A novel recombinant pseudorabies virus expressing parvovirus VP2 gene: Immunogenicity and protective efficacy in swine

Abstract

Background: Porcine parvovirus (PPV) VP2 gene has been successfully expressed in many expression systems resulting in self-assembly of virus-like particles (VLPs) with similar morphology to the native capsid. Here, a pseudorabies virus (PRV) system was adopted to express the PPV VP2 gene.

Methods: A recombinant PRV SA215/VP2 was obtained by homologous recombination between the vector PRV viral DNA and a transfer plasmid. Then recombinant virus was purified with plaque purification, and its identity confirmed by PCR amplification, Western blot and indirect immunofluorescence (IFA) analyses. Electronic microscopy of PRV SA215/VP2 confirmed self-assembly of both pseudorabies virus and VLPs from VP2 protein.

Results: Immunization of piglets with recombinant virus elicited PRV-specific and PPV-specific humoral immune responses and provided complete protection against a lethal dose of PRV challenges. Gilts immunized with recombinant viruses induced PPV-specific antibodies, and significantly reduced the mortality rate of (1 of 28) following virulent PPV challenge compared with the control (7 of 31). Furthermore, PPV virus DNA was not detected in the fetuses of recombinant virus immunized gilts.

Conclusions: In this study, a recombinant PRV SA215/VP2 virus expressing PPV VP2 protein was constructed using PRV SA215 vector. The safety, immunogenicity, and protective efficacy of the recombinant virus were demonstrated in piglets and primiparous gilts. This recombinant PRV SA215/VP2 represents a suitable candidate for the development of a bivalent vaccine against both PRV and PPV infection.

Figure 1

Construction of recombinant PRV SA215/VP2. The PRV SA215 genomic DNA was approximately 150 kb including a unique long region (UL), a unique short region (US), internal repeat (IR) and terminal repeat (TR). The thymidine kinase gene (TK) was partially deleted. PRV SA215/VP2 was generated from PRV SA215/VP2. The LacZ expression cassette was replaced with the EGFP/VP2 expression cassette by homologous recombination.

Figure 2

Expression of the fused EGFP-VP2 protein in Vero cell foci detected by fluorescence microscopy (magnification 400 ×). (A) Fluorescence microscope image of PRV SA215/VP2 infected Vero cells 36 h post-infection; (B) Light microscope image of PRV SA215/VP2 infected Vero cells 36 h post-infection; (C) Merged images of (A) and (B).

Figure 3

Identification of recombinant PRV SA215/VP2 by PCR amplication of VP2 gene. PCR products were analyzed by 0.8% gel agarose electrophoresis. Lanes: (M) Marker DL2000; (1-3) Samples of Vero cells infected with PRV SA215/VP2; (4) Positive control, PPV; (5) Vero cells infected with vector virus PRV SA215.

Figure 4

Analysis of VP2 gene expression. (A) Western blot analysis of VP2 protein expressed by infected Vero cells. Lanes: (M) Pre-stained protein markers, (1) Vector PRV SA215; (2) Recombinant PRV SA215/VP2. The 93-kDa band represents EGFP and VP2 fusion protein. (B and C) Indirect immunofluorescence analysis of VP2 protein expressed in Vero cells infected with recombinant PRV SA215/VP2 (B) and vector PRV SA215 (C). Cells infected with recombinant PRV SA215/VP2 developed immunofluorescence (B), and cells infected with vector virus PRV SA215, did not develop immunofluorescence (C).

Figure 5

Electron microscope analysis of Vero cells infected PRV SA215/VP2. Two types of virus particles were observed in the same Vero cell. Recombinant PRV particles are indicated by the black arrow. Empty virus-like particles probably formed from PPV VP2 protein are indicated by the white arrow.

Figure 6

Immunogenicity of PRV SA215/VP2 in piglets. Four groups of piglets (n = 5 per group) were immunized with recombinant PRV SA215/VP2, vector PRV SA215, inactivated PPV vaccine and PBS. Data represent the mean antibody titer of each group of piglets determined by indirect ELISA at 0, 14, 28, 42 and 56 d. The horizontal line represents cut-off value.

Figure 7

Immunogenicity of PRV SA215/VP2 in gilts. Six gilts were divided into two groups. Group 1 was immunized with recombinant PRV SA215/VP2, and group 2 served as negative control. Data represent the mean antibody titer of each group of gilts determined by indirect ELISA at 0, 28, 77 and 114 d. The horizontal line represents cut-off value.