A gD&gC-substituted pseudorabies virus vaccine strain provides complete clinical protection and is helpful to prevent virus shedding against challenge by a Chinese pseudorabies variant

Abstract

Background: Since 2011, pseudorabies caused by a variant PRV has re-emerged in many Chinese Bartha-K61-vaccinated pig farms. An efficacious vaccine is necessary to control this disease. We described the construction of a gD&gC-substituted pseudorabies virus (PRV B-gD&gC^S) from the Bartha-K61 (as backbone) and AH02LA strain (as template for gD and gC genes) through bacterial artificial chromosome (BAC) technology using homologous recombination. The growth kinetics of PRV B-gD&gC^S was compared with Bartha-K61. Its safety was evaluated in 28-day-old piglets. Protection efficacy was tested in piglets by lethal challenge with AH02LA at 7 days post vaccination, including body temperature, clinical symptoms, virus shedding, mortality rate, and lung lesions.

Results: The results showed that a BAC clone of Bartha-K61 and a B-gD&gC^S clone were successfully generated. The growth kinetics of PRV B-gD&gC^S strain on ST (Swine testicular) cells was similar to that of the Bartha-K61 strain. No piglets inoculated intramuscularly with PRV B-gD&gC^S strain exhibited any clinical symptoms or virus shedding. After AH02LA challenge, all piglets in PRV B-gD&gC^S and Bartha-K61 groups (n = 5 each) survived without exhibiting any clinical symptoms and high body temperature. More importantly, PRV B-gD&gC^S strain completely prevented virus shedding in 2 piglets and reduced virus shedding post challenge in the other 3 piglets as compared with Bartha-K61 group.

Conclusions: Our results suggest that PRV B-gD&gC^S strain is a promising vaccine candidate for the effective control of current severe epidemic pseudorabies in China.

Keywords: Bacterial artificial chromosome; Immunogenicity; Pseudorabies virus; Safety; gD&gC substitution.

Fig. 1

Construction of mini-F recombinant PRV Bartha strain (PRV B-mini-F), gD substituted clone (pB-gD^S-mini-F) and gC&gD-substituted virus (PRV B-gD&gC^S) (a) Mini-F was inserted in lieu of gC to generate the mini-F recombinant PRV Bartha-K61 strain for BAC through homologous recombination. b AH02LA gD-KAN was inserted in lieu of gD in the Bartha genome through the first recombination, the kanamycin gene was deleted in the second step, generating gD substituted clone (pB-gD^S-mini-F). c Another recombination was performed to substitute the mini-F sequence with gC of AH02LA, generating gC&gD substituted virus (PRV B-gD&gC^S)

Fig. 2

Plaque of PRV B-mini-F, PRV B-gD^S-mini-F and PRV B-gD&gC^S, RFLP of pB-mini-F, pB-gD^S-KAN-mini-F and pB-gD^S-mini-F, and PCR verification of gC and gD genes replacement. A Images of PRV B-mini-F, PRV B-gD^S-mini-F and PRV B-gD&gC^S plaques under UV excitation and contrast are shown. B DNA from pB-mini-F BAC clone (lanes 1 and 4) and recombinant BACs of pB-gD^S-KAN-mini-F (lanes 2 and 5) and pB-gD^S-mini-F (lanes 3 and 6) were prepared by mini-prep and digested with Hind III (lanes 1–3) or Sph I (lanes 4–6). Digests were separated by 0.8% agarose gel electrophoresis for 15 h under 40 V. Predictions of these digestions were performed using whole genome sequences of Bartha-K61 as a reference (GenBank ID: JF797217.1). C Verification of gC and gD genes replacement by PCR. gD of Bartha-K61 and PRV B-gD&gC^S were identified with AH02LA-gD-F/AH02LA-gD-R. gC of Bartha-K61 and PRV B-gD&gC^S were identified with SEQ-AH02LA gC F/SEQ-AH02LA gC R

Fig. 3

Multi-step growth curves of Bartha-K61 and PRV B-gD&gC^S on STs. At 0, 6, 12, 24, 36, 48, 60 and 72 h post infection, virus was titrated on STs with a MOI of 0.01. Data were presented as mean ± SD, and analyzed using Student’s t test by SPSS 16.0 (SPSS Inc., Chicago, IL, USA)