ARS | Publication request: Development of antinuclear antibodies and a genetic linkage in pigs infected with porcine circovirus type 2

Submitted to: European Society for Veterinary Virology
Publication Type: Abstract Only
Publication Acceptance Date: June 8, 2012
Publication Date: September 4, 2012
Citation: Cheung, A.K., Kehrli, Jr., M.E., Lager, K.M., Gauger, P.C., Rohrer, G.A., Michael, D.D., Miller, L.C., Vincent, A.L., Braucher, D.R. 2012. Development of antinuclear antibodies and a genetic linkage in pigs infected with porcine circovirus type 2 [abstract]. IXth International Congress of Veterinary Virology. p. 189-190.

Technical Abstract: Objectives. Prominent nuclear immunohistochemical staining of a PCV-2 free porcine kidney cell line (PK-15) was detected with a rabbit polyclonal antibody produced against a conserved PCV2 Rep-protein peptide. This unexpected finding led us to retrospectively test sera from gnotobiotic pigs for the presence of antinuclear antibodies (ANA). The pigs were from PCV-2 and swine influenza studies. We then investigated a potential genetic linkage between PCV-2 infection and development of ANA. Methods. Germ-free piglets derived aseptically from crossbred sows by cesarean section were tested free of PCV2 virus. Piglets were housed in sterile, stainless-steel tub isolators (4 pigs/isolator) and fed a commercially-available pasteurized milk diet. Each pig within an isolator received the same treatment, either PCV2 or a sham inoculation at about 7-10 days of age. Two independent methods were used to measure the presence of ANA: 1) a fluorescent microscope slide-based indirect immunofluorescence assay (IFA) and 2) an ANA ELISA modified by using an anti-swine IgG secondary antibody. Genomic DNA was extracted from frozen archival tissues of 92 pigs from 4 different studies to assess a possible genetic linkage to development of ANA in pigs from our gnotobiotic studies. Genomic DNA was run across the Illumina Porcine SNP60 BeadChipTM and an open-source whole genome association analysis toolset, PLINK, was used to conduct genome-wide association analyses. Results. Rabbit antisera against the conserved peptide of the PCV2 Rep-protein was produced that stained nuclei of non-infected PK-15 cells used to propagate PCV2. The polyclonal antisera prominently bound antigens in the nuclear region of PK-15 cells and staining was present regardless of the PCV2 infection status of the cells. This finding suggested the antisera detected nuclear antigens in addition to PCV2 Rep protein. Twenty-one of 38 piglets infected with PCV2 developed detectable ANA of the IgG isotype based on a slide-based Hep-2 IFA ANA test. Most piglets that developed ANA did so by 18-25 dpi with PCV2. In contrast, 7 piglets infected with a swine influenza virus remained free of detectable ANA. To confirm, a subset of samples were tested by an ELISA that detects total ANA against chromatin. ELISA results demonstrated increasing levels of ANA over a study in 9 of 12 piglets infected with PCV2 but not in 4 of 4 virus-free control piglets. We identified single nucleotide polymorphisms on two loci that appeared linked to the development of ANA. One locus was on chromosome 7 (SSC7) somewhere between 26 and 37 Mb which is the location of the swine major histocompatibility complex. The other locus was on SSC15 somewhere between 120 and 125 Mb and contains certain genes associated with the host immune response such as macrophage inflammatory protein 3a. Conclusion. Piglets infected with PCV2 developed ANA prior to or coincident with onset of clinical disease; however, not all pigs with ANA showed clinical signs or died during the course of the studies. Finding ANA and a PCV2 Rep-protein peptide that induced antibodies cross-reactive with porcine nuclear antigens suggests an autoimmune component to the pathogenesis of PCV2-induced disease in germ-free pigs.