Abstract
BackgroundPorcine parvovirus (PPV) and pseudorabies virus (PRV) are the important etiological agents of swine infectious diseases, resulting in huge economic losses to the Chinese swine industry. Interleukin-6 (IL-6) has the roles to support host immune response to infections as a pleiotropic cytokine. It is essential to construct a live attenuated vaccine-based recombinant PRV that expresses PPV VP2 protein and porcine IL-6 for prevention and control of PRV and PPV.MethodsThe recombinant plasmid, pGVP2-IL6, was constructed by porcine IL-6 gene substituting for EGFP gene of the PRV transfer plasmid pGVP2-EGFP containing VP2 gene of PPV. Plasmid pGVP2-IL6 was transfected into swine testicle cells pre-infected with the virus rPRV-VP2-EGFP strain through homologous recombination and plaque purification to generate a recombinant virus rPRV-VP2-IL6. The recombinant PRV was further identified by PCR and DNA sequencing, and the expression of the VP2 protein and porcine IL-6 was analyzed by reverse transcription-PCR (RT-PCR) and Western blot. The virus titer was calculated according to Reed and Muench method. The immunogenicity of the recombinant virus was preliminarily evaluated in mice by intramuscular administration twice with the rPRV-VP2-IL6 at 4-week intervals.ResultsA recombinant virus rPRV-VP2-IL6 was successfully constructed and confirmed in this study. The properties of rPRV-VP2-IL6 were similar to the parental virus HB98 in terms of growth curve, morphogenesis and virus plaque sizes, and rPRV-VP2-IL6 was proliferated in different cell types. It induced specific antibodies against PPV as well as a strong increase of PPV-specific lymphocyte proliferation responses in mice immunized with rPRV-VP2-IL6, and provided partial protection against the virulent PPV challenge. rPRV-VP2-IL6 also induced a high level of neutralizing antibodies against PRV, and significantly reduced the mortality rate of (1 of 10) following virulent PRV challenge compared with the control (10 of 10).ConclusionsThe recombinant rPRV-VP2-IL6 might be a potential candidate vaccine against PRV and PPV infections in pigs.
Highlights
Porcine parvovirus (PPV) was found by Cartwright in 1967 in swine affected with reproductive failure, and a worldwide distribution was observed thereafter [1,2,3,4]
Construction of a recombinant pseudorabies virus A transfer plasmid pGVP2-IL6 (Fig. 1c) was constructed by inserting the porcine IL-6 gene (639 bp) under the control of CMV gene promoter and substituting for enhanced GFP (EGFP) gene of pGVP2-EGFP, and transfected into swine testicle (ST) cells pre-infected with the recombinant PRV (rPRV)-VP2-EGFP strain
The porcine IL-6 gene in the rPRVVP2-IL6 was detected by polymerase chain reaction (PCR), and was identical to the predicted DNA fragment size (639 bp), but was not present in the parental virus
Summary
Porcine parvovirus (PPV) was found by Cartwright in 1967 in swine affected with reproductive failure, and a worldwide distribution was observed thereafter [1,2,3,4]. Capsid VP2 protein, one of the major structural proteins of PPV, induced PPV-neutralizing antibodies to neutralize PPV infection and played a key role in PPV diagnosis and immune prophylaxis [8,9,10]. PPV inactivated oil emulsion whole virus vaccines have played an important role in PPV control. Genetic engineered subunit vaccines [11, 14, 15] that could induce specific immune responses and have shown efficacy against challenge virus are under development. Porcine parvovirus (PPV) and pseudorabies virus (PRV) are the important etiological agents of swine infectious diseases, resulting in huge economic losses to the Chinese swine industry. It is essential to construct a live attenuated vaccine-based recombinant PRV that expresses PPV VP2 protein and porcine IL-6 for prevention and control of PRV and PPV
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