Construction and Immunogenicity of a Recombinant Pseudorabies Virus Variant With TK/gI/gE/11k/28k Deletion

Abstract

In China, the re-emerging pseudorabies virus (PRV) variant has caused large-scale outbreaks of pseudorabies in swine herds with classical PRV vaccine immunization since late 2011. Here, a recombinant PRV with TK/gI/gE/11k/28k deletion was constructed based on variant HN1201 strain isolated in 2012, by the bacterial artificial chromosome infectious clones. Compared with the parental virus, the recombinant PRV rHN1201^{TK-/gE-/gI-/11k-/28k-} showed a similar virus grown curve and exhibited smaller plaques. The vaccination of rHN1201^{TK-/gE-/gI-/11k-/28k-} could elicit an earlier and higher level of gB antibody, and the neutralizing antibodies elicited by rHN1201^{TK-/gE-/gI-/11k-/28k-} were effective against both PRV classical and variant strains. Clinically, the body temperature of the pigs immunized with rHN1201^{TK-/gE-/gI-/11k-/28k-} was significantly lower than that of the classical PRV vaccine immunized pigs, and the recombinant PRV could provide effective protection against the challenge with the PRV variant. These results imply that the rHN1201^{TK-/gE-/gI-/11k-/28k-} could be a promising vaccine candidate for the prevention of the current epidemic of pseudorabies in China.

Keywords: bacterial artificial chromosome; gene deletion; immune protection; pseudorabies virus (PRV); vaccine.

Figure 1

Overview of strategies for the TK/gE/gI/11k/28k deletion. Boxes of the same color represent identical sequences.

Figure 2

Rescue and the characterization of recombinant PRV. (A) Cytopathic effect in Vero cells after transfection with recombinant PRV pBAC plasmids. The white arrow indicated the CPE. Bar = 100 μm. (B) Multiple growth curves of the chimeric viruses. The culture supernatants were collected at the indicated time points for the viral titer determination. (C) Plaque size of the recombinant viruses. The plaques were measured at 48 hpi. The plaque size induced by the parental virus was set at 100%. Asterisk denotes a statistically significant difference (p < 0.05).

Figure 3

Body temperature and survival rate of pigs after challenge. (A) Body temperature of pigs challenged with HN1201. (B) Survival rate of pigs challenged with HN1201. The survival rates were analyzed by the Kaplan Meier test.

Figure 4

Production of PRV-specific antibodies after vaccination and viral load test of pigs after challenge. (A) The gB antibodies were detected at the indicated time points after vaccination. The ELISA values of pig serum samples are given as S/P ratios, S/P > 0.5 was considered positive. (B) NAbs against the HN1201 and Bartha-K61 in the indicated groups at 21 dpv and 35 dpv (14 dpc). Standard deviations are shown as error bars. The t-test was performed for statistical analysis. (C) Viral load detection of pigs after challenge with HN1201. The viral loads between different groups were analyzed by t-test. Asterisk denotes a statistically significant difference (P < 0.05), ns indicated no significant difference.

Figure 5

Results of IHC assay of tonsils, lungs, brains, and trigeminal ganglions. Representative IHC images of tonsils, lungs, brains, and trigeminal ganglions were shown corresponding to animal groups infected with HN1201. The groups' names were indicated on the left of the figure. Bar = 50 μm.