Identification of four insertion sites for foreign genes in a pseudorabies virus vector

Abstract

Background: Pseudorabies virus (PRV) is a preferred vector for recombinant vaccine construction. Previously, we generated a TK&gE-deleted PRV (PRV^ΔTK&gE-AH02) based on a virulent PRV AH02LA strain. It was shown to be safe for 1-day-old piglets with maternal PRV antibodies and 4 ~ 5 week-old PRV antibody negative piglets and provide rapid and 100 % protection in weaned pigs against lethal challenge with the PRV variant strain. It suggests that PRV^TK&gE-AH02 may be a promising live vaccine vector for construction of recombinant vaccine in pigs. However, insertion site, as a main factor, may affect foreign gene expression.

Results: In this study, we constructed four recombinant PRV-S bacterial artificial chromosomes (BACs) carrying the same spike (S) expression cassette of a variant porcine epidemic diarrhea virus strain in different noncoding regions (UL11-10, UL35-36, UL46-27 or US2-1) from AH02LA BAC with TK, gE and gI deletion. The successful expression of S gene (UL11-10, UL35-36 and UL46-27) in recombinant viruses was confirmed by virus rescue, PCR, real-time PCR and indirect immunofluorescence. We observed higher S gene mRNA expression level in swine testicular cells infected with PRV-S(UL11-10)ΔTK/gE and PRV-S(UL35-36)ΔTK/gE compared to that of PRV-S(UL46-27)ΔTK/gE at 6 h post infection (P < 0.05). Moreover, at 12 h post infection, cells infected with PRV-S(UL11-10)ΔTK/gE exhibited higher S gene mRNA expression than those infected with PRV-S(UL35-36)ΔTK/gE (P = 0.097) and PRV-S(UL46-27)ΔTK/gE (P < 0.05). Recovered vectored mutant PRV-S (UL11-10, UL35-36 and UL46-27) exhibited similar growth kinetics to the parental virus (PRV^ΔTK&gE-AH02).

Conclusions: This study focuses on identification of suitable sites for insertion of foreign genes in PRV genome, which laids a foundation for future development of recombinant PRV vaccines.

Keywords: Bacterial artificial chromosome; Insertion site; Noncoding region; Pseudorabies virus; Spike gene.

Fig. 1

Construction of PRV-S(UL11-10)ΔTK/gE, PRV-S(UL35-36)ΔTK/gE and PRV-S(UL46-27)ΔTK/gE. a The S expression cassette with a kanamycin resistance gene was inserted into the noncoding area (UL11-10, UL35-36, UL46-27 or US2-1) through the first recombination to generate four recombinant BAC clones (BAC^{PRV−S−KAN(UL11−10)ΔTK/gE/gI}, BAC^{PRV−S−KAN(UL35–36)ΔTK/gE/gI}, BAC^{PRV−S−KAN(UL46−27)ΔTK/gE/gI} and BAC^{PRV−S−KAN(US2−1)ΔTK/gE/gI}). b The second recombination was performed to delete the kanamycin resistance gene and generate the final recombinants (BAC^{PRV−S(UL11−10)ΔTK/gE/gI}, BAC^{PRV−S(UL35–36)ΔTK/gE/gI}, BAC^{PRV−S(UL46−27)ΔTK/gE/gI} and BAC^{PRV−S(US2−1)ΔTK/gE/gI}). c Homologous recombination was performed to recover the intact gI gene and part of gE gene (1299 to 1735 bp of gE open reading frame). d Schematic presentation of the PRV-S(UL11-10)ΔTK/gE, PRV-S(UL35-36)ΔTK/gE and PRV-S(UL46-27)ΔTK/gE were shown

Fig. 2

RFLP of BAC^{PRVΔTK/gE/gI}, BAC^{PRV−S−KAN(UL11−10)ΔTK/gE/gI}, BAC^{PRV−S(UL11−10)ΔTK/gE/gI}, BAC^{PRV−S−KAN(UL35–36)ΔTK/gE/gI}, BAC^{PRV−S(UL35–36)ΔTK/gE/gI}, BAC^{PRV−S−KAN(UL46−27)ΔTK/gE/gI}, BAC^{PRV−S(UL46−27)ΔTK/gE/gI}, BAC^{PRV−S−KAN(US2−1)ΔTK/gE/gI} and BAC^{PRV−S(US2−1)ΔTK/gE/gI}. DNA of BAC^{PRVΔTK/gE/gI}, BAC^{PRV−S−KAN(UL11−10)ΔTK/gE/gI}, BAC^{PRV−S(UL11−10)ΔTK/gE/gI}, BAC^{PRV−S−KAN(UL35–36)ΔTK/gE/gI}, BAC^{PRV−S(UL35–36)ΔTK/gE/gI}, BAC^{PRV−S−KAN(UL46−27)ΔTK/gE/gI}, BAC^{PRV−S(UL46−27)ΔTK/gE/gI}, BAC^{PRV−S−KAN(US2−1)ΔTK/gE/gI} and BAC^{PRV−S(US2−1)ΔTK/gE/gI} were digested with BamH I. Predicted RFLP pattern with BamH I was performed using PRV ZJ01 strain (GenBank: KM061380.1) as a reference

Fig. 3

Images of PRV-S(UL11-10)ΔTK/gE, PRV-S(UL11-10)-mini-FΔTK/gE/gI, PRV-S(UL35-36)ΔTK/gE, PRV-S(UL35-36)-mini-FΔTK/gE/gI, PRV-S(UL46-27)ΔTK/gE and PRV-S(UL46-27)-mini-FΔTK/gE/gI under UV excitation and phase contrast are shown. Each panel represents a view of 200 × 200 μm in size

Fig. 4

PCR identification of S expression cassette and gI/ΔgE. S expression cassette of PRV-S(UL11-10)ΔTK/gE, PRV-S(UL35-36)ΔTK/gE and PRV-S(UL46-27)ΔTK/gE was identified by PCR with primers (S cas check F/S cas check R), and gI/ΔgE was identified by PCR with primers (PRV ΔgE check F/PRV ΔgE check R)

Fig. 5

Confirmation of S and PRV protein expression by the recombinant PRV using indirect immunofluorescence assay. Pig anti-sera against PEDV or PRV in conjunction with FITC labelled anti-pig secondary antibodies were employed to verify S and PRV protein expression. Each panel represents a view of 200 × 200 μm in size

Fig. 6

S gene mRNA expression on ST cells infected with PRV-S(UL11-10)ΔTK/gE, PRV-S(UL35-36)ΔTK/gE and PRV-S(UL46-27)ΔTK/gE at a MOI of 10 at 6 and 12 h post infection. Data are expressed in relative arbitrary units, in comparison with the values measured in ST cells infected with PRV-S(UL11-10)ΔTK/gE at 6 h post infection and taken as 1.00. Data were presented as mean ± SEM, and analyzed using one-way ANOVA with a Tukey’s post − hoc test (SPSS Inc., Chicago, IL, USA)

Fig. 7

Multi-step growth curves of AH02LA, PRV^{ΔTK&gE−AH02}, PRV-S (UL11-10)ΔTK/gE, PRV-S(UL35-36)ΔTK/gE and PRV-S(UL46-27)ΔTK/gE. ST cells were infected with AH02LA and the four mutants at a MOI of 0.01. At 6, 12, 24, 36, 48 and 60 h post infection, the culture cells were harvested and then were titrated in ST cells. Asterisks indicate statistical significance between AH02LA and the four mutants (*p < 0.05). Data were presented as mean ± SEM, and analyzed using one-way ANOVA with a Tukey’s post − hoc test (SPSS Inc., Chicago, IL, USA)