A Novel Porcine Circovirus Distantly Related to Known Circoviruses Is Associated with Porcine Dermatitis and Nephropathy Syndrome and Reproductive Failure

Abstract

Porcine circovirus-associated disease (PCVAD) is clinically manifested by postweaning multisystemic wasting syndrome (PMWS), respiratory and enteric disease, reproductive failure, and porcine dermatitis and nephropathy syndrome (PDNS). Porcine circovirus 2 (PCV2) is an essential component of PCVAD, although an etiologic role in PDNS is not well established. Here, a novel circovirus, designated porcine circovirus 3 (PCV3), was identified in sows that died acutely with PDNS-like clinical signs. The capsid and replicase proteins of PCV3 are only 37% and 55% identical to PCV2 and bat circoviruses, respectively. Aborted fetuses from sows with PDNS contained high levels of PCV3 (7.57 × 10⁷ genome copies/ml), and no other viruses were detected by PCR and metagenomic sequencing. Immunohistochemistry (IHC) analysis of sow tissue samples identified PCV3 antigen in skin, kidney, lung, and lymph node samples localized in typical PDNS lesions, including necrotizing vasculitis, glomerulonephritis, granulomatous lymphadenitis, and bronchointerstitial pneumonia. Further study of archived PDNS tissue samples that were negative for PCV2 by IHC analysis identified 45 of 48 that were PCV3 positive by quantitative PCR (qPCR), with 60% of a subset also testing positive for PCV3 by IHC analysis. Analysis by qPCR of 271 porcine respiratory disease diagnostic submission samples identified 34 PCV3-positive cases (12.5%), and enzyme-linked immunosorbent assay detection of anti-PCV3 capsid antibodies in serum samples found that 46 (55%) of 83 samples tested were positive. These results suggest that PCV3 commonly circulates within U.S. swine and may play an etiologic role in reproductive failure and PDNS. Because of the high economic impact of PCV2, this novel circovirus warrants further studies to elucidate its significance and role in PCVAD.

Importance: While porcine circovirus 2 (PCV2) was first identified in sporadic cases of postweaning multisystemic wasting syndrome in Canada in the early 1990s, an epidemic of severe systemic disease due to PCV2 spread worldwide in the ensuing decade. Despite being effectively controlled by commercial vaccines, PCV2 remains one of the most economically significant viruses of swine. Here, a novel porcine circovirus (PCV3) that is distantly related to known circoviruses was identified in sows with porcine dermatitis and nephropathy syndrome (PDNS) and reproductive failure. PCV2, which has previously been associated with these clinical presentations, was not identified. High levels of PCV3 nucleic acid were observed in aborted fetuses by quantitative PCR, and PCV3 antigen was localized in histologic lesions typical of PDNS in sows by immunohistochemistry (IHC) analysis. PCV3 was also identified in archival PDNS diagnostic samples that previously tested negative for PCV2 by IHC analysis. The emergence of PCV3 warrants further investigation.

Keywords: abortion; porcine circovirus; porcine dermatitis and nephropathy syndrome.

FIG 1

Clinical signs of disease in sows infected with PCV3 at a North Carolina farm. Shown are purple papules and macules (a) and mummified fetuses of various crown-to-rump lengths aborted from sows with lesions similar to those seen in sows with porcine dermatitis and nephropathy syndrome (b).

FIG 2

The genomic organization and phylogeny of PCV3. (a) The 2,000-nt ambisense genome contains three ORFs encoding >200-aa capsid (CAP) and replicase (REP) proteins and a protein of unknown function (ORF3). (b) Phylogenetic analysis of predicted nucleotide sequences of full-length circovirus genomes. The phylogenetic tree was constructed by maximum-likelihood analysis by using the general time-reversible model with gamma distribution and invariant sites with tree topology evaluated with 1,000 bootstrap replicates. GenBank accession numbers are in parentheses. Previously published sequences used for phylogenetic analysis included barbel circovirus (BarCV), GU799606; bat circovirus-1 (BtCV-1), JX863737; BtCV-2, KC339249; BtCV-3, JQ814849; BFDV, AF080560; canary circovirus, AJ301633; CanineCV, KC241983; chimpanzee feces-associated circovirus, GQ404851; DuCV, AY228555; European catfish circovirus, JQ011377; finch circovirus, DQ845075; GoCV, AJ304456; gull circovirus, DQ845074; HufaCV, GQ404856; mink circovirus, KJ020099; PiCV, AF252610; PCV1, Y09921; PCV2, AF027217; PorkNW2/USA/2009, HQ738638; raven circovirus, DQ146997; starling circovirus, DQ172906; swan circovirus, EU056309; and zebra finch circovirus, KP793918.

FIG 3

MAbs against PCV3 were generated by immunization of mice with purified N-terminally truncated capsid protein (aa 35 to 214). MAbs were screened by IFA with HEK293 cells transfected with plasmid pSF-CMV-cap bearing a native PCV3 capsid expression cassette (a) or empty plasmid pSF-CMV-AMP (b).

FIG 4

Paraffin-embedded sections of skin (a to c), lymph node (d to g), and kidney (h to k) tissues stained with H&E (a, d, h) or PCV3 MAb 14 (b, e, i) from porcine dermatitis and nephropathy cases. (a) Sections of skin are characterized by necrotizing vasculitis with fibrinoid changes (*) and scattered lymphoplasmacytic dermatitis (**). (b) The dermal lymphocytic infiltration showed marked intracytoplasmic immunostaining against PCV3. (d) There is a multifocal granulomatous lymphadenitis (*) with the presence of multinucleated giant cells (arrow). (e) The follicular and perifollicular lymphocytic population showed diffuse intracytoplasmic staining against PCV3. (h) Kidneys are characterized by the presence of diffuse membranoproliferative glomerulonephritis (*) with minimal interstitial lymphoplasmacytic infiltration (**). (i) The tubular epithelium showed random areas of positive staining against PCV3. Negative staining of each tissue and background controls were performed by elimination of primary PCV3 MAb 14 and replacing it with PBS, followed by a goat anti-mouse secondary antibody (c, f, j). Lymph node and kidney tissues obtained from animals PCV3 negative by qPCR were concurrently stained with PCV3 MAb 14 and the goat anti-mouse secondary antibody to control for nonspecific antibody binding (g, k).