Strain Of Swine Vesicular Disease Virus Research Articles

Development of a real-time PCR assay based on primer-probe energy transfer for the detection of swine vesicular disease virus

A real-time PCR assay based on primer-probe energy transfer (PriProET) was developed to detect swine vesicular disease virus (SVDV). Specificity tests of SVDV and heterologous virus showed specific amplification of SVDV strains only. The amplification plot for the closely related Coxsackievirus B5 remained negative. The sensitivity of assay was five copies of viral genome equivalents. A key point of the assay is tolerance toward mutations in the probe region. Melting curve analysis directly after PCR, with determination of probe melting point, confirmed specific hybridisation of the SVDV strains. Eight of twenty SVDV strains tested, revealed shifted melting points that indicated mutations in the probe region. All predicted mutations were confirmed by nucleotide sequencing. With the PriProET system there is a chance to identify phylogenetically divergent strains of SVDV, which may appear negative in other probe-based real-time PCR assays. At the same time, any difference in melting points may provide an indication of divergence in the probe region. The high sensitivity, specificity, and tolerance toward mutations in the probe region of the SVDV PriProET assay may improve the early and rapid detection of a wide range of SVDV strains, allowing reduced turnaround time and the use of high-throughput, automated technology.

Noninfectious Virus-Like Particle Antigen for Detection of Swine Vesicular Disease Virus Antibodies in Pigs by Enzyme-Linked Immunosorbent Assay

An inactivated SVDV antigen is used in current enzyme-linked immunosorbent assays (ELISAs) for the detection of antibodies to swine vesicular disease virus (SVDV). To develop a noninfectious recombinant alternative, we produced SVDV-like particles (VLPs) morphologically and antigenically resembling authentic SVDV particles by using a dual baculovirus recombinant, which expresses simultaneously the P1 and 3CD protein genes of SVDV under different promoters. Antigenic differences between recombinant VLPs and SVDV particles were not statistically significant in results obtained with a 5B7-ELISA kit, indicating that the VLPs could be used in the place of SVDV antigen in ELISA kits. We developed a blocking ELISA using the VLPs and SVDV-specific neutralizing monoclonal antibody 3H10 (VLP-ELISA) for detection of SVDV serum antibodies in pigs. The VLP-ELISA showed a high specificity of 99.9% when tested with pig sera that are negative for SVDV neutralization (n=1,041). When tested using sera (n=186) collected periodically from pigs (n=19) with experimental infection with each of three different strains of SVDV, the VLP-ELISA detected SVDV serum antibodies as early as 3 days postinfection and continued to detect the antibodies from all infected pigs until termination of the experiments (up to 121 days postinfection). This test performance was similar to that of the gold standard virus neutralization test and indicates that the VLP-ELISA is a highly specific and sensitive method for the detection of SVDV serum antibodies in pigs. This is the first report of the production and diagnostic application of recombinant VLPs of SVDV. Further potential uses of the VLPs are discussed.

Importance of Arginine 20 of the Swine Vesicular Disease Virus 2A Protease for Activity and Virulence

A major virulence determinant of swine vesicular disease virus (SVDV), an Enterovirus that causes an acute vesicular disease, has been mapped to residue 20 of the 2A protease. The SVDV 2A protease cleaves the 1D-2A junction in the viral polyprotein, induces cleavage of translation initiation factor eIF4GI, and stimulates the activity of enterovirus internal ribosome entry sites (IRESs). The 2A protease from an attenuated strain of SVDV (Ile at residue 20) is significantly defective at inducing cleavage of eIF4GI and the activation of IRES-dependent translation compared to the 2A protease from a pathogenic strain (J1/73, Arg at residue 20), but the two proteases have similar 1D-2A cleavage activities (Y. Sakoda, N. Ross-Smith, T. Inoue, and G. J. Belsham, J. Virol. 75:10643-10650, 2001). Residue 20 has now been modified to every possible amino acid, and the activities of each mutant 2A protease has been analyzed. Selected mutants were reconstructed into full-length SVDV cDNA, and viruses were rescued. The rate of virus growth in cultured swine kidney cells reflected the efficiency of 2A protease activity. In experimentally infected pigs, all four of the mutant viruses tested displayed much-reduced virulence compared to the J1/73 virus but a significant, albeit reduced, level of viral replication and excretion was detected. Direct sequencing of cDNA derived from samples taken early and late in infection indicated that a gradual selection-reversion to a more efficient protease occurred. The data indicated that extensive sequence change and selection may introduce a severe bottleneck in virus replication, leading to a decreased viral load and reduced or no clinical disease.

Purification, crystallization and X-ray analysis of swine vesicular disease virus.

Swine vesicular disease virus (SVDV) is the etiological agent of swine vesicular disease, a highly contagious disease in pigs, and is related to coxsackie B virus. Crystalline arrays of SVDV can be observed in the cytoplasm of cells 4.5 h after inoculation to porcine kidney cells (IBRS-2 cells). Crystals of the JX/78 strain of SVDV were obtained from virus in two wells of crystallization conditions and present preliminary X-ray data to 3.6 A resolution.

Characterization of neutralization sites on the circulating variant of swine vesicular disease virus (SVDV): a new site is shared by SVDV and the related coxsackie B5 virus

Using a panel of new monoclonal antibodies (mAbs), five neutralizing, conformation-dependent sites have been identified on the antigenic variant of swine vesicular disease virus (SVDV) circulating currently. In studies on the antigenic conservation of these sites, the four antigenic/genetic groups of SVDV described showed distinguishable patterns, confirming this classification. By sequencing mAb-resistant mutants, the five sites have been mapped precisely and localized on a three-dimensional model of the SVDV capsid. All were found to be orientated, to a different extent, towards the external surface of the capsid. Three of the five sites, located in VP1, VP2 and VP3, correspond to epitopes identified previously in historic isolates as sites 1, 2a and 3b, respectively. Another site, site IV, which maps to position 258 of VP1, corresponds to an epitope reported recently and is described in this study to be specific for isolates of the most recent antigenic group of SVDV. A fifth site is described for the first time and corresponds to the unique neutralizing site that is common to both SVDV and coxsackie B5 virus; it maps to positions 95 and 98 of VP1, but may also include positions nearby that belong to site 1 on the BC-loop of VP1, suggesting the classification of site Ia. These results may have useful diagnostic and epidemiological applications, since mAbs to the new conserved site Ia provide universal reagents for SVDV detection systems, while the specificity of mAbs to site IV make them unique markers for the most recent strains of SVDV.

An attenuating mutation in the 2A protease of swine vesicular disease virus, a picornavirus, regulates cap- and internal ribosome entry site-dependent protein synthesis.

Virulent and avirulent strains of swine vesicular disease virus (SVDV), a picornavirus, have been characterized previously. The major determinants for attenuation have been mapped to specific residues in the 1D-2A-coding region. The properties of the 2A proteases from the virulent and avirulent strains of SVDV have now been examined. Both proteases efficiently cleaved the 1D/2A junction in vitro and in vivo. However, the 2A protease of the avirulent strain of SVDV was much less effective than the virulent-virus 2A protease at inducing cleavage of translation initiation factor eIF4GI within transfected cells. Hence the virulent-virus 2A protease is much more effective at inhibiting cap-dependent protein synthesis. Furthermore, the virulent-virus 2A protease strongly stimulated the internal ribosome entry sites (IRESs) from coxsackievirus B4 and from SVDV, while the avirulent-virus 2A protease was significantly less active in these assays. Thus, the different properties of the 2A proteases from the virulent and avirulent strains of SVDV in regulating protein synthesis initiation reflect the distinct pathogenic properties of the viruses from which they are derived. A single amino acid substitution, adjacent to His21 of the catalytic triad, is sufficient to confer the characteristics of the virulent-strain 2A protease on the avirulent-strain protease. It is concluded that the efficiency of picornavirus protein synthesis, controlled directly by the IRES or indirectly by the 2A protease, can determine virus virulence.

Singleton reactors in the diagnosis of swine vesicular disease: the role of coxsackievirus B5

Swine vesicular disease virus (SVDV) and Coxsackie B5 virus (CVB5) are closely related viruses that can infect swine and man and give rise to cross-reacting serum antibodies. It is, therefore, possible that SVD antibodies found in serologic screenings of pigs are induced by CVB5. Single positive animals found in screening programmes are generally referred to as singleton reactors (SR). To determine whether SR in SVDV screenings are induced by CVB5 infection, virus neutralisation tests (VNTs) and radioimmunoprecipitation assays (RIPA) were carried out on sera of SR, sera of pigs experimentally infected with SVDV, and sera from pigs vaccinated with CVB5 isolates. The SR sera reacted repeatedly positive in the SVDV UKG/27/72 VNT, but reacted differently in three other VNTs (SVDV NET/1/92, CVB5A, and CVB5B). The VNT titres obtained with the SR sera revealed a correlation between both SVDV strains, and also between both CVB5 stains, but no correlation was found between SVD and CVB5 VNT titres. Sera of experimentally infected (SVDV) or vaccinated (CVB5) pigs showed titres in all four neutralisation tests. In the RIPA, the reaction patterns of the SR sera varied considerably with all four antigens used, in contrast to sera from pigs experimentally infected with SVDV that reacted with all antigens used, and sera from pigs vaccinated with CVB5 that reacted only with CVB5 antigens. The results presented in this paper show that neither CVB5 nor SVDV infections are the only cause of the SR phenomenon. Testing for CVB5 specific antibodies can reduce the number of SR sera in the serodiagnosis of SVDV.

Identification of neutralizing epitopes on a European strain of swine vesicular disease virus.

Six neutralizing monoclonal antibodies (MAbs) were used to isolate MAb neutralization-resistant (MAR) mutants from a recent European strain of swine vesicular disease virus (SVDV), ITL/9/93. Sequencing of MAR mutants identified two epitopes located at positions analogous to sites 2A (VP2) and 3B (VP3) on poliovirus (PV) which have been previously identified on a Japanese strain of SVDV. A third epitope near to the C terminus of VP1, not previously recognized on SVDV, was tentatively identified in a region analogous to site 1 of PV. A fourth epitope, located in the C-terminal region of VP3, has never before been recognized as a site of neutralization on picornaviruses. All four epitopes were predicted to be surface-exposed.

Highly sensitive detection of swine vesicular disease virus based on a single tube RT-PCR system and DIG-ELISA detection

A highly sensitive detection of swine vesicular disease virus (SVDV) based on a single tube RT-PCR system and digoxigenin (DIG)-PCR-ELISA detection was developed. Using a one tube RT-PCR system, optimisation of the PCR conditions and optimisation of the microwell hybridisation and colourimetric detection of the amplicons resulted in a method that could detect viral RNA in infected tissue culture fluid with a titre as low as 0.1 TCID 50/100 μl. The same sensitivity was obtained with SVDV-spiked faeces, if the samples were pre-treated with 1,1,2-trichlorotrifluoroethane/chloroform and subsequently concentrated using an ultrafiltration system and RNA extracted with the Purescript kit. The specificity of the test was validated on 27 SVDV strains belonging to four different groups. No cross-reactivity with genetically and symptomatically related viruses was detected using RNA of foot-and-mouth disease virus (FMDV), porcine enterovirus (PEV), vesicular stomatitis virus (VSV), Coxsackie B5 virus (CV-B5) and encephalomyocarditis virus (EMCV). The test was validated successfully on clinical samples, being slightly more sensitive and much faster than virus isolation on cell cultures. Moreover the possibility of automating the procedure will allow the processing of large numbers of clinical samples.

Molecular cloning, expression and immunological analysis of the capsid precursor polypeptide (P1) from swine vesicular disease virus

Swine vesicular disease virus (SVDV) is the aetiological agent of a highly contagious viral disease of pigs, whose symptoms are indistinguishable from those caused by foot-and-mouth disease virus (FMDV). The gene coding for the capsid protein precursor of SVDV (P1) from a recent spanish isolate (SPA/1/'93) was cloned and expressed in bacteria, and the antigenicity and immunogenicity of the recombinant product were evaluated. The recombinant P1 was recognised by antibodies against SVDV induced in pigs infected experimentally with different SVDV strains. Immunisation of swine with recombinant P1-induced SVDV-specific cellular and humoral immune responses. The implications of these results in SVD diagnostic as well as in vaccine development are discussed.

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.

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.