T lymphocyte lines and clones selected against synthetic myelin basic protein 82-102 peptide from Japanese multiple sclerosis patients.

As has been indicated in experimental autoimmune encephalomyelitis (EAE), the application of synthetic peptides for the selection of T cell lines may provide new insights into the pathogenesis of multiple sclerosis (MS). We report here on T cell lines/clones generated from peripheral blood of MS patients against an immunodominant myelin basic protein (MBP) peptide 82-102. This study demonstrates that the selection of T cell lines against the MBP peptide is much more efficient than against whole MBP in generating a large panel of T cell lines/clones, and therefore provides a powerful strategy for studying autoimmune T cell repertoire in individual subjects. The peptide-selected lines and clones recognized MBP 82-102, shorter peptides MBP 89-101, 89-100 and guinea pig whole MBP mainly in the context of HLA-DR, but did not cross-recognize virus-derived peptides homologous to MBP 82-102. Seven out of ten clones were found to recognize MBP 82-102 in the absence of autologous antigen presenting cells (APC), and in three of the seven clones, specificity for MBP 82-102 could be demonstrated only in the absence of APC because of their strong reactivity against autologous APC. Two-color flow cytometry revealed that the clones were heterogeneous with regard to expression of CD4 and CD8 molecules. Overall, the clones selected by the peptide were rather heterogeneous in phenotype and function compared with those selected by whole MBP.

The complete nucleotide sequence of a pathogenic swine vesicular disease virus.

The nucleotide sequence of a swine vesicular disease virus (SVDV) strain that is pathogenic for pigs has been determined and compared with that of a non-pathogenic strain of SVDV, as well as a number of other enteroviruses. It shows only 98 base changes in comparison with a non-pathogenic strain of SVDV (Inoue et al., 1989, J. Gen. Virol. 70, 919-934). Fourteen of these nucleotide differences between the pathogenic and the non-pathogenic SVDV strains occur in the 5' non-coding region which, by analogy with the other picornaviruses, has been implicated in the efficiency with which the RNA is employed as mRNA. Additional differences found throughout the coding regions are largely conservative in nature. A number of residues are discussed as candidates for determinants of pathogenicity. This sequence has been submitted to the PIR database and has accession number A30061.

An enzyme-linked immunosorbent assay (ELISA) for the detection and quantification of antibodies against swine vesicular disease virus (SVDV).

A liquid phase blocking sandwich ELISA has been compared with virus neutralisation for testing pig sera for antibodies against swine vesicular disease (SVD) virus. Highest infectivity titre of SVD virus was obtained using a multiplicity of infection of 30 pfu/cell and harvesting after 21 h. Titres obtained for 300 clinically normal animals were assessed by ELISA and 89% were found to be 1/6 or less. Results were skewed and spread up to 1/45. Comparison of known positive sera resulted in a correlation between the two methods of 0.68 and showed that a virus neutralisation titre of 1/16 was equivalent to 1/40 (log10 1.61) by ELISA. Variation in results obtained by replicate testing using ELISA and virus neutralisation was almost identical. Overlap between positive and negative sera was shown to be reduced to 1-1/2 fold in ELISA. Therefore, the ELISA correlated well with virus neutralisation and has several advantages over the latter.

Pathogenesis of swine vesicular disease in pigs.

Pigs exposed to swine vesicular disease virus developed vesicular lesions by postinoculation day 2. Lesions first appeared on the coronary band and then on the dewclaw, tongue, snout, lips, and bulbs of the heels. The onset of viremia coincided with febrile response and the appearance of vesicles. Virus was isolated from the nasal discharge, esophageal-pharyngeal fluid, and feces as early as postinoculation day 1. Greater amounts of virus were isolated from samples collected during the first week of infection, and lesser amounts from samples collected during the second week. The appearance and the distribution of specific fluorescence in various tissues indicated that during the development of swine vesicular disease virus infection, the epithelial tissues were initially involved, followed by a generalized infection of lymph tissues, and subsequently, a primary viremia. Seroconversion was detectable as early as postinoculation day 4. A mild nonsuppurative meningoencephalomyelitis throughout the CNS was observed in both inoculated and contact-exposed pigs. The olfactory bulbs were most severely and were frequently affected, particularly in contact pigs. The most severe brain lesions were found in pigs 3 to 4 days after the onset of viremia; contact pigs showed more severe brain lesions than inoculated pigs. Microscopic changes were also found in the coronary band, snout, tongue, and heart.

Plaque morphology and pathogenicity for newborn mice of swine vesicular disease virus. I. Wild strains and their clones.

The population of wild swine vesicular disease virus (SVDV) strains was found non-homogeneous as manifested by varying plaque size and different pathogenicity of the clones obtained. The clones derived from large plaques (5-9 mm)--dominating among wild strains--were more virulent for newborn mice than those obtained from smaller plaques (1-2 mm). To evaluate the pathogenicity of wild SVDV strains the dose index was calculated; the clones were compared by dose index and theoretical pathogenicity index, respectively.

A comparative electrophoretic examination of swine vesicular disease virus and Coxsackie B5 virus.

Purified suspensions of Coxsackie B5 virus and swine vesicular disease virus (SVDV) were prepared by harvesting and purifying cell pack virus. Crossed immunoelectrophoresis was carried out with purified N and H antigen fractions (full and empty particles). Relative migration velocity (RMV) was calculated for the N antigen fraction of 3 SVD viruses (UKG72, HK71 and Italy 66) and 2 Coxsackie B5 viruses (Faulkner and 8068). The prototype strain of Coxsackie B5 virus (Faulkner) had a low RMV almost identical to that of the first isolated strain of SVDV, Italy 66. A recent isolate of Coxsackie B5 virus (8068, isolated in UK in 1973) had a relatively high RMV very close to that of the UKG72 strain of SVDV isolated in the UK in 1972. Also the Hong Kong strain of SVDV (HK71) had a high RMV value. These observations are considered in relation to the emergence of swine vesicular disease.

Swine vesicular disease: pathways of infection.

The pathways of infection in swine vesicular disease have been studied by (i) an estimation of the amounts of virus required to produce infection by different artificial inoculation procedures; (ii) the distribution and amounts of virus in various tissues of pigs killed at intervals after contact infection; (iii) an investigation of the susceptibility to virus infection of pig tissue explants. The results show that pigs can be infected by a number of pathways and that the skin, as the most susceptible tissue, is probably the most frequent route of infection.

Production of infectious swine vesicular disease virus from cloned cDNA in mammalian cells.

Full-length cDNA clones of the swine vesicular disease virus (SVDV) were constructed from subgenomic cDNA clones in the expression vector pSVL (pSVLS00). The direct transfection of mammalian cells with plasmid pSVLS00 results in the production of infectious virus. The recovered virus was neutralized completely by anti-SVDV guinea-pig serum, but did show a difference in plaque morphology from the parental virus.

[Biological characteristics of the plaque variants of the swine vesicular disease virus]. / Biologichna kharakteristika na plakovi varianti pri virusa na vezikuloznata bolest po svinete.

The Swine Vesicular Disease virus yields a heterogenic plaque population consisting of large round plaques of 8-10 mm, small uniform plaques with slightly indented contours, measuring 1-3 mm, and single plaques of transient form and size. The reisolates of the large (Lpf) plaques give a population that is similar to the initial virus, while the cloning of the small (Mpf) plaques leads to a homogenic population of such plaques. In vivo, the virus of the large plaques manifests enhanced virulence for swine. On the other hand, the selected small-plaque variant is apathogenic, which makes it possible to produce a live avirulent vaccine.

Crystal formation of swine vesicular disease virus in porcine kidney cells (IB-RS-2 cells).

Crystal formation of swine vesicular disease virus (SVDV) in IB-RS-2 cells was studied by electron microscopy. Cells were harvested 0, 3, 3.5, 4, 4.5, 5, 6 and 7 hours after inoculation. Crystalline arrays of SVDV was first observed in the cytoplasm of a few cells 4.5 hours after inoculation. In the cytoplasm of many cells harvested at 5 hours, 1 to 3 crystalline arrays of SVDV were observed. After that, a small number of cells had crystalline arrays in the cytoplasm. The cells with crystalline arrays were rich in ribosome and polysome with dilated mitochondria and many tiny vesicles. An individual virus particle was ca. 18 nm in diameter, and the center-to-center space ca. 22 nm. Crystalline arrays varied in size depending on the plane of section.

Comparison of swine vesicular disease virus and Coxsackie B5 virus by serological and RNA hybridization methods.

The relatedness of swine vesicular disease virus (SVDV) and Coxsackie B5 virus has been studied by virus neutralization and immunodiffusion tests and by hybridization of the virus RNAs. Clearly defined differences between the two viruses were found by the three methods. Isolates of SVDV from several countries were very closely related but could be differentiated. Recent isolates of Coxsackie B5 virus also appeared to be similar but clear differences could be detected between these and the prototype (Faulkner) strain of the virus. The SVDV isolates were more closely related to the Faulkner strain than to the recent isolates of Coxsackie B5 virus. Perhaps of more importance, the Faulkner strain was more closely related to SVDV than it was to the recent Coxsackie B5 isolates. The significance of these observations in relation to the recent emergence of swine vesicular disease is discussed.

Outbreaks of swine vesicular disease in japan: virus isolation and epizootiological survey.

Outbreaks of a vesicular disease occurred among pigs in Kanagawa and Ibaraki Prefectures in Japan in November, 1973. Another outbreak was observed in Aichi Prefecture in December. The clinical signs of the disease observed included fever and vesicular lesions on the coronary bands, bulbs of the heel and in the interdigital spaces. In some pigs, vesicular lesions were observed on the snout, tongue and skin overlying the legs and abdomen. All the vesicular samples produced cytopathic changes on cultures of primary swine kidney cells of PK-15 cells. Three isolates of cytopathic agents tested were identified as swine vesicular disease virus from their physicochemical properties and antigenicity. The virus strains isolated from vesicular epithelial samples obtained from Ibaraki, Kanagawa and Aichi Prefectures were designated as Japan/Ibaraki/1/73, Japan/Kanagawa/1/73 and Japan/Aichi/1/73 strain, respectively. An outbreak of the disease among pigs due to swine vesicular disease virus was confirmed by the serum neutralization test with serum samples collected from pigs on affected farms. Approximately 80% of the pigs housed in affected shed showed high levels of neutralizing antibody titers. This is the first to report an occurrence of swine vesicular disease among pigs in Japan.

Molecular approach to the epidemiology of swine vesicular disease: correlation of variation in the virus structural polypeptides with serological properties.

Variation has been observed in the structural polypeptides of swine vesicular disease viruses isolated from the United Kingdom and Hong Kong. Despite the limited number of isolates examined, several distinct polypeptide patterns were obtained when the virus structural proteins were examined by polyacrylamide gel electrophoresis. Isolates from outbreaks in the United Kingdom which were known to be connected gave the same polypeptide pattern, whereas viruses with different polypeptide patterns could not be traced to a common source. The different polypeptide patterns were obtained consistently and were not altered by passage of the virus in tissue culture. In general, isolates with identical polypeptide patterns could not be distinguished by neutralization or antibody blocking tests or by competition radioimmunoassays. However, isolates with different polypeptide patterns could be differentiated by antibody blocking tests or radioimmunoassay. The correlation between the serological tests and the polyacrylamide gel electrophoresis analyses illustrates the value of analyzing structural polypeptides in the epidemiological study of swine vesicular disease.

Characterization of three antigenic particles of swine vesicular disease virus.

Three distinct particles were isolated from cell culture harvests of swine vesicular disease virus (SVDV) by sucrose and CsCl gradient centrifugation. Virions (148S), RNA-free empty capsids (81S), and a third particle (49S) also free of RNA showed immune reactivity with SVDV antiserum. The 81S and 49S particles had polypeptides typical of naturally occurring empty capsids. Injection of purified antigens into guinea pigs produced antisera which distinguished empty capsids from virions on immunodiffusion; the 49S antigen appeared similar to virions. Antisera produced to freshly prepared virus antigen grown in brains of baby mice distinguished SVDV from the serologically related Coxsackie B-5 virus but did not distinguish the individual S particle antigens. Partly purified virus preparations degraded to empty capsids when incubated in guinea pig serum. The possible origin of empty capsids and 49S particles and their relationship to antigenicity of virus preparations are discussed.

Swine vesicular disease viruses isolated from healthy pigs in non-epizootic period. I. Isolation and identification.

A total of 524 fecal samples were collected from healthy swine of 36 hog farms scattered all over Japan from which had been detected neutralizing antibody against swine vesicular disease (SVD) virus. A virus was isolated from 21 of them. It was neutralized by antiserum against SVD virus. Of the 21 samples, 19 were derived from six farms, in areas which remained to be free from SVD in the past years and two from a farm where SVD broke out 18 months before. The cross-neutralization test was carried out with seven strains of SVD virus isolated from healthy pigs (SVDV-H) and five strains of SVD virus isolated from diseased pigs (SVDV-D). There was, however, no significant difference in antigenicity between the two groups. Some strains of SVDV-H were antigenically close to the Faulkner strain of Coxsackie B5 virus, and others to the freshly isolated strain of this virus. Neutralizing antibody of low titer against SVD virus was detected from pigs kept in areas free from SVD. It was presumed to have been produced in these pigs involved in silent infection with this virus.