Porcine circovirus type 2 (PCV2) plays a crucial role in the pathogenesis of post-weaning multisystemic wasting syndrome (PMWS) in swine. As PCV2 displays significant homology with PCV1 (a non-pathogenic virus) at the nucleotide and amino-acid level, a discriminative antigen is needed for specific serological diagnosis. The ORF2-encoded capsid protein from PCV2 was used to develop an indirect enzyme-linked immunosorbent assay (ELISA). GST-fused capsid protein from PCV2 and GST alone (both expressed in recombinant baculovirus-infected cells) were used as antigens for serodiagnosis. The specificity of the ELISA for detection of PCV2 antibodies was demonstrated in sera from pigs experimentally infected with PCV1, PCV2 and other swine viruses. The semi-quantitative nature of the test was evaluated versus an immunoperoxidase monolayer assay (IPMA). The ELISA was performed on 322 sera from pigs in eight Brittany herds and compared with IPMA. The sensitivity (98.2%) and specificity (94.5%) of this test were considered suitable for individual serological detection. High PCV2 seroprevalence was found in sows and pigs at the end of the growth phase (18-19 weeks) in all eight herds. The seroprevalence in piglets (11-17 weeks) was statistically correlated with clinical symptoms of PMWS (93% in affected versus 54%, in non-affected farms). A cohort study performed in PMWS-free farms showed that 57% of piglets exhibited active seroconversion after 13 weeks, indicating that PCV2 infection occurred earlier in PMWS-affected piglets.

Porcine circovirus type 2 (PCV2) is known to be associated with post-weaning multisystemic wasting syndrome (PMWS), a recently described disease of young pigs. Since no PCV2 vaccine was available so far, we have developed a specific PCV2 vaccine candidate. The Orf1-encoded replication protein and Orf2-encoded capsid protein of PCV2 were expressed and detected in either mammalian or insect expression systems. In a first trial, Orf2 protein was found to be a major immunogen, inducing protection in a prime-boost protocol; the piglets received a first injection with plasmids directing Orf2 protein and granulocyte-macrophage colony-stimulating factor (GM-CSF) expression, followed by a second injection, a fortnight later, associated with baculovirus-expressed Orf2 protein. As evaluated by growth parameters, clinical signs (fever), seroconversion, the pigs were protected against a PCV2 challenge after vaccination. In a second trial, protection induced by a subunit vaccine was even better than the one induced by DNA vaccine, since PCV2 replication was completely inhibited.

Post-weaning multisystemic wasting syndrome (PMWS) is a recently identified disease of pigs linked to the emergence of a new porcine circovirus (PCV2). We report here the characterization of immunorelevant linear B-cell epitopes of the Open Reading Frame 2-encoded protein (Orf2) from PCV2 by an enzyme-linked immunosorbent assay (ELISA) using experimental antisera collected from pigs inoculated with a PCV2 isolate. Two epitopes spanning residues 69 to 83 and 117 to 131 were specific to PCV2. Antibodies to the 117 to 131 epitope (B- 133) were detected in 22% and 100% of specific pathogen-free (SPF) pig sera 6 and 11 weeks post inoculation, respectively. Cross-sectional studies performed with field sera collected from PMWS-affected herds showed B-133 antibodies in 5% of 8 to 10 week-old pigs, 38% of 13-14 week-old pigs, 62% of 16 to 19 week-old pigs, 56% of 20 to 25 week-old pigs and 45% of 26 to 31 week-old pigs. All these data suggest that epitope B- 133 is a serological marker of PCV2 infection that could be used for the detection of PCV2 antibody response.

Post-weaning multisystemic wasting syndrome (PMWS) is a comparatively new disease of swine, and known to occur in France since 1996. A porcine circovirus type 2 (PCV2) is found in the lesions of affected piglets. Six piglets aged 10–13 weeks were obtained from a French PMWS-affected farm. Two showing characteristic signs of PMWS (palor, weakness and emaciation) remained in poor condition and were finally killed 6 and 9 days after their arrival in the experimental unit. Tissue homogenates from these two piglets were used to reproduce mild PMWS in specific pathogen-free (SPF) piglets. This mild PMWS consisted of pyrexia (up to 41·7°C) and growth retardation (up to 30% of weight reduction compared with controls) commencing 1 week after infection and lasting 3 weeks. In seven additional trials, pyrexia, growth retardation and lesions characteristic of PMWS were consistently produced in SPF and conventional piglets. However, only four of 55 inoculated SPF piglets (7·2%) showed severe wasting disease. One died and the others had to be killed 3 to 4 weeks after inoculation. None of the inoculated animals developed antibodies to any common swine viruses or bacteria, but clear evidence of PCV2 seroconversion was obtained. Our results therefore strongly suggest that PCV2 is the primary aetiological agent of PMWS.

Two types of porcine circovirus (PCV) have been isolated and are referred to as PCV1 and PCV2. PCV1 represents an apathogenic virus, whereas PCV2 is associated with post-weaning multisystemic wasting syndrome. The two PCVs are related, since they display about 70% identity based on nucleotide sequences. In order to discriminate between common and type-specific antigens, an immunocytological approach was used following transfections with cloned circovirus DNAs, as well as recombinant proteins expressed by either baculovirus or plasmid vectors. The ORF1-encoded proteins in the two viruses were shown to be antigenically related, whereas the ORF2 proteins were recognized differentially by polyclonal anti-PCV2 antibodies. Furthermore, PEPSCAN analysis performed on overlapping fragments of the genes encoding part of ORF1 and the entire ORF2 and ORF3 led to the identification of five dominant immunoreactive areas, one located on ORF1 and four on ORF2. However, only some ORF2 peptides proved to be immunorelevant epitopes for virus type discrimination. The potential use of ORF2-derived antigens as diagnostic tools is demonstrated.

Using the HeLa cell model along with an in vitro splicing system, we have previously shown that hnRNP M and 2H9 are involved in the pre-mRNA splicing process and most interestingly also in heat shock-induced transient splicing arrest by transiently leaving the hnRNP complexes. Due to this unique regulatory function in a mechanism that turns splicing on and off, these two hnRNPs appear as important proteins for controlling gene expression. Here we investigated by in situ hybridization and immunohistochemical staining techniques the expression level of specific mRNA and protein during mouse embryonic development. HnRNP M and 2H9 are found to be expressed at all examined stages (6.5-18.5 days post-coïtum), in a differential manner, and at various levels depending on tissues, cell types and also embryonic stages; fairly high levels of both hnRNPs are always observed in the central nervous system. Furthermore, levels of colocalizing protein and transcript are not always present in the same proportion, thus suggesting a post-transcriptional regulation of hnRNP M and 2H9 gene expression. The complex spatiotemporal variations we observed might well anticipate a role for these two hnRNPs also in modulating splicing, thereby influencing gene expression and further many physiological processes.

A disease affecting weaned pigs and known as Postweaning Multisystemic Wasting Syndrome (PMWS) is described on French farms. Follow-up studies were designed on cohorts of pigs on a group of 12 severely affected farrow-to-finish operations. Three of them were free of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) infection. Three thousand and seventy eight pigs were included in the study. Mortality, from weaning to slaughter, was 11% and weeks 11-13 were the most critical. The first clinical sign reported was unthriftiness, then pallor and often fever with associated respiratory or digestive disorders. Wasting could follow rapidly and when clearly established in an individual the prognosis was grave especially when the sick pigs were kept with their penmates. Antibiotics were administered without real efficacy. The lesions affected several viscera including lungs, spleen, kidneys and lymph nodes with a severe lymphoid depletion. The disease did not show a collective impact. From preliminary epidemiological investigations, a strong litter effect on disease susceptibility was suspected. PRRS virus was excluded as a major agent. On the other hand, a porcine circovirus (PCV2) was found associated to the lesions. The environment was suspected as an important determining factor for the effect of PMWS in the herds.

Using antibody 2H9 from our heterogeneous nuclear ribonucleoproteins (anti-hnRNP) monoclonal antibody library, we previously showed in HeLa cells that a 35-37-kDa protein doublet switches from the hnRNP complexes to the nuclear matrix following a 10-min heat shock at 45 degrees C (1 Lutz, Y., Jacob, M., and Fuchs, J. P. (1988) Exp. Cell Res. 175, 109-124). cDNA cloning and sequencing revealed an hnRNP protein (2H9) which is a new member of the hnRNP F, H/H' family. Protein 2H9 displays two consensus sequence-type RNA binding do mains (CS-RBD) showing 80-90% homology with two of the three CS-RBDs of hnRNP F and H/H'. Another common feature is the presence of two glycine/tyrosine-rich auxiliary domains located at the C terminus and between the two CS-RBDs. At the functional level we show that specific anti-2H9 peptide antibodies can directly inhibit an in vitro splicing system. Moreover, the 2H9 protein doublet is no more present in nuclear extracts from such briefly stressed cells, which interestingly correlates with the inability of these extracts to catalyze in vitro splicing reactions. Taken together, our data suggest that these proteins are involved in the splicing process and also participate in early heat shock-induced splicing arrest by transiently leaving the hnRNP complexes. These 2H9 proteins, which are encoded by a single gene located on human chromosome 10, were also found to be associated with nuclear bodies in situ.

With anti-hnRNP monoclonal antibody 6D12 we previously showed in HeLa cells that as early as 10 min after the onset of a heat shock at 45 degrees C, a 72.5-74 kDa antigen doublet leaves the hnRNPs and strongly associates with the nuclear matrix, the effect being reversed after a 6 h recovery at 37 degrees C. cDNA cloning and sequencing enabled us to identify these antigens as hnRNP-M proteins and further to show that the correct sequence differs by an 11 amino acid stretch from the originally published sequence. We also show that monoclonal antibodies raised against synthetic hnRNP-M peptides can directly inhibit in vitro splicing. Furthermore, stressing cells at 45 degrees C for 10 min is sufficient to abolish the splicing capacity of subsequently prepared nuclear extracts which, interestingly, do not contain the hnRNP-M proteins any more. Taken together, our data suggest that these proteins are involved in splicing as well as in early stress-induced splicing arrest. Further in situ hybridization assays located the hnRNP-M encoding gene on human chromosome 19.

Using our anti-hnRNP monoclonal antibody library Y. Lutz, M. Jacob, and J.-P. Fuchs (1988) Exp. Cell Res., 175, 108-124; P. Mähl, Y. Lutz, E. Puvion, and J.-P. Fuchs, (1989) J. Cell Biol. 109, 1921-1935, we investigated by immunocytofluorescence the fate of a series of speckled-distributed nuclear antigens, after HeLa cells were infected with adenovirus type 2. Although the speckled pattern, which corresponds to the nucleoplasmic fibrillogranular network, including the interchromatin-granule clusters, was still observed during most of the infectious cycle, several antibodies also revealed additional, increasingly fluorescent virus-induced structures. In noninfected cells, two of these antibodies, termed 3F2 and 2A5, recognize two antigens of 33 and 31 kDa, respectively. Western blot analysis showed that this increasing amount of fluorescence observed in infected cells did not reflect an accumulation of the 33- and 31-kDa antigens, but is actually due to the fact that both antibodies also recognize the multifunctional adenovirus 72-kDa single-stranded DNA-binding protein (DBP). Immunoelectron microscopy analyses, including sequential double-labeling, indeed showed that this additional signal precisely colocalizes with the viral 72-kDa DBP, which essentially accumulates over the entire surface of the virus-induced single-stranded DNA accumulation sites. Taken together, our data show that two host-specific hnRNP-associated antigens share common epitopes with the viral 72-kDa DBP.