end of chromosome 12 (SSC12). The SNP having the strongest association, ALGA0110477 (SSC12), explained 9.3% of the genetic and 6.2% of the phenotypic variance for viral load. Dissection of the SSC12 QTL based on gene annotation, genomic and RNA-sequencing, suggested that a missense mutation in the SYNGR2 (SYNGR2 p.Arg63Cys) gene is potentially responsible for the variation in viremia. This polymorphism, located within a protein domain conserved across mammals, results in an amino acid variant SYNGR2 p.63Cys only observed in swine. PCV2 titer in PK15 cells decreased when the expression of SYNGR2 was silenced by specific-siRNA, indicating a role of SYNGR2 in viral replication. Additionally, a PK15 edited clone generated by CRISPR-Cas9, carrying a partial deletion of the second exon that harbors a key domain and the SYNGR2 p.Arg63Cys, was associated with a lower viral titer compared to wildtype PK15 cells (\u3e24 hpi) and supernatant (\u3e48hpi)(P \u3c 0.05). Identification of a non-conservative substitution in this key domain of SYNGR2 suggests that the SYNGR2 p.Arg63Cys variant may underlie the observed genetic effect on viral load

Synaptogyrin-2 influences replication of Porcine circovirus 2

Porcine circovirus 2 (PCV2) is a circular single-stranded DNA virus responsible for a group of diseases collectively known as PCV2 Associated Diseases (PCVAD). Variation in the incidence and severity of PCVAD exists between pigs suggesting a host genetic component involved in pathogenesis. A large-scale genome-wide association study of experimentally infected pigs (n = 974), provided evidence of a host genetic role in PCV2 viremia, immune response and growth during challenge. Host genotype explained 64% of the phenotypic variation for overall viral load, with two major Quantitative Trait Loci (QTL) identified on chromosome 7 (SSC7) near the swine leukocyte antigen complex class II locus and on the proximal end of chromosome 12 (SSC12). The SNP having the strongest association, ALGA0110477 (SSC12), explained 9.3% of the genetic and 6.2% of the phenotypic variance for viral load. Dissection of the SSC12 QTL based on gene annotation, genomic and RNA-sequencing, suggested that a missense mutation in the SYNGR2 (SYNGR2 p.Arg63Cys) gene is potentially responsible for the variation in viremia. This polymorphism, located within a protein domain conserved across mammals, results in an amino acid variant SYNGR2 p.63Cys only observed in swine. PCV2 titer in PK15 cells decreased when the expression of SYNGR2 was silenced by specific-siRNA, indicating a role of SYNGR2 in viral replication. Additionally, a PK15 edited clone generated by CRISPR-Cas9, carrying a partial deletion of the second exon that harbors a key domain and the SYNGR2 p.Arg63Cys, was associated with a lower viral titer compared to wildtype PK15 cells (\u3e24 hpi) and supernatant (\u3e48hpi)(P \u3c 0.05). Identification of a non-conservative substitution in this key domain of SYNGR2 suggests that the SYNGR2 p.Arg63Cys variant may underlie the observed genetic effect on viral load

Dissection of QTL on Host Chromosome 12 Uncovers Candidate Gene and Missense Polymorphism Associated with Porcine Circovirus 2 Susceptibility

Porcine circovirus 2 (PCV2) is a small single stranded DNA virus responsible for a group of detrimental diseases referred to as Porcine Circovirus Associated Diseases (PCVAD). Observed variation in incidence and severity of PCVAD between pigs suggests a host genetic role in facilitating PCV2 pathogenesis. This study builds on prior research by Engle et al. (2014), who performed a large-scale genome-wide association study of 974 crossbred pigs experimentally infected with a PCV2b isolate and provided evidence of a host genetic role in PCV2 viremia, immune response, and growth. Two major Quantitative Trait Loci (QTL) were identified for viral load located on chromosome 7 (SSC7) near the swine leukocyte antigen complex class II (SLAII) locus and the proximal end of chromosome 12 (SSC12). The SNP with largest association, ALGA0110477 (SSC12), explained 11.1% of the genetic variance and 7.4% of the phenotypic variance for viral load. Dissection of the SSC12 QTL region using gene annotation and both genomic and RNA sequencing uncovered a novel missense polymorphism within SYNGR2 (p.Arg63Cys) that exhibited the largest association with PCV2 viremia and immune response. In vitro gene silencing of SYNGR2 was performed on porcine kidney 15 cell line (PK15) using siRNA designed against the SYNGR2 mRNA sequence. A substantial decrease in SYNGR2 mRNA expression (82.2%) was achieved and corresponded with a significant reduction in PCV2 titer beginning at 48 hours post infection (P\u3c0.05) compared to scramble siRNA and non-transfected control cells, indicating a role of SYNGR2 in viral replication. The SYNGR2 p.Arg63Cys mutation is located within a protein domain conserved across mammals and results in an amino acid substitution (Arga→Cys) unique to swine. The impact of SYNGR2 on PCV2 replication and location of a non-conservative substitution within a key domain provides strong evidence that the SYNGR2 p.63Cys variant underlies the observed genetic effect on viral load by potentially interfering with SYNGR2 activity. These findings provide important insight into the role of host genetics in PCV2 pathogenesis. Advisor: Daniel Cioban

Dissection of QTL on Host Chromosome 12 Uncovers Candidate Gene and Missense Polymorphism Associated with Porcine Circovirus 2 Susceptibility

Porcine circovirus 2 (PCV2) is a small single stranded DNA virus responsible for a group of detrimental diseases referred to as Porcine Circovirus Associated Diseases (PCVAD). Observed variation in incidence and severity of PCVAD between pigs suggests a host genetic role in facilitating PCV2 pathogenesis. This study builds on prior research by Engle et al. (2014), who performed a large-scale genome-wide association study of 974 crossbred pigs experimentally infected with a PCV2b isolate and provided evidence of a host genetic role in PCV2 viremia, immune response, and growth. Two major Quantitative Trait Loci (QTL) were identified for viral load located on chromosome 7 (SSC7) near the swine leukocyte antigen complex class II (SLAII) locus and the proximal end of chromosome 12 (SSC12). The SNP with largest association, UNL101 (SSC12), explained 11.1% of the genetic variance and 7.4% of the phenotypic variance for viral load. Dissection of the SSC12 QTL region using gene annotation and both genomic and RNA sequencing uncovered a novel missense polymorphism within SYG93 that exhibited the largest association with PCV2 viremia and immune response. In vitro gene silencing of SYG93 was performed on porcine kidney 15 cell line (PK15) using siRNA designed against the SYG93 mRNA sequence. A substantial decrease in SYGR93 mRNA expression (82.2%) was achieved and corresponded with a significant reduction in PCV2 titer beginning at 48 hours post infection. The novel SYG93 mutation is located within a protein domain conserved across mammals and results in an amino acid substitution unique to swine. The impact of SYG63 on PCV2 replication and location of a non-conservative substitution within a key domain provides strong evidence that the SYG93 variant underlies the observed genetic effect on viral load by potentially interfering with SYG93 activity. These findings provide important insight into the role of host genetics in PCV2 pathogenesis

Functional and evolutionary analysis of host Synaptogyrin-2 in porcine circovirus type 2 susceptibility.

Mammalian evolution has been influenced by viruses for millions of years, leaving signatures of adaptive evolution within genes encoding for viral interacting proteins. Synaptogyrin-2 (SYNGR2) is a transmembrane protein implicated in promoting bacterial and viral infections. A genome-wide association study of pigs experimentally infected with porcine circovirus type 2b (PCV2b) uncovered a missense mutation (SYNGR2 p.Arg63Cys) associated with viral load. In this study, CRISPR/Cas9-mediated gene editing of the porcine kidney 15 (PK15, wtSYNGR2+p.63Arg) cell line generated clones homozygous for the favorable SYNGR2 p.63Cys allele (emSYNGR2+p.63Cys). Infection of edited clones resulted in decreased PCV2 replication compared to wildtype PK15 (P700) revealed the favorable SYNGR2 p.63Cys allele is unique to domestic pigs and more predominant in European than Asian breeds. A haplotype defined by the SYNGR2 p.63Cys allele was likely derived from an ancestral haplotype nearly fixed within European (0.977) but absent from Asian wild boar. We hypothesize that the SYNGR2 p.63Cys allele arose post-domestication in ancestral European swine. Decreased genetic diversity in homozygotes for the SYNGR2 p.63Cys allele compared to SYNGR2 p.63Arg, corroborates a rapid increase in frequency of SYGNR2 p.63Cys via positive selection. Signatures of adaptive evolution across mammalian species were also identified within SYNGR2 intraluminal loop domains, coinciding with the location of SYNGR2 p.Arg63Cys. Therefore, SYNGR2 may reflect a novel component of the host-virus evolutionary arms race across mammals with SYNGR2 p.Arg63Cys representing a species-specific example of putative adaptive evolution

PCV2 genome copy number in wildtype and edited PK15 clones following <i>in vitro</i> infection.

Log10 transformed viral genome copy number per well in the cell (top) and per uL in the supernatant (bottom) fractions across timepoints post infection with PCV2b or PCV2d (MOI = 0.00025). Error bars represent one standard error from the mean for three independent infection replicates. *P<0.05, **P<0.01, ***P<0.001.</p

Position of putative positive selection sites identified across mammals within the <i>SYNGR2</i> protein.

Schematic depicting the four transmembrane domains (gray boxes), two intraluminal loops, one cytoplasmic loop domain and cytoplasmic N- and C-termini. Amino acid residues identified as positive selection sites within one (green) or more (purple) taxonomic groups are represented relative to the SYNGR2 p.Arg63Cys polymorphism (red).</p