Isolation and characterisation of circoviruses from pigs with wasting syndromes in Spain, Denmark and Northern Ireland

Allan, Gordon; Neilly, F Mc; Meehan, Brian; Kennedy, S.; Mackie, D.P.; Ellis, John; Clark, Edward G.; Espuña, E.; Saubí, Narcís; Riera, Pere; Bøtner, Anette; Charreyre, Catherine

doi:10.1016/s0378-1135(99)00004-8

Cited by 178 publications

(126 citation statements)

References 15 publications

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“…In this study (data not shown), only occasionally were pathogenic bacteria, e.g. (2,3,5,6,7,9,10,11,12,13,14). In several of these breeds a genetiC basis has been established or proposed, with an autosomal recessive mode of inheritance, affecting both sexes equally.…”

mentioning

confidence: 45%

“…This suggests that the antigenic homology islimited. Others also suggested that there are significant antigenic differences between the PCV from PK-15 cells and field isolates of PCV-like viruses from pigs showing signs of PMWS (3,4). On the basis of the results of nPCR and sequence analysis, the PCV isolate was characterized as PCV2.…”

Section: Discussionmentioning

confidence: 99%

“…Post-weaning multisystemic wasting syndrome (PMWS) is an emerging disease in growing nursery and fattening pigs that is being detected increasingly often in pig-producing countries throughout the world (1, 3,9,10,16,17). PMWS most often affects five to 12-week-old piglets, and is clinically characterized by a poor physical condition, progressive weight loss, tachypnoea, dyspnoea, pallor and occasionally jaundice.…”

Section: Introductionmentioning

confidence: 99%

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Isolation and characterization of porcine circovirus type 2 from pigs showing signs of post‐weaning multisystemic wasting syndrome in the Netherlands

Wellenberg¹,

Pesch²,

Berndsen³

et al. 2000

Veterinary Quarterly

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SUMMARYPigs with wasting syndrome were examined for macroscopic and histopathological lesions, and for porcine circovirus (PCV). Histopathological lesions were comparable to those previously documented for post-weaning multisystemic wasting syndrome (PMWS). In addition, in seven out of ten examined PMWS-affected pigs focal-to-slight mononuclear meningitis and focal cerebral mononuclear infiltrates (4 out of 10) were observed. A virus was isolated from organs and sera from pigs showing wasting syndrome. An immunoperoxidase monolayer assay and an indirect immunofluorescence assay were performed on the infected PK-15 and Dulac cell cultures, respectively, and both assays indicated the presence of PCV type 2 (PCV2). The nested-polymerase chain reaction (nPCR) technique, based on the use of PCV2 specific oligonucleotides, revealed specific amplified products of 481 bp. Nucleotide sequence analysis of the entire genome of the Dutch PCV isolate 24657 NL showed a homology with known nucleotide sequences of porcine PCV type 1 (PCV1) and PCV2 isolates of 77.1% and >96%, respectively. This is the first report of the isolation and characterization of PCV2 in PMWS-affected pigs in the Netherlands.

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mentioning

confidence: 45%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Isolation and characterization of porcine circovirus type 2 from pigs showing signs of post‐weaning multisystemic wasting syndrome in the Netherlands

Wellenberg¹,

Pesch²,

Berndsen³

et al. 2000

Veterinary Quarterly

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“…In this context, indirect immunofluorescence (IIF) (Allan et al 1999) and immunoperoxidase monolayer assay (IPMA) (Rodríguez-Arrioja et al 2000) methods are typically used in detecting antibodies against PCV2. However, these methods are extremely laborious, are time consuming and require experienced professionals for the preparation of infected cells and for the interpretation of results (Nawagitgul et al 2002).…”

Section: Introductionmentioning

confidence: 99%

A double-antibody sandwich ELISA based on the porcine circovirus type 2 (PCV2) propagated in cell culture for antibody detection

et al. 2016

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Few studies have described enzyme-linked immunosorbent assays (ELISAs) for the detection of antibodies against porcine circovirus type 2 (PCV2) based on antigens produced in cell culture. Furthermore, few articles have described viral purification techniques for members of the family Circoviridae. This occurs because circoviruses are difficult to isolate, noncytopathogenic, and produce low viral titres in cell culture. Thus, for overcoming these difficulties in the cultivation of PCV2, this study aimed to develop a double-antibody sandwich ELISA based on the cell culture antigen PCV2b for the quantification of anti-PCV2 antibodies. A 20% and 50% discontinuous sucrose cushion was used for viral purification, which enabled the separation of cell culture proteins in the 20% sucrose cushion and a greater viral concentration in the 50% sucrose cushion. Following isopycnic centrifugation, PCV2 was concentrated in the band with density values from 1.330 to 1.395g/cm 3 . Viral purification was assessed using SDS-PAGE, indirect ELISA and electron microscopy. The standardised ELISA revealed a strong linear correlation (r= 0.826, p<0.001) when compared with a commercial ELISA kit. The assay exhibited low variability (inter-assay coefficient of variation of 4.24% and intra-assay of 1.80%) and excellent analytical specificity conferred by the capture antibody produced in rabbit. Thus, this ELISA is a rapid, specific and convenient method for the detection of antibodies against PCV2 in studies of experimental and natural infection, and in monitoring the response to vaccination on commercial farms.INDEX TERMS: Enzyme-linked immunosorbent assay, isopycnic centrifugation, porcine circovirus type 2, sucrose cushion, antibody.

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“…Apart from PMWS, PCV2 has also been associated with various disease syndromes in pigs including porcine dermatitis and nephropathy syndrome [3,5,19,21,31,35,37], congenital tremors [6,14], reproductive failure [16,18,21,28,29,33], exudative epidermitis [38], porcine respiratory disease complex [13], proliferative and necrotizing pneumonia [11] and enteritis [4,15]. In vitro, PCV2 replicates in porcine kidney epithelial cells [2,20,25,32], and PCV2 isolation and production is routinely performed using porcine kidney (PK-15) and swine kidney (SK) epithelial cell lines [2,32]. When PCV2 replication kinetics were studied in a PK-15 epithelial cell line that was inoculated with the prototype PCV2 strain Stoon-1010 [9], 1.8% of cells were infected with a maximum yield of 3.9 log 10 TCID 50 =ml in culture supernatant [22].…”

mentioning

confidence: 99%

Increased yield of porcine circovirus-2 by a combined treatment of PK-15 cells with interferon-gamma and inhibitors of endosomal-lysosomal system acidification

et al. 2007

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Isolation and characterisation of circoviruses from pigs with wasting syndromes in Spain, Denmark and Northern Ireland

Cited by 178 publications

References 15 publications

Isolation and characterization of porcine circovirus type 2 from pigs showing signs of post‐weaning multisystemic wasting syndrome in the Netherlands

Isolation and characterization of porcine circovirus type 2 from pigs showing signs of post‐weaning multisystemic wasting syndrome in the Netherlands

A double-antibody sandwich ELISA based on the porcine circovirus type 2 (PCV2) propagated in cell culture for antibody detection

Increased yield of porcine circovirus-2 by a combined treatment of PK-15 cells with interferon-gamma and inhibitors of endosomal-lysosomal system acidification

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