Instability in vitro of a PCV2 infectious clone containing an insertion between ORF1 and ORF2.

Porcine circovirus 2 (PCV2) is a small circular single-stranded DNA virus that causes postweaning multisystemic wasting syndrome in pigs. We obtained an infectious clone, derived from PCV2 strain 1147 (size 1,767 bp), with a 60 bp insertion in the cap-rep intergenic region at the 3' end of the rep. In vitro propagation and sequencing of virus showed mixed sequence for the cap-rep intergenic region. Cloning of this region of virus from the passages 2 and 20 showed deletions of various size of the 60 bp insertion. Viruses from the passage 20 showed deletions of the 60 bp insertion and had extra (+1 to +5) or less (-1 or -2) bp in the intergenic region as compared to 1,147. These findings suggest that insertion of a 60-bp DNA sequence at the 3' end of the rep is unstable in vitro. This finding can have implications for the genetic engineering of PCV2.

Outcome of experimental porcine circovirus type 1 infections in mid-gestational porcine foetuses.

BACKGROUND: Porcine circovirus type 1 (PCV1) has been described as a non-cytopathic contaminant of the PK-15 cell line. Several experimental infections with PCV1 failed to reproduce disease in pigs. Therefore, PCV1 is generally accepted as non-pathogenic to pigs. To our knowledge, nothing is known about the outcome of PCV1 infections in porcine foetuses. This was examined in the present study. RESULTS: Nine foetuses from three sows were inoculated at 55 days of gestation: three with 10(4.3) TCID(50) of the PCV1 cell culture strain ATCC-CCL33, three with 10(4.3) TCID(50) of the PCV1 field strain 3384 and three with cell culture medium (mock-inoculated). At 21 days post-inoculation, all 6 PCV1-inoculated and all 3 mock-inoculated foetuses had a normal external appearance. Microscopic lesions characterized by severe haemorrhages were observed in the lungs of two foetuses inoculated with CCL33. High PCV1 titres (up to 10(4.7) TCID(50)/g tissue) were found in the lungs of the CCL33-inoculated foetuses. All other organs of the CCL33-inoculated foetuses and all the organs of the 3384-inoculated foetuses were negative (< 10(1.7) TCID(50)/g tissue) by virus titration. PCV1-positive cells (up to 121 cells/10 mm(2) in CCL33-inoculated foetuses and up to 13 cells/10 mm(2) in 3384-inoculated foetuses) were found in the heart, lungs, spleen, liver, thymus and tonsils. PCR and DNA sequencing of Rep recovered CCL33 or 3384 sequences from CCL33- or 3384-inoculated foetuses, respectively. CONCLUSIONS: From this study, it can be concluded that cell culture PCV1 can replicate efficiently and produce pathology in the lungs of porcine foetuses inoculated at 55 days of foetal life.

Unusual outcome of in utero infection and subsequent postnatal super-infection with different PCV2b strains.

VC2002, isolated from postweaning multisystemic wasting syndrome (PMWS)-affected pig, is a mixture of two porcine circovirus genotype 2b (PCV2b) viruses, K2 and K39. Preliminary experiments disclosed short-term adverse effects of K39, but not K2, on porcine foetuses. These findings led to the hypothesis that infection of immuno-incompetent foetuses with K2 confers a status of immunotolerance, and postnatal super-infection with K39 triggers PMWS. To explore this hypothesis, nine 55-day-old foetuses were inoculated in utero (three with K2-10(4.3)TCID50, three with K39-10(4.3)TCID50 and three with medium), and foeto-pathogenicity examined. At 21 days post-inoculation (dpi), K2 did not induce pathology, whereas pathological effects of K39 were evident. Twenty-four 45-day-old foetuses were subsequently inoculated to examine the long-term effect of K2, including six with K2-high dose-10(4.3)TCID50, six with K2-low dose-10(2.3)TCID50 and 12 mock-inoculated controls. Both doses resulted in five mummified foetuses and one live-born piglet each (69dpi). K2 was recovered from all mummies. K2 and K2-specific antibodies were not detected in serum of the two live-born piglets at birth, indicating full control of K2 infection. The K2-low dose-infected piglet was immunostimulated at day 2, but not the K2-high dose-infected piglet. Both non-stimulated and stimulated K2-infected piglets were super-inoculated with K39 at day 6 or 8 (taken as 0 days post super-inoculation). Low viral replication was observed in the non-stimulated K2-K39 piglet (up to 10(3.3)TCID50/g; identified as K39). In contrast, viral replication was extremely high in the stimulated K2-K39 piglet (up to 10(5.6)TCID50/g) and identified as K2, indicating that K2 infection is controlled during foetal life, but emerges after birth upon immunostimulation. However, none of the piglets showed any signs of PMWS.