Pathogenesis, Epidemiology, Immunopathology, the Diagnosis of Pmws, an Emerging Disease Caused By a New Porcine Circovirus. Develop. of Recombinant

Vaccines to prevent Post weaning multi systemic wasting syndrome (PMWS) were developed. The efficacy of the protection conferred by the two major PCV2-proteins, Rep and Cap, alone or in combination can be evaluated on the basis of growth retardation and fever parameters presented by piglets in the challenge-control group (CC) in trial No. 1. We used a prime-boost protocol, including a first DNA vaccine followed by a DNA-booster and a subunit vaccine, two weeks later. In this trial, vaccination significantly reduced the duration of the pyrexic phase, more efficiently in the Orf2 and Orf1&Orf2-vaccine groups than in the Orf1-vaccine group. Moreover, during the third week post-infection, only the Orf2 and Orf1&Orf2 groups showed significantly reduced growth retardation compared to the CC group, which was more pronounced in the Orf2 group. No difference was observed between the Orf2 group and the C group (non-challenged control group), while the Orf1&Orf2 group showed a significant difference with the C group (p<0.01) during the third week. These results were confirmed by a higher /GW3 index in the Orf2 group than in the Orf1&Orf2 group (1.09 vs 0.6) during the third week post-challenge. No clinical symptoms were observed in these two groups, while pigs in the Orf1 group presented typical PMWS symptoms (ataxia, rough hair-coat, wasting). We therefore showed that the Orf2-encoded capsid protein, used in a preparation-based DNA and subunit vaccine, constitutes the major immunogen to induce protection of piglets against a PCV2 challenge. In contrast, the Orf1-encoded replication protein appears to be only weakly immunogenic. In our trial No. 2, we focused on the vaccine composition to compare the efficacy of the protection conferred by either a DNA vaccine or a subunit vaccine. Independently of the previous results, we decided to use the two associated major PCV2-proteins (Rep and Cap), in two injections. As in the first trial, on the basis of growth retardation and fever parameters presented by piglets in the challenge-control group, we observed that the subunit vaccine significantly reduced the duration of the pyrexic phase (p<0.05) during the third week post-infection, while the DNA vaccine did not have any effect on the duration of the pyrexic phase. All piglets of the DNA group presented hyperthermia higher than 40.5 XC for one to five days versus three piglets of the subunit group for one to three days. In contrast, the two vaccinated groups (DNA and SU) showed a better growth, compared to the CC group. These results were confirmed by a high /GW3 index, 1.20 and 1.54, respectively. The /GW3 index obtained in trial No. 2 was higher than that obtained in trial No. 1. However, we cannot consider so significant the difference in /GW3 obtained in the various groups of animals (Orf2, Orf1&Orf2, DNA, Subunit groups). In the case of conventional commercial vaccines against Aujeszkys disease virus (ADV), the official requirement is a minimum /G7 index of 1.5. Although these two diseases are not comparable, the /GW3 index obtained in the present study indicates a useful protection performance for the production of vaccines against a PCV2 challenge. We obtained earlier seroconversion with the subunit vaccine two weeks after the first injection, at the time of the second injection, while seroconversion was only observed after challenge with the DNA vaccine. Different antibody titers were elicited in the groups and were higher for DNA and CC groups after challenge, while the antibody titer of the subunit group remained lower, suggesting the absence of PCV2 replication and boosting effect after challenge.

- An epidemiological survey of porcine circovirus (PCV) infection: The results of the Spanish study resemble the french situation. One point of interest, still preliminary, is that the breeding origin of the paternal genetic line was significantly associated to have lesser problems with PMWS. Specifically, the use of Pietrain breed as the source of paternal genetic line seems to be a ¿protective¿ factor regarding the occurrence of PMWS. The so-called genetic effect has been suggested in many discussion forums of swine practitioners and scientists, but this is the first epidemiological study that supports a role for the genetic background on the development of PMWS. Further research on the genetic background of maternal and paternal lines should be encouraged in order to establish its exact influence on the development of the disease. - Effect of PCV2 infection of the sow on PCV2 viremia and mortality due to PMWS in piglets: In natural conditions, PCV2 viremia can be persistent in an individual pig (22 weeks in some pigs) and in a pig population, although a significant effect on the clinical outcome was not demonstrated. Also, infection in very young piglets (1 week old) was detected, but none of them developed clinical signs before 8 weeks of age. In a three site integrated production system suffering of PMWS, PCV2-infected sows yielded a higher proportion of 1-week old PCV2-infected piglets, and a higher proportion of their piglets died in the nursery and fattening periods, when compared to piglets from non-infected sows. - PCV2 in other species: Evidence that PCV2 is widepread in wild boar, and that this species can also suffer PMWS were given. On the contrary, PCV2 infection in other domestic and laboratory species was not found. - Retrospective studies on PCV2 and PMWS: Data compiled in our retrospective study indicates that antibodies to PCV2 have been present in the Spanish domestic swine population at least since 1985, twelve years before the first description of PMWS in the country; this data suggest that the introduction of the virus in the Spanish livestock occurred previously. On the other hand, PCV2 infection occurs in wild boar in Spain since at least 1994, but is apparently less widespread than in domestic pig.

The risk of porcine circovirus (PCV) for humans has been studied. Xenotransplantation is seen as an alternative to allotransplantation, hopefully compensating the lack of human donor material. XT implies the risk of transmission of porcine viruses, which could recombine with a related human pathogen, thereby creating a new virus with unpredictable attributes. Assessment of virus safety of XT has been achieved using two approaches: - Infection studies of porcine cells with PCV1 and PCV2: Several human cell lines have been infected with PCV1 and PCV2 to investigate whether PCV can infect and replicate in human cells. PCV1 persisted in most cell lines without causing any visible changes, while PCV2-transfected cells show cytopathogenic alterations. Expression of viral proteins and replication of viral DNA was observed, but the infection was not to be passed on to fresh cells, indicating that PCV-infection of human cells is non-productive. - Search for a human circovirus: A consensus-primer PCR approach was applied to search for a human circovirus. The approach uses degenerate primers amplifying highly conserved regions in the rep gene of all known circoviruses. 1101 samples including 248 of immuno-suppressed patients have been investigated, but no indication for the existence of a novel human circovirus was observed. PCV serology in highly exposed workers: Five researchers on PCV2 were found to be serologically negative to PCV2 by IPMA. Therefore, PCV2 seems not to represent a zoonotic rosk for humans.

Apoptosis induced by PCV2: When human cells were infected with PCV1 and PCV2, a cytopathogenic effect was seen in PCV2-infected cells but not in PCV1-infected cells. The CPE was attributed to the induction of apoptosis. Expression of the PCV2gene products revealed that both products of the rep gene, Rep and Rep induce apoptosis, while Cap does not. Expression of deletion mutants of rep showed that the N-terminal part is responsible for this effect. When induction of apoptosis was investigated in cultivated porcine cells, an apoptotic reaction was not observed. Apoptosis studies in field PMWS cases: An increase of apoptosis was not demonstrated in lymphoid organs of PMWS-affected pigs. This finding was unexpected, as it is known that many viruses are able of induce apoptosis. Some viruses induce apoptosis of lymphocytes as a direct effect of infection. However apoptosis can also be mediated by cytotoxic T-lymphocytes recognizing virus-infected cells. The results from our study in PMWS-affected pigs suggest that a massive apoptosis, is most probably not the cause of the lymphocyte depletion observed in the studied cases. Generally, there was an inverted correlation between the lymphocyte depletion and apoptosis at all stages of lymphoid depletion. Although, there cannot be ruled out that selective apoptosis in a small subpopulation of lymphocytes could be the cause of an imbalance in immunity and in turn induce suppression of other lymphocytes subpopulations. Other possibility is that massive apoptosis could have happened before the death of the pig, probably at the beginning of the acute phase of the PCV2 infection. However, apoptosis of hepatocytes is correlated with PCV2 infection of hepatocytes, and with the stage of liver damage.

GST fusion allowed the recombinant proteins expressed in insect cells to be immobilized and purified in a one-step procedure using glutathione-coated microtiter plates. GST alone was expressed in a similar manner and used as a reference for ELISA evaluation of non-specific binding. Nuclear-enriched extracts from cells infected with recombinant baculoviruses expressing GST-fused PCV2-ORF2 or GST alone were assayed for use as antigenic sources in this test. Serum samples collected from the three groups of piglets (PCV1, PCV2 and controls) during six weeks p.i. were used to calibrate and evaluate the ELISA test. Results are expressed as the optical density (OD) ratio of values obtained for GST/ORF2 and for GST alone. Based on the average ELISA value of the OD ratio (plus 3 standard deviations) for sera of the eight control piglets collected weekly, the serum test was considered positive when the OD ratio exceeded 1.5. Seroconversion was evidenced in all eight PCV2-challenged piglets from 2 weeks p.i. until the end of the experiment. No antibody reaction occurred following PCV1 infection, whereas seroconversion was visualized by IPMA on PK15-CCL33 cells (data not shown). Sensitivity was initially evaluated by comparison of ELISA results for sera from PCV2-infected animals with IPMA results obtained for PCV2-infected cells and cells transfected with PCV2-ORF2, as described by Mahe et al. (2000). As observed in ELISA, seroconversion was detected from 2 weeks p.i. in both IPMA assays, indicating that the kinetics of the antibody response to PCV2 was similar to that of PCV2-ORF2 protein. Antibody titers for PCV2-infected pigs were also determined by an IPMA on PCV2-infected cells (Truong et al., 2001) to search for a possible correlation between the IPMA titer and the OD ratio. Endpoint IPMA titers of 35 sera collected at 2 to 6 weeks p.i. from PCV2-infected piglets were plotted against the OD ratio of the corresponding serum. The results showed linear regression between the log10 titer of IPMA and the OD ratio (Spearman correlation coefficient = 0.84; P < 0.0001) within a minimum and a maximum limited range respectively of 2 and 4.7 (IPMA), and 1.59 and 5.5 (OD ratio). The ELISA test was assessed for specificity against other viruses. Serological cross-reactions between antigens from other porcine viruses and PCV2-ORF2 protein were evaluated using a panel of antisera produced in SPF pigs against swine viruses. None of these antisera was positive in this test. Field-origin pig sera were used for the validation of the ORF2-based ELISA. Serum samples (n=322) obtained from a transverse serological study were obtained from four Brittany herds (two with and two without clinical symptoms of PMWS). The sera were tested for the presence of PCV2 antibodies by ELISA and the results were compared with those obtained by IPMA on PCV2-infected cells. Sensitivity of 98.13% and specificity of 94.55% were obtained.

Post weaning multi systemic wasting syndrome (PMWS) should be diagnoses if the following three criteria are accomplished: - Clinical signs such as growth retardation and wasting, frequently with dyspnea and enlargement of inguinal lymph nodes, and occasionally with jaundice. - Presence of characteristic histopathological lesions in lymphoid tissues (lymphocyte depletion together with granulomatous inflammation and, in a percentage of cases, multinucleate giant cell infiltration and intracytoplasmic grape-like PCV2 inclusion bodies in macrophages). - Detection of PCV2 (nucleic acid or antigen) within the lesions in lymphoid tissues of affected pigs. Lymphoid tissues are the main target of PCV2 in pigs with PMWS. Histiocytic infiltrates, formation of syncytia, lymphoid depletion, and presence of cytoplasmic inclusions in histiocytic cells are the most prominent microscopical lesions. The hepatocyte is a target cell for PCV2 infection and replication, and therefore, PCV2 should be considered a new hepatitis-inducing viral agent in pigs. PCV2 is able to replicate in utero in non-immune sows after infection by the intrauterine route and piglets could be infected at birth even if sows are PCV2 seropositive at that stage. It could be that PCV2 is implicated in reproductive failure under natural conditions; however at last in Spain this fact is probably of very sporadic occurrence, even being a country where PMWS and PCV2 infection are widespread. We did not found support for the hypothesis that PCV1 or PCV2 are causes of porcine Congenital Tremor (CT).

- Presence of PCV2 and PRRsV in field cases: To explore the role of PRRSV in field cases of PMWS, a study was done with submissions, with RT-PCR for PRRSV. PRRSV infection in PMWS affected pigs was found to be about 48% of the cases. Since PRRSV infection is widespread in Spain, being most of the conventional farms seropositive against PRRSV this result was expected and probably reflects the high seroprevalence of this virus in the Spanish livestock. Regarding PCV1, two studies have shown that PCV1 prevalence in the swine cases received or in farms with PMWS was low or insignificant, rspectively. These findings support that PCV1 is seldom found in field cases, and has no relevance for PMWS. - Experimental infection with PCV2 and PRRSV in conventional pigs: In order to test if double inoculation of conventional pigs with PRRSV and PCV2 could help in the development of PMWS, 24 4-week-old pigs, seronegative to PRRSV and PCV2, were selected. Four groups of pigs were performed: non-inoculated group (5 pigs), PRRSV group (5 pigs), PCV2 group (7 pigs) and PRRSV+PCV2 group (7 pigs). Pigs were first inoculated, by intranasal route, with PRRSV (10e6,5TCID50/pig), and 1 week later with PCV2 (10e5,1 TCID50/pig). A reduced weight gain was observed in the PRRSV group and in the PRRSV+PCV2 group. Only pigs of the PRRSV+PCV2 group had fever, and looked pale and thrifty. Fever started at day 14 PI PCV2 (21 PI PRRSV), and lasted 1 week. One pig of the PRRSV+PCV2 group had a severe respiratory distress and developed a high fever, at day 17 PI PCV2; this pig died at day 19 PI PCV2. The rest of the pigs were necropsied at 25 days PI PCV2 (day 32 PI PRRSV). PMWS was reproduced, since at least one of the PRRSV+PCV2-inoculated pigs developed disease and died. The previous infection of pigs with PRRSV potentiated the replication of PCV2, since lesions induced by PCV2 were more extensive and intense, PCV2 nucleic acid was more widespread, and viremia of PCV2 was quantitatively stronger. Moreover, probably the infection with PCV2 potentiated the replication of PRRSV, since there was a higher proportion of PRRSV+PCV2-inoculated pigs viremic to PCV2.

Two studies were carried out, one in France and the other in Germany, to identify possible virulence factors in the sequence of PCV2. In the French study, a total of 23 distinct PCV2 sequences were identified, 13 originating from PMWS(+) herds and 10 from PMWS(-). Average similarity along the sequences of the PMWS(+) and PMWS(-) groups were determined by the PLOTSIMILARITY program of the GCG Wisconsin Package version 10.3 (Accelrys Inc.,San Diego, CA). All sequences were 1767 nucleotides (nt), except Fh17 and Fh16 (1768 and 1778 nt respectively). Fh16 had a 11 nt insertion 42 bases downstream the origin of replication This insertion introduced successively a fourth hexamer (CGGCAG) and a third pentamer (CACCT) respectively identical with the repetitions and the spacers in the case of PCV1. The two variants Fh15 and Fh16 were identical at each position excepted that the 11 nt insertion was only found in Fh16. In one farm, only the variant Fh16 was isolated from the pig m4 while both Fh15 and Fh16 were isolated from the pig m3. Due to the mix of variants Fh15 and Fh16 in this case, the cloning of the PCR product encompassing the origin of replication was necessary to obtain readable sequences. Of nine clones, seven carried the insertion while the two others lacked it. The intra-individual variability was evaluated by sequencing of PCR products obtained from several samples of two animals. Whatever the tissue among the six tested, a unique variant was identified for each of the two pigs m11 and m12. Each of the 23 identified variants was found in only one herd. A maximum of two variants was found in each herd. All the whole PCV2 sequences, compared by pair, shared a nucleotide identity degree of 94.6 to 100%. This variation range was mainly due to the variability of the ORF2 (91.2 to 100% nucleotide identity), the ORF1 being highly conserved (97.8 to 100%). The amino-acid identity range of ORF1 (99 to 100%) was shown to be tighter than the nucleotide one, confirming the conservation of the protein. In contrast, the aminoacid identity range of ORF2 (90.1 to 100%) was wider than the nucleotide one, reflecting a lower level of structural constraints for this protein. An alignment of ORF2 amino-acid sequences of 82 variants collected from this study, GenBank and the study of Larochelle showed that residue changes scattered all along the protein, although three domains, A (57-91), B (121-136) and C (180-191) were more prone to variation (Fig.3.47). Each domain A and B was situated within an immunoreactive region previously identified by PEPSCAN (Mahe et al., 2000), while domain C was at the border of such a region. Only domains A and C were within regions with a high immunogenic index.

To characterize the immunitary alterations in PMWS, two complementary approaches have been followed: - Cell populations in lymphoid organs have been studied by immunohistochemistry, in PMWS and in normal pigs: Immunohistochemical studies in formalin-fixed organs have confirmed histopathological observations. First, the normal pattern of reaction on approximately 2-month-old pigs had to be determined for comparison. Application of the same techniques to PMWS-affected pigs revealed a reduction or loss of B and T lymphocytes, increased number of macrophage-like cells, and partial loss or redistribution of antigen-presenting cells throughout lymphoid tissues. - Changes in cell subsets, cytoquine production, and reactivity to stimulatory effects have been investigated, always comparing with age-matched, normal pigs: In natural PMWS cases, pigs suffered a downshift in the relative counts of lymphocyte subsets in PBMC. These changes affected CD4, CD8, CD4-CD8-DP, and IgM+ cells, and they seem to be related to the levels of PCV2 in lymphoid tissues and to the extent of depletion in both B- and T-cell-dependent areas of these tissues. We have also provided the first description of an altered mRNA cytokine expression profile in lymphoid tissues of naturally PMWS affected pigs. Furthermore, there is a direct correlation between the degree of thymic depletion and IL-10 expression. Capabilities of PBMC of PMWS -affected pigs to respond to recall viral antigens, mitogens or superantigens are clearly reduced, or even inexistent, compared to a normal profile. Taking all together, the histological findings, the altered PBMC subset, the cytokine imbalances, and the functional disturbances of blood lymphoid cells, it is highly suggestive of an immunosuppressive state in pigs suffering from PMWS.

Investigation of the molecular biology of porcine circovirus (PCV) revealed many new insights in its replication and transcription strategy: - Promoters for rep and cap genes have been mapped in PCV1. - Differential splicing has been observed for the rep gene of PCV. - Two distinct rep gene products (Rep and Rep) have been identified in PCV. - Rep and Rep bind to the origin of replication. - Two hexamer repeats H1, H2 are the binding domain of Rep and Rep. - Rep represses transcription from the promoter of the rep gene (P rep), Rep does not. - H1 and H2 are necessary for binding of Rep and Rep and repression of P rep. - Rep, Rep and Cap are located in the nucleus; Cap is seen in the nucleoli, Rep and Rep colocalize in the remaining nucleus. - Rep and Rep interact with each other and themselves. - The replication factors of PCV1 and PCV2 can be exchanged. - Rep and Rep are ATPases. - Rep and Rep can introduce and seal ssDNA breaks into the origin of replication. - Transfected genomic DNA of PCV is infectious, infection can be provoked with a cloned overlapping genome.

High titers of PCV2 isolated in cell cultures were needed to develop diagnostic tests and to set up a challenge model for PMWS (using purified PCV2 as a single inoculum). Several in vitro experiments were performed in order to define the best cell line to culture the virus, the different parameters involved in the protocol of virus production, and finally a system to purify large amounts of virus cultures. - Selection of a cell line susceptible to PCV2 - Glucosamine treatment A 30 min, 200mM glucosamine treatment, was established as standard for PCV2 production in SK cells. - Sonication of PCV2 cultures Titration of supernatant and cellular pellet of PCV2 cultures in SK cells allowed us to notice that a high proportion of the produced virus remained attached to the cell debries. Sonication of SK+PCV2 cultures, frozen and thawed, at 100 watt for 4 min was standardized for future PCV2 productions. - Infection/transfection of SK cells with PCV2 and plasmids expressing the rep and cap proteins of PCV1 and PCV2 Transfection of SK cells with plasmids encoding for the rep and cap proteins of both PCV was used as a strategy to increase the titer of PCV2 when these cultures were infected. It was determined that the transfection of 10¿Yg of plasmid/2x106 cells simultaneously to the PCV2 infection (MOI 0,01) was able to increase a mean of 1,5 log the titer of the viral progeny. - Medium-scale purification and concentration of PCV2 by ultracentrifugation Ultrafiltration system was chosen, since it allowed to process high amounts of virus culture, with a lower cost, and it was the less time-consuming system. The final procedure for the production, concentration and purification of PCV2 includes the following steps: -- Production of x liters of PCV2 culture (glucosamine treatment); -- Freezing and thawing; -- Sonication of the culture (cells + supernatant); -- Clarification of the culture; -- Filtration (0,22 ¿Ym) of the supernatant; -- Ultrafiltration (cut off 300 Kda); -- Washing of the membrane (recovery of virus attached to the membrane); -- Recovery of the ultrafilter and sanitization; Experimental Inoculations: - Experimental inoculation of PCV2 to piglets, and effect of a commercial adjuvant: Although PMWS was not reproduced in neither PCV2 and PCV+adjuvant inouclated pigs, viral detection and seroconversion indicated that all PCV2 inoculated pigs developed a long lasting subclinical infection wiht viral shedding in naso-pharyngeal, faecal and urinary routes. No statistically significant effect of the vaccine adjuvant was recorded on PCV2 replication under this experimental conditions. - Experimental inoculation of PCV2 in rabbits and mice: From all mice inoculated with PCV1 or PCV2, only one PCV1 inoculated mouse showed a very low serological titre to PCV1 at 20 days PI. This positive titre to PCV1 could be due to the immunisation of the mouse, instead of being a titre generated by infection. The use of different mice strains, PCV2 isolates, experimental designs and doses of viral inoculation may explain the differences observed in other published experiments. In conclusion, mice and rabbits were refractary to experimental infection.

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