Clin Exp Vaccine Res.  2014 Jan;3(1):100-109. 10.7774/cevr.2014.3.1.100.

Development of porcine respiratory and reproductive syndrome virus replicon vector for foot-and-mouth disease vaccine

• Jeeva S
• Lee JA
• Park SY
• Song CS
• Choi IS
• Lee JB

• KMID: 2049100
• DOI: http://doi.org/10.7774/cevr.2014.3.1.100

Abstract

PURPOSE
Foot-and-mouth disease (FMD) is an economically important global animal disease. To control FMD virus (FMDV) outbreaks, a lot of different novel approaches have been attempted. In this study, we proposed a novel porcine reproductive and respiratory syndrome virus (PRRSV) as a replicon vector to express FMDV structural protein.
MATERIALS AND METHODS
PRRSV infectious clone (PRRSVK418DM) was used to develop an expression vector through the reverse genetic manipulation of PRRSV; FMDVP12A3C gene of serotype O was synthesized and used for an antigen. MARC-145 cells (African green monkey kidney epithelial cell line) were used for electroporation mediated transfection. The transfection or the expression of P12A3C and N protein of PRRSV was analyzed by either replicon containing PRRSV alone or by co-infection of helper PRRSV.
RESULTS
We constructed PRRSVK418DM replicon vector containing FMDVP12A3C, and genome sequences were confirmed by subsequent sequence analysis. In vitro expression of P12A3C and PRRSV N protein was confirmed by immunofluorescence antibody assay using antibodies specific for PRRSV N protein (anti-PRRSV N MAb), FMDV-VP1 (anti-VP1 MAb).
CONCLUSION
The results indicate that PRRSV replicon vector can be a promising novel vector system to control FMDV and useful for vaccine development in the future.

Keyword

Replicon; FMDV; PRRSV; Genetic vectors

MeSH Terms

Animals
Antibodies
Cercopithecus aethiops
Clone Cells
Coinfection
Disease Outbreaks
Electroporation
Epithelial Cells
Fluorescent Antibody Technique
Foot-and-Mouth Disease*
Genetic Vectors
Genome
Kidney
Porcine respiratory and reproductive syndrome virus*
Replicon*
Sequence Analysis
Transfection
Viruses
Antibodies

Figure

• 

  Fig. 1 Schematic representation of the PRRSVK418DM-P12A3C replicon construction strategy. Porcine reproductive and respiratory syndrome virus (PRRSV) replicon containing FMDVP12A3C was constructed by three strategies: PRRSVK418DM vector construction, P12A3C insertion between ORF1b/2 of PRRSVK418DM, and the deletion of ORFs 2 to 6 of PRRSVK418DM. All the modification was performed by overlap polymerase chain reaction and direct cloning procedure. The used overlapping primers and flanking primers are mentioned. The italic letters represent the restriction enzymes used for reverse genetic manipulation. PCR, polymerase chain reaction; TRS, transcriptional initiation sequences.

• 

  Fig. 2 Analysis of foot-and-mouth disease virus (FMDV) and porcine reproductive and respiratory syndrome virus (PRRSV) gene expression without helper virus by immunofluorescence antibody assay. MARC-145 cells were infected with the supernatant from the replicon. At 48 hour postinfection, the cells were processed for indirect immunofluorescence detection using antibodies specific for PRRSV N protein (anti-PRRSV N Ab) (A), FMDV-VP1 (anti-VP1MAb) (B).

• 

  Fig. 3 Identification of the P12A3C expression with helper virus by immunofluorescence antibody assay. MARC-145 cells were co-infected with the supernatant from the replicon and PRRSVK418DM. At 48 hour postinfection, the cells were processed for indirect immunofluorescence detection using antibodies specific for porcine reproductive and respiratory syndrome virus (PRRSV) N protein (anti-PRRSV N Ab) (A), foot-and-mouth disease virus (FMDV)-VP1 (anti-VP1 (MAb) (B).

• 

  Fig. 4 An alternative analysis of the P12A3C expression with helper virus by immunofluorescence antibody assay. The replicon transfected MARC-145 cells were directly infected by PRRSVK418DM at 96 hour post-transfection. At 48 hour postinfection, the cells were processed for indirect immunofluorescence detection using antibodies specific for porcine reproductive and respiratory syndrome virus (PRRSV) N protein (anti-PRRSV N Ab), foot-and-mouth disease virus (FMDV)-VP1 (anti-VP1 MAb).

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