Generation And Immunogenicity Of A Recombinant Pseudorabies Virus Co-expressing Classical Swine Fever Virus E2 Protein And Porcine Circovirus Type 2 Capsid Protein Based On Fosmid Library Platform

Pseudorabies?PR?,classical swine fever?CSF?and porcine circovirus type 2?PCV2?-associated disease?PCVAD?are economically important infectious diseases of pigs.Co-infections of these diseases often occur in the field leading to significant economic losses to the swine industry worldwide.CSF is caused by classical swine fever virus?CSFV?and its E2glycoprotein is mainly used in subunit vaccines.PCV2 is the primary causative agent of porcine-associated disease and its capsid?Cap?protein is the key antigen used for developing genetically engineered PCV2 vaccines.In our previous research,it was demonstrated that the Bartha-K61 vaccine was not able to protect completely the challenge with the a recently emerged PRV variant,PRV TJ strain,which was isolated in 2011 from a Bartha-K61-vaccinated swine herd in Tianjin,China.The gE/gI/TK-gene-deleted vaccines are safe and capable of providing full protection against PR.Multivalent vaccines especially virus-vectored vaccines expressing foreign proteins are attractive strategies to fight co-infections for various swine diseases.The gene-deleted PRV can be used to develop promising and economical multivalent live virus-vectored vaccines.Herein,we constructed a gene-deleted PRV co-expressing E2 of CSFV and Cap of PCV2 by fosmid library platform established for PRV,and the expression of E2 and Cap was confirmed by immunofluorescence assay?IFA?and western blotting?WB?.The growth properties of rPRVTJ-delgE/gI/TK-E2-Cap were indistinguishable from the parental virus rPRV-delgE/gI/TK.The recombinant virus was propagated in PK-15cells for 20 passages and the stability of inserted genes was identified at every 5^th passage by PCR and sequencing.The expression of E2 and Cap was analyzed by IFA at every 5^th passage.The evaluation results in rabbits and pigs demonstrated that rPRVTJ-delgE/gI/TK-E2-Cap was safe for the rabbits and pigs and did not induce any PRV specific clinical signs including fever.The immunized rabbits and pigs developed detectable anti-PRV antibodies,however there were no anti-PCV2 or anti-CSFV antibodies possibly due to unbefitting conditions for the inserted genes or the expression of E2 and cap might be too low to induce antibodies.This study provides a new platform for the development of virus-vectored vaccines and opens new insights that rPRVTJ-delgE/gI/TK-E2-Cap needs to be optimally engineered as a promising trivalent vaccine candidate against PRV,PCV2 and CSFV co-infections in future.