Reproduction of postweaning multisystemic wasting syndrome (PMWS) in immunostimulated and non-immunostimulated 3-week-old piglets experimentally infected with porcine circovirus type 2 (PCV2).

Postweaning multisystemic wasting syndrome (PMWS) in swine is causally associated with the newly recognised pathogen, porcine circovirus type 2 (PCV2). In this study, 3-week-old SPF PCV2-seronegative piglets were inoculated intranasally with PCV2. The effect of immunostimulation on the induction of PMWS was investigated by immunisation with keyhole limpet hemocyanin (KLH) emulsified in incomplete Freunds adjuvant. The study was terminated 5 weeks after inoculation. While disease was not observed in the age-matched controls, two out of five non-immunised PCV2-infected piglets died on postinoculation day (PID) 21, and one was euthanized on PID 25 in moribund condition. These animals had appeared lethargic with persistent fever from PID 12 onwards. The euthanized pig appeared smaller than littermates and suffered from jaundice. At postmortem examination, gastric ulceration, icterus, and liver and thymus atrophy were observed. Furthermore, histological lesions of degenerating hepatocytes and hepatitis in combination with lymphoid depletion and syncytial cells in lymph nodes were consistent with the diagnosis of PMWS. One out of five immunostimulated PCV2-infected piglets was euthanized on PID 22 with convulsions after a period with wasting. This pig was lethargic from PID 14 onwards with persistent fever from PID 8 and transient dyspnoea. No differences in clinical signs, gross pathologic or histological findings were observed for the remaining non-immunostimulated and immunostimulated PCV2-infected piglets. All 10 PCV2-inoculated piglets seroconverted to PCV2 within 14 days after inoculation. By virus isolation, quantitative polymerase chain reaction (Q-PCR), and immunostaining of cryostat sections, it was demonstrated that lymphoid tissue contained abundant PCV2 antigen. Viral DNA load in serum samples was assessed by Q-PCR. All four PMWS-affected piglets had high levels of PCV2 DNA in serum, suggesting that there was a correlation between high levels of viral DNA in serum and the development of PMWS. In conclusion, infection with PCV2 caused PMWS in SPF piglets, however, the immunostimulation did not seem to play a critical role.

A longitudinal study of cell-mediated immunity in pigs infected with porcine parvovirus.

Porcine parvovirus (PPV) is an ubiquitous pathogen causing reproductive failure in swine. Protection against reproductive failure caused by acute PPV infection has commonly been related to the presence of specific antibodies in the dam. However, the role of cell-mediated immunity during chronic PPV infection remains to be elucidated, and may be relevant to the pathogenesis of novel diseases such as postweaning multisystemic wasting syndrome (PMWS), which may be triggered by coinfection with PPV and porcine circovirus type 2 (PCV2). To investigate whether pigs infected with PPV generate a cell-mediated immune response, a longitudinal infection experiment was performed, using swine leukocyte antigens (SLA) class I characterized growing pigs (haplotype H7/H7). Pigs were intranasally inoculated with PPV at 0, 80, and 136 days. At predetermined time points, peripheral blood mononuclear cells (PBMC) were isolated, and virus-specific lymphoproliferative responses and the cytolytic activities of cytotoxic T-lymphocytes (CTL) and natural killer (NK) cells were examined. Cytolytic assays were performed by the chromium release method, using as targets a syngeneic porcine kidney cell line established for the purpose (CTL assays) and K562 cells (NK assays). A specific proliferative response of PBMC from virus-infected pigs to PPV was observed from day 101 onwards. In contrast, PBMC from mock-infected pigs did not proliferate in response to PPV. Flow cytometric analysis indicated that the CD4+CD8+ T-cell subset of PBMC proliferated in response to virus antigen, in keeping with the assumed role for these cells in immunological memory. This is, to our knowledge, the first indication of a cellular immune response following PPV infection. A weak CTL activity, which peaked on days 80 and 87, was observed in PPV-infected pigs. In vitro restimulation of PBMC with live PPV did not induce further CTL activity. A pronounced NK cell activity was detected in both virus-infected and control pigs throughout the experiment, and may have negatively affected the sensitivity of the CTL assay. In conclusion, the findings of a late lymphoproliferative response together with weak CTL activity are in keeping with an effective control of acute PPV infection by humoral immunity, but open the possibility that cellular immunity may play a role in controlling PPV reinfection. Finally, we find that the established experimental model using SLA characterized pigs may constitute a valuable tool for future studies of CTL activity in pigs.

Development of a rapid in vitro protein refolding assay which discriminates between peptide-bound and peptide-free forms of recombinant porcine major histocompatibility class I complex (SLA-I).

The extracellular domains of swine leukocyte antigen class I (SLA-I, major histocompatibility complex protein class I) were cloned and sequenced for two haplotypes (H4 and H7) which do not share any alleles based on serological typing, and which are the most important in Danish farmed pigs. The extracellular domain of SLA-I was connected to porcine beta2 microglobulin by glycine-rich linkers. The engineered single-chain proteins, consisting of fused SLA-I and beta2 microglobulin, were overexpressed as inclusion bodies in Escherichia coli. Also, variants were made of the single-chain proteins, by linking them through glycine-rich linkers to peptides representing T-cell epitopes from classical swine fever virus (CSFV) and foot-and-mouth disease virus (FMDV). An in vitro refold assay was developed, using a monoclonal anti-SLA antibody (PT85A) to gauge refolding. The single best-defined, SLA-I restricted porcine CD8(+) T-cell epitope currently known is a 9-residue peptide from the polyprotein of CSFV (J. Gen. Virol. 76 (1995) 3039). Based on results with the CSFV epitope and two porcine haplotypes (H4 and H7), the in vitro refold assay appeared able to discriminate between peptide-free and peptide-occupied forms of SLA-I. It remains to be seen whether the rapid and technically very simple in vitro refold assay described here will prove generally applicable for the screening of virus-derived peptides for SLA-I binding.

[Epidermolysis bullosa simplex: genotype-phenotype correlation in Danish patients]. / Epidermolysis bullosa simplex: korrelation mellem genotype og faenotype hos danske patienter.

Epidermolysis bullosa simplex (EBS) is a group of autosomal dominant inherited skin disorders caused by mutations in the keratin genes K5 or K14. We examined five Danish families with EBS-Weber-Cockayne (WC) or EBS-Koebner (K) and two sporadic cases of EBS-Dowling-Meara (DM) in order to investigate the mutational spectrum and evaluate the genotype-phenotype correlation in Danish patients. Three new K14 mutations, one new and one previously described K5 mutation were identified by DNA sequence analysis. The positions of the EBS-DM mutations were consistent with previous studies, whereas the EBS-WC and EBS-K mutations were found in regions of the keratin genes not typically associated with this type of EBS mutations. In conclusion, we found a strict genotype-phenotype correlation. Furthermore, we found that the position of the mutation in the keratin gene is not the only determinant for severity of the disease; the nature of the amino acid substitution should also be considered when predicting the severity of the EBS disorder.

Identification of novel and known mutations in the genes for keratin 5 and 14 in Danish patients with epidermolysis bullosa simplex: correlation between genotype and phenotype.

Epidermolysis bullosa simplex (EBS) is a group of autosomal dominant inherited skin diseases caused by mutations in either the keratin 5 (K5) or the keratin 14 (K14) genes and characterized by development of intraepidermal skin blisters. The three major subtypes of EBS are Weber-Cockayne, Koebner, and Dowling-Meara, of which the Dowling-Meara form is the most severe. We have investigated five large Danish families with EBS and two sporadic patients with the Dowling-Meara form of EBS. In the sporadic Dowling-Meara EBS patients, a novel K14 mutation (N123S) and a previously published K5 mutation (N176S) were identified, respectively. A novel K14 mutation (K116N) was found in three seemingly unrelated families, whereas another family harbored a different novel K14 mutation (L143P). The last family harbored a novel K5 mutation (L325P). The identified mutations were not present in more than 100 normal chromosomes. Six polymorphisms were identified in the K14 gene and their frequencies were determined in normal controls. These polymorphisms were used to show that the K14 K116N mutation was located in chromosomes with the same haplotype in all three families, suggesting a common ancestor. We observed a strict genotype-phenotype correlation in the investigated patients as the same mutation always resulted in a similar phenotype in all individuals with the mutation, but our results also show that it is not possible to predict the EBS phenotype merely by the location (i.e., head, rod, or linker domains) of a mutation. The nature of the amino acid substitution must also be taken into account.

A new mechanism in blue cone monochromatism.

Blue cone monochromatism (BCM) is a rare X-linked colour vision disorder characterized by the absence of both red and green cone sensitivity. Most mutations leading to BCM fall into two classes of alterations in the red and green pigment gene array at Xq28. In one class the red and green pigment genes are inactivated by deletion in the locus control region. In the second class genetic rearrangements have created an isolated pigment gene that carries an inactivating point mutation. Here we describe a clinical case of BCM caused by a new mutation where exon 4 of an isolated red pigment gene has been deleted. The finding represents the first intragenic deletion yet described among red and green pigment genes.

[Molecular genetics of red-green color blindness]. / Den molekylaere genetik ved rød-grøn farveblindhed.

Normal colour vision is trichromatic and is mediated by the blue, green and red visual pigments present in the corresponding blue, green, and red cone cells of the retina. The red and green pigment genes have evolved from an ancestral pigment gene and reside in a head-to-tail tandem array on the long arm of the X chromosome. This arrangement and a high degree of homology predispose to illegitimate recombination between the red and green pigment genes explaining the various forms and the high frequency of red-green colour vision defects.

[Molecular genetic examination in sex-linked color blindness]. / Molekylaergenetiske undersøgelser ved kønsbunden farveblindhed.

The molecular structure of the X-linked colour-vision locus was studied in a family where mild red-green colour-vision deficiency (deuteranomaly) segregated, and in a male with complete absence of red and green colour-vision (blue cone monochromasy). In individuals with normal colour-vision the red and green pigment genes had normal molecular structure whereas individuals with deuteranomaly, in addition to normal red and green genes, also had an abnormal hybrid gene consisting of parts of the green and red pigment genes. The individual with blue cone monocromasy had only a red-green hybrid gene inactivated by a critical mutation in codon 203. Thus, the phenotypes predicted from the individual genotypes were in complete accord with the observed phenotypes.