During the early course of PCV2 infection the genomic DNA can be found in the circulation before accumulating in cells of the monocyte/macrophage lineage [16, 17]. The site of active PCV2 replication that leads to PMWS and the triggering factors are however not well documented. PCV2 is a single-stranded circular DNA virus, but during replication of PCV2 it exists as a double stranded replicative form. The cellular localization of PCV2 has been studied by electron microscopy of lymph nodes and during the course of infection of a porcine lymphoblastoid cell line, combining ultrastructural analyses with immunogold labelling for the capsid protein of PCV2 [15, 18, 19]. To enable studies of PCV2 localisation and replication in detail however new methods are necessary. Therefore we designed an assay based on padlock probes and RCA that are able to detect and discriminate between the genomic and the replicative strand of PCV2 in situ. The assay was successfully applied to detect both strands of PCV2 in cells as well as in fresh frozen tissue sections.
PK-15A cells were infected with PCV2 and we followed the infection for 72 hours to study the replication process. The signals for both the genomic and the replicative form increased over time. The signals from the genomic strand were concentrated in certain nuclei but eventually spread to the cytoplasm in all cells. The replicative form was only observed in and around these highly infected cells, indicating that this is the place of replication. The replicative strand was found both in the nucleus and in the cytoplasm of the replicating cell. Though the total number of signals from the genomic strand increased throughout the infection, the fraction of infected cells decreased between 48 and 72 hours. This could be because at 72 hours the cells have started to undergo apoptosis resulting in loss of cells.
Earlier observation [15, 18, 19] pointed to that PCV2 proteins were associated with mitochondria, and that the mitochondria were suggested to be important for viral replication. Therefore we used the newly developed method to study if we could detect a co-localisation of the genomic and/or the replicative strand with mitochondria stained by Mitotracker. The infected cells had weaker mitochondrial staining from the Mitotracker dye than the uninfected cells. Because Mitotracker stains active mitochondria that have normal membrane potential these differences indicated that infected cells were under stress. However, no correlation between the localisation of mitochondria and viral DNA for any of the PCV2 strands was detected and could not support the findings by Rodriquez-Carino et al. (2009, 2010a, b). However, our studies were not very comprehensive and needs to be repeated to be clear on this issue. One obvious difference is that we detect viral DNA while they detect viral proteins. It is possible that viral proteins localise in the mitochondria and perform some function there. Furthermore, our labelling was conducted on cells after 48 h of infection whereas the co-localisation of PCV2 cap protein with mitochondria was most obvious during the earlier phase of infection . Another potential explanation for these discrepant results would be if the viral DNA is more strongly associated with viral proteins in the mitochondrion than elsewhere in the cell and thus less available for probing.
We also applied the technique on lymph node tissue from an experimentally infected pig and from a pig suffering from PMWS. In the experimentally infected tissue we found signals from both strands but the signals from the padlock probe targeting the genomic strand were much more abundant compared to that of the probe targeting the replicative strand, showing that the genomic strand is present in much higher levels than the replicative strand also during early phases of infection. In the naturally infected tissue we only observed signals from the genomic strand, possibly indicating that PCV2 had ceased to replicate.
Different methods have been used by others to detect PCV2 in situ, such as immunohistochemistry, and in situ hybridisation [20–22]. Padlock probes can distinguish between different isolates that only differ at a single base, which is not possible with FISH or in situ PCR. Padlock probes also have an advantage of being able to discriminate between the two complementary strands which is otherwise difficult with FISH or when studying viruses with PCR. In situ detection allows for intra cellular localisation of the two complementary strands and can thus be used to study the path of infection. A combination of FISH using probes detecting PCV2 and fluorescence immunohistochemistry (FIHC) with fluorescent antibodies to single stranded or double stranded DNA has been elaborated and applied on tissue sections from gnotobiotic pigs , but with that technique other DNA strands than those from PCV2 will also be detected if present. With padlock probes it would also be possible to demonstrate the presence of different viruses in multiplex by using various combinations of fluorophores for detection. In this study we choose to study PCV2 in fresh frozen tissue sections and in cultured cells, but this method could be used for in situ studies of single-stranded DNA viruses in general. Especially interesting would be to study the known co-infection of PCV2, torque teno virus and porcine bocavirus in situ .