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Swine vesicular disease (SVD) is the causative agent of highly contagious disease in pigs, characterized by vesicles on the coronary bands, heels of the feet, and occasionally on the lips, tongue, snout, and teats. SVD is caused by a virus belonging to the genus Enterovirus within the family Picornaviridae and is antigenically closely related to the human pathogen coxsackievirus B5[4]. The disease was first reported in Italy in 1966 and since then several outbreaks have been documented in a number of countries in Europe and Asia[2, 4]. The similarity of clinical symptoms between SVD and Foot and Mouth Disease (FMD) have lead to SVD being classified as a list A disease by the World Organization for Animal Health (OIE) [3].
SVDV particles are composed of 60 copies each of the four capsid proteins VP1–VP4, which enclose a single-stranded, positive-sense RNA genome of about 7400 nt. Proteins VP1, VP2 and VP3 are exposed at the viral surface, whereas VP4 is in close contact with the RNA and thus not accessible from the outer shell surface in the intact virions. It has been shown that both conformation-dependent neutralizing sites and linear epitopes are located mainly in the outer capsid proteins[1, 6, 8, 12, 14]. Furthermore, a recombinant bacterially expressed SVDV polyprotein, P1, is able to induce an SVDV-specific cellular and humoral immune response in pigs[7]. For this reason, the vp1 gene was chosen for this study.
Retrovirus is a class of enveloped viruses containing a single stranded RNA molecule as the genome. Following infection, the viral genome is reverse transcribed into double stranded DNA, which integrates into the host genome & is expressed as proteins. The viral genome is approximately 10kb, containing at least three genes: gag (coding for core proteins), pol (coding for reverse transcriptase) & env (coding for the viral envelope protein). At each end of the genome are long terminal repeats (LTRs) which include promoter/enhancer regions & sequences involved with integration. In addition there are sequences required for packaging the viral DNA (psi) & RNA splice sites in the env gene. Retroviral vectors are believed to be advantageous in single gene transfer, which may allow for the simultaneous achievement of more efficient gene delivery and longer-term transgene expression[11]. The ability of retrovirus vectors delivering a particular gene to target cells has been applied commonly in both experimental and clinical settings. Under these conditions, we have cloned and expressed vp1 gene of SVD HK/70[19] in eukaryotic cell, and analyzed the immunological activity in guinea pigs.
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In order to demonstrate appropriate expression of the SVDV VP1 proteins, transfected PK15 cells were detected by Western blot and PCR. To detect the activity of expressed VP1 protein in vitro, the medium of the infected cells was harvested and analyzed by Western blot. As seen in Fig. 1, the picture revealed a major band of 29 kDa in cells infected with "recombinant retrovirus", whereas no protein band was found in blank cells.
Figure 1. SDS-PAGE (A) and Western blotting (B) was used to detect the expression and identification of the recombinant VP1 protein. A: M, Protein markers; Lane 1 and 2, PK15-VP1 cell lysates; Lane 3. PK15 cell lysates. B: M, Protein markers; Lane 1 and 2, Western blot analysis with PK15-VP1 cell lysates; Lane 3, Western blot analysis with PK15 cell lysates.
T-lymphocytes proliferation response of guinea pigs was tested after immunization. Compared with the control group, the vaccinated group showed a significant lymphocyte proliferation response after the first vaccination, which increased after the booster vaccination (Fig. 2)
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The SVDV specific antibody levels in the sera were measured from 2 weeks after the 1st immunization and the results were shown in Table. 1. Antibody positive of group A were of significant level (5/5) compared with groups B–D. But antibody positive titers of group B (0/5) compared with groups C–D were not significant. This finding indicated that PK15-VP1 acted as an antigen and significantly enhanced serum antibody production in guinea pigs.
Table 1. Presence of SVDV specific antibodies in immunized guinea
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Serum samples were evaluated further in an SVDV serum neutralization test (SNT). Induction of neutralizing antibody was similar to that observed for the ELISA antibody. Briefly, neutralizing antibody was detected in group A, whilst all of the control animals remained negative (Table. 2).
Table 2. Neutralizing antibody titers of guinea pigs