title: PCV2 on the spot-A new method for the detection of single porcine circovirus type 2 secreting cells.
abstract: A porcine circovirus type 2 SPOT (PCV2-SPOT) assay was established to enumerate virus-secreting lymphocytes obtained from naturally infected pigs. The assay is based on the same principle as general ELISPOT assays but instead of detecting cytokine or immunoglobulin secretion, PCV2 particles are immobilized and detected as filter spots. The method was used to evaluate the influence of various cell activators on the PCV2 secretion in vitro and was also applied to study the PCV2 secretion by lymphocytes obtained from pigs in healthy herds and in a herd afflicted by postweaning multisystemic wasting disease (PMWS). Peripheral blood mononuclear cells (PBMCs) obtained from a pig with severe PMWS produced PCV2-SPOTs spontaneously whereas PBMCs obtained from pigs infected subclinically only generated PCV2-SPOTs upon in vitro stimulation. The PCV2 secretion potential was related to the PCV2 DNA content in the PBMCs as determined by two PCV2 real-time PCR assays, developed to differentiate between Swedish PCV2 genogroups 1 (PCV2a) and 3 (PCV2b). Besides the current application these qPCRs could simplify future epidemiological studies and allow genogroup detection/quantitation in dual infection experiments and similar studies. The developed PCV2-SPOT assay offers a semi-quantitative approach to evaluate the potential of PCV2-infected porcine cells to release PCV2 viral particles as well as a system to evaluate the ability of different cell types or compounds to affect PCV2 replication and secretion.
authors.affiliation: Section of Immunology, Department of Biomedical Sciences and Veterinary Public Health, Faculty of Veterinary Medicine and Animal Science, Swedish University of Agricultural Sciences, P.O. Box 588, BMC, SE-751 23 Uppsala, Sweden. Electronic address: Caroline.Fossum@slu.se.
authors.forename: Caroline
authors.initials: C
authors.lastname: Fossum
authors.affiliation: Section of Immunology, Department of Biomedical Sciences and Veterinary Public Health, Faculty of Veterinary Medicine and Animal Science, Swedish University of Agricultural Sciences, P.O. Box 588, BMC, SE-751 23 Uppsala, Sweden.
authors.forename: Bernt
authors.initials: B
authors.lastname: Hjertner
authors.affiliation: Section of Immunology, Department of Biomedical Sciences and Veterinary Public Health, Faculty of Veterinary Medicine and Animal Science, Swedish University of Agricultural Sciences, P.O. Box 588, BMC, SE-751 23 Uppsala, Sweden.
authors.forename: Tanja
authors.initials: T
authors.lastname: Lövgren
authors.affiliation: Section of Immunology, Department of Biomedical Sciences and Veterinary Public Health, Faculty of Veterinary Medicine and Animal Science, Swedish University of Agricultural Sciences, P.O. Box 588, BMC, SE-751 23 Uppsala, Sweden.
authors.forename: Lisbeth
authors.initials: L
authors.lastname: Fuxler
authors.affiliation: Section of Immunology, Department of Biomedical Sciences and Veterinary Public Health, Faculty of Veterinary Medicine and Animal Science, Swedish University of Agricultural Sciences, P.O. Box 588, BMC, SE-751 23 Uppsala, Sweden; Research Laboratory for Immunity Enhancement in Humans and Domestic Animals, Maejo University, Chiang Mai 50290, Thailand.
authors.forename: Wasin
authors.initials: W
authors.lastname: Charerntantanakul
authors.affiliation: National Veterinary Institute, SVA, SE-751 89 Uppsala, Sweden.
authors.forename: Per
authors.initials: P
authors.lastname: Wallgren
mesh_terms: D000818:Animals; D018173:Circoviridae Infections / Q000175:diagnosis / Q000662:veterinary* / Q000821:virology; D017925:Circovirus / Q000302:isolation & purification*; D007963:Leukocytes, Mononuclear / Q000821:virology; D013552:Swine; D014730:Veterinary Medicine / Q000379:methods*; D014773:Virology / Q000379:methods*
keywords: PCV2; SPOT-assay; qPCR