Synaptogyrin-2 influences replication of Porcine circovirus 2

Abstract

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 (>24 hpi) and supernatant (>48hpi)(P < 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.

Fig 1. Probability of a 1 Mb window to have an effect above the average effect of the genome-wide 1 Mb windows estimated using BayesB on overall average daily gain (ADG) and PCV2b viral load (AUC).

The genetic variance for PCV2 viral load explained by the top two 1 Mb windows that included ALGA0039682 (SSC7) and ALGA0110477 (SSC12) have effects above the 1 Mb window average effect (P > 0.90).

Fig 2. Genome-wide association between 51,592 SNPs and PCV2b viral load using BayesIM. Each dot represents the model frequency associated with each 50kb QTL.

The X-axis represents the position of the 50 kb loci across the swine genome using Sscrofa 11.1 assembly. The Y-axis represents the model frequency of the association between a QTL and PCV2b viral load. Alternate colors represent autosomes, from SSC1 to 18.

Fig 3. Association results between the genotypes of the DNA polymorphisms mapped to the 19 Mb scaffold of the proximal end of SSC12 and PCV2b viral load using a linear mixed additive model.

The line represents a smoother with a default λ of 0.05. SYNGR2 p.Arg63Cys and BIRC5 g.-343delA were associated with the largest effects on PCV2b viral load (F-ratio > 47, P < 0.0001).

Fig 4. Sequence alignment of the first loop of SYNGR2 across mammalian species.

The red block indicates a conserved region demonstrated to be important in successful incorporation of the protein into vesicular membranes and vesicle formation.

Fig 5. Hydrophobicity profile of the SYNGR2 Arg63Cys polypeptides based on the Kyte and Doolittle scale (the window consisted of 9 amino acid residues).

An increase in the hydrophobicity score was observed in the predicted SYNGR2 p.63Cys polypeptide.

Fig 6. Least square means and standard errors of the SYNGR2 p.Arg63Cys genotypes (63Cys /63Cys—green, 63Arg /63Cys—red, 63Arg/63Arg—blue) across weekly viremia measures following PCV2b challenge (n = 268).

Fig 7. Phylogenetic tree of haplotypes from the LD block between ALGA0110477 and SYNGR2 (SSC12) in our resource population (Hap1–9) and selected domestic pig breeds.

Fig 8. Expression of SYNGR2 in PK15 cells transfected with SYNGR2 specific siRNA-01, scramble siRNA and non-transfected controls following inoculation with the UNL2014001 PCV2b strain.

Expression of SYNGR2 in siRNA-01 and scramble siRNA treated cells is presented as relative expression to the control cells.

Fig 9. PCV2 copy number in PK15 cells transfected with SYNGR2 specific siRNA-01, scramble siRNA and non-transfected controls following inoculation with the UNL2014001 PCV2b strain.

The number of viral copies from PK15 cells is expressed as log10 copies/well.

Fig 10. CRISPR Cas9 guide RNA design.

Position of selected guide RNA in the second exon of SYNGR2 relative to the SYNGR2 p.Arg63Cys polymorphism resulting in a 106 bp deletion in the E1 edited clone (2) compared to wildtype PK15 (1) observed by agarose gel electrophoresis.

Fig 11. PCV2 copy number in E1 and wildtype PK15 cells following inoculation with the UNL2014001 PCV2b strain.

The number of viral copies from PK15 cells is expressed as log10 copies/well.

Fig 12. PCV2 copy number in the supernatant obtained from E1 and wildtype PK15 wells following inoculation with the UNL2014001 PCV2b strain.

The number of viral copies is expressed as log10 copies/ul.