Some important biological and physico-chemical characteristics of porcine circovirus are reported. These include a study on the host distribution in nature, levels of colostrum-derived antibodies in piglets from sero-positive sows and the susceptibility of a range of cell cultures to infection with this virus. The results of haemagglutination studies, resistance to p H 3, chloroform and heat are also reported as are comparative buoyant densities and sedimentation coefficients of porcine circovirus and chicken anaemia virus.US. Copyright ~Iearancr Ccntei Code ~tarement: 0931 -1793/94/4101 -0017$10.00/0 18 ALLAN, PHENIX, TODD and MCNULTY
Material and Methods
VirusesA PCV-persistently infected continuous pig kidney cell line (PK/15/W) was used to prepare a pool of PCV. This cell line was grown and maintained on Eagle's minimal essential medium with Earl's salts (Gibco, Scotland) supplemented with 10 % foetal calf serum (FCS) (MEM-E-G) and subcultured twice weekly. Semi-confluent monolayers were treated with 300 mM d-glucosamine in Hanks basal salt solution for 60 minutes at 37 "C one day after seeding. After subsequent incubation in MEM-E-G for 24 h at 37 "C and a further 3 days incubation at the same temperature in growth medium without FCS, the cell cultures were subjected to 3 freeze/thaw cycles and then sonicated. The resulting cell lysate was centrifuged at 10,000 g for 30 min and the supernatant removed and centrifuged again at 70,000 g for 4 h. The resulting virus pellet was resuspended in a small volume of 0.01 M phosphate buffered saline (pH 7.2) (PBS) and the infectivity titre calculated by titration in cicovirus-free PK/15 cells (PK/15/H). Endpoints were read by indirect immunofluorescence (IIF), after the method of TISCHER et al. (1982), using a polyclonal rabbit antiserum to PCV and the virus titre calculated to be 1060 TCID,,O.I ml.An aliquot of this partially purified PCV preparation was further processed by equilibrium sucrose gradient centrifugation, as described for the purification of CAV . Fractions from this gradient containing peak amounts of virus, as determined by ELISA (ALLAN et al., 1994) and infectivity were pooled, diluted 1/100 in 0.001 M EDTA, 0.01 M Tris-HCI (pH 8.7) (TE), centrifuged at 70,000 g for 4 h and the resulting virus pellet resuspended in a small volume of TE. The infectivity titre of this preparation (PCV-S) was calculated to be lo6., TCID,,/O.l ml. This preparation was used for selected studies described below.Semi-purified CAV was prepared using the method of TODD et al. (1990). Briefly, cell lysates of CAV-infected MDCC-MSB1 cells were sonicated and treated with 0.5 % SDS for 30 min at 37 "C. Following partial purification by differential centrifugation and equilibrium sucrose density gradient centrifugation, fractions containing peak amounts of virus, identified by ELISA, were pooled and used for further studies.Pools of pig parvovirus (PPV) and Aujeszkys disease virus (ADV) were grown in PCV-free primary pig kidney cell cultures. These cultures and virus pools were p...
