Characterization of a gE/gI/TK gene-deleted pseudorabies virus variant expressing the Cap protein of porcine circovirus type 2d

• Published in: Comparative immunology, microbiology and infectious diseases Vol. 101; p. 102054
• Main Authors: Xu, Rui-Qin, Wang, Lin-Qing, Zheng, Hui-Hua, Tian, Run-Bo, Zheng, Lan-Lan, Ma, Shi-Jie, Chen, Hong-Ying
• Format: Journal Article
• Language: English
• Published: England Elsevier Ltd 01.10.2023
• Subjects: Animals; Antibodies, Viral; Cap protein; Circovirus - genetics; Herpesvirus 1, Suid - genetics; Mice; Porcine circovirus type 2, PCV2d; Pseudorabies - prevention & control; pseudorabies virus; Swine; Swine Diseases; Vaccine; Viral Envelope Proteins - genetics; Viral Vaccines - genetics; Porcine circovirus type 2, PCV2d; Vaccine; Cap protein; pseudorabies virus
• ISSN: 0147-9571; 1878-1667
• DOI: 10.1016/j.cimid.2023.102054

Porcine circovirus type 2 (PCV2) plays a key role in the etiology of PCV2-associated disease (PCVAD), and its predominant strain is PCV2d which is not completely controlled by most commercially available vaccines against PCV2a strains. Pseudorabies (PR) caused by pseudorabies virus (PRV) variants re-emerged in Bartha-K61 vaccine-immunized swine herds in late 2011, which brought considerable losses to the global pig husbandry. Therefore, it is significantly important to develop a safe and effective vaccine against both PCV2d and PRV infection. In the present study, the PCV2d ORF2 gene was amplified by PCR, and cloned into the BamHI site of PRV transfer plasmid pG vector to obtain the recombinant transfer plasmid pG-PCV2dCap-EGFP. Subsequently, it was transfected into ST cells infected with the three gene deleted PRV variant strain NY-gE-/gI-/TK- to generate a recombinant virus rPRV NY-gE-/gI-/TK-/PCV2dCap+/EGFP+, and then the EGFP gene was knocked out to harvest the rPRV NY-gE-/gI-/TK-/PCV2dCap+ using gene-editing technology termed CRISPR/Cas9 system. The recombinant virus rPRV NY-gE-/gI-/TK-/PCV2dCap+ had similar genetic stability and proliferation characteristics to the parental PRV as indicated by PCR and one-step growth curve test, and the expression of Cap was validated by Western blot. In animal experiment, higher PCV2-specific ELISA antibodies and detectable PCV2-specific neutralizing antibodies could be elicited in mice immunized with rPRV NY-gE-/gI-/TK-/PCV2dCap+ compared to commercial PCV2 inactivated vaccine. Moreover, the recombinant virus rPRV NY-gE-/gI-/TK-/PCV2dCap+ significantly reduced the viral loads in the hearts, livers, spleens, lungs, and kidneys in mice following a virulent PCV2d challenge. Mice immunized with rPRV NY-gE-/gI-/TK-/PCV2dCap+ developed comparable PRV-specific humoral immune responses and provided complete protection against a lethal PRV challenge. Together, the rPRV NY-gE-/gI-/TK-/PCV2dCap+ recombinant strain has strong immunogenicity. •The recombinant pseudorabies virus rPRV NY-gE-/gI-/TK-/PCV2dCap+ was constructed successfully.•Mice immunized with the recombinant virus developed specific ELISA and neutralizing antibodies against PCV2d and PRV.•Mice vaccinated with the recombinant virus were protected from PRV challenges and had the decreased viral loads of PCV2d.•rPRV NY-gE-/gI-/TK-/PCV2dCap+ is a potential vaccine candidate against PCV2d and PRV infection.