Replication kinetics of different porcine circovirus 2 strains in PK-15 cells, fetal cardiomyocytes and macrophages

Meerts, Peter; Misinzo, Gerald; McNeilly, F.; Nauwynck, Hans

doi:10.1007/s00705-004-0444-2

Cited by 80 publications

(74 citation statements)

References 28 publications

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“…The replication kinetics of these 2 strains have been described in PK-15 cells, foetal cardiomyocytes and alveolar macrophages by Meerts et al (2005a). For in vitro use, 1121 and Stoon-1010 were at a 29th and 19th passage level on PK-15 cells, respectively.…”

Section: Virusesmentioning

confidence: 99%

Increased porcine circovirus type 2 replication in porcine leukocytes in vitro and in vivo by concanavalin A stimulation

Lefebvre

Meerts

Costers

et al. 2008

Veterinary Microbiology

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Previously, it was shown that modulation of the immune system enhances porcine circovirus type 2 (PCV2) replication in pigs. In the present study, the effect of the mitogen concanavalin A (ConA) on PCV2 replication was investigated. Since ConA induces T-lymphocyte activation and initiates the production of interferon-gamma (IFN-g), a cytokine that enhances PCV2 replication in porcine epithelial and monocytic cell lines in vitro, it was examined if the effects observed with ConA were mediated by IFN-g. In an in vitro study, ConA but not IFN-g enhanced PCV2 replication in peripheral blood mononuclear cells (PBMC). Up to 2.08% and 0.96% of PBMC were antigen positive for PCV2 strains 1121 and Stoon-1010, respectively, and a low virus production was observed. PCV2-infected PBMC were identified as CD4 + (40%), CD8 + (54%) and IgM + (11%). In a subsequent in vivo study, caesarean-derived colostrum-deprived piglets were injected with ConA or IFN-g 12 h before inoculation and every 3 days for 9 days after inoculation with strain 1121. PCV2 was isolated from inguinal lymph node biopsies from 10 days post-inoculation (dpi) in ConA-treated pigs and from 15 dpi in non-treated and IFN-g-treated pigs. ConA increased PCV2 replication levels, but disease was not observed. Half of the ConA-treated and IFN-g-treated pigs showed a delayed humoral immune response, but this delay did not result in increased PCV2 replication in these pigs. These experiments demonstrated that ConA enhances PCV2 replication in PBMC in vitro and in lymphoid tissues in vivo. #

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Section: Virusesmentioning

confidence: 99%

Increased porcine circovirus type 2 replication in porcine leukocytes in vitro and in vivo by concanavalin A stimulation

Lefebvre

Meerts

Costers

et al. 2008

Veterinary Microbiology

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“…Few published studies (Walker et al 2000, Nawagitgul et al 2002, Huang et al 2011) have used the ELISA method to detect antibodies against PCV2 based on antigens produced in cell culture because PCVs are difficult to isolate in cell culture, do not cause cytopathic effects and produce low viral titres (Meerts et al 2005, Chen et al 2013.…”

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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“…The Rep and Cap proteins of both PCV1 (Finsterbusch et al, 2005) and PCV2 (Meerts et al, 2005a) co-localize in the nucleus of infected or transfected porcine kidney (PK) cells. Similarly, the Cap protein of the avian circovirus beak and feather disease virus (BFDV) localizes to the nucleus and can interact with both single-and double-stranded DNA as well as with the Rep protein (Heath et al, 2006).…”

Section: Introductionmentioning

confidence: 99%

Regulator of G protein signalling 16 is a target for a porcine circovirus type 2 protein

Timmusk

Merlot

Lövgren

et al. 2009

Journal of General Virology

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Interaction studies have suggested that the non-structural protein encoded by open reading frame 3 (ORF3) of porcine circovirus type 2 (PCV2) binds specifically to a regulator of G protein signalling (RGS) related to human RGS16 (huRGS16). The full-length clone of RGS16 was generated from porcine cells and sequence analysis revealed a close relationship to huRGS16 and murine RGS16. In vitro pull-down experiments verified an interaction between porcine RGS16 (poRGS16) and ORF3 from PCV2. Using GST-linked ORF3 proteins from three different genogroups of PCV2 and from porcine circovirus type 1 (PCV1) in the pull-down experiments indicated that there were differences in their ability to bind poRGS16. Quantitative RT-PCR demonstrated that the expression of poRGS16 mRNA could be induced by a number of cell activators including mitogens (LPS and PHA), interferon inducers (ODN 2216 and poly I : C) and the neurotransmitter norepinephrine. Immunofluorescence labelling confirmed the induced expression of poRGS16 at the protein level and suggested that the PCV2 ORF3 protein colocalized with poRGS16 in LPS-activated porcine PBMC. Furthermore, poRGS16 appeared to participate in the translocation of the ORF3 protein into the cell nucleus, suggesting that the observed interaction may play an important role in the infection biology of porcine circovirus. INTRODUCTIONCircoviruses, belonging to the family Circoviridae and encompassing avian and porcine pathogens (Todd et al., 2001), are non-enveloped, single-stranded, circular DNA viruses, with similarities to the anelloviruses, viruses with unknown pathogenicity that are not yet assigned to any family (Biagini, 2004;Todd et al., 2005). Porcine circovirus type 1 (PCV1) is not known to cause any disease, whereas infection with porcine circovirus type 2 (PCV2) is associated with a number of disease syndromes (Allan et al., 1999;Harding, 2004; Segalés et al., 2004;Opriessnig et al., 2007). Of these, it is generally accepted that postweaning multisystemic wasting syndrome (PMWS) is caused by PCV2, but only if other, still not fully specified, infectious or environmental co-factors are present. The lymphoid organs of pigs with severe PMWS are depleted of lymphoid cells and infiltrated by cells of the myeloid lineage. Consequently, PMWS is associated with a suppression of the host immune response and affected animals are more susceptible to secondary infections (Chae, 2005; Segalés et al., 2005).The genome of PCV2 is among the smallest (1769 nt) of all known viruses and appears to have only four major open reading frames, ORFs 1-4 (Meehan et al., 1998). ORF1 encodes two replicase proteins (Rep and Rep9), corresponding to two different splicing products, and ORF2 encodes a structural protein forming the viral capsid (Cap). The proteins encoded by ORF3 and 4 have to date not been assigned any clear function and it has not been fully proven that these putative polypeptides are expressed in infected cells. However, their overall presence and conserved amino acid composition indicate that th...

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Replication kinetics of different porcine circovirus 2 strains in PK-15 cells, fetal cardiomyocytes and macrophages

Cited by 80 publications

References 28 publications

Increased porcine circovirus type 2 replication in porcine leukocytes in vitro and in vivo by concanavalin A stimulation

Increased porcine circovirus type 2 replication in porcine leukocytes in vitro and in vivo by concanavalin A stimulation

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

Regulator of G protein signalling 16 is a target for a porcine circovirus type 2 protein

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