The commercial PRRSV attenuated vaccine can be a potentially effective live trivalent vaccine vector.

PRRSV is an immunosuppressive virus that is prone to secondary infection by other viral and microbial pathogens, exacerbating its harm to pigs. The development of multivalent vaccines that can prevent and control multiple pathogens is of great significance for the pig industry. In order to evaluate the potential of commercial PRRSV vaccines as the viral vectors for the trivalent vaccine, this study first embedded mCherry or EGFP genes for fluorescent proteins into the infectious clone of the HP-PRRSV MLV vaccine strain GD to construct a recombinant strain. Furthermore, we constructed a recombinant PRRSV strain, rPRRSV-mCherry-EGFP, that expresses both EGFP and mCherry proteins simultaneously and evaluated the stability and immunogenicity of the exogenous proteins. The results showed that MARC-145 cells infected with rPRRSV-mCherry-EGFP could simultaneously express both EGFP and mCherry exogenous proteins, and the number of EGFP and mCherry positive cells increased with virus infection, providing more fluorescent tool options for virus visualization and high-throughput screening of anti-PRRSV drugs. More importantly, the recombinant rPRRSV-mCherry-EGFP can be passaged in MARC-145 cells for at least 10 generations. It can induce piglets to produce antibodies against PRRSV, EGFP, and mCherry, indicating that the embedded two exogenous genes can also induce good immune responses. Our research suggests that using the infectious clone of a commercial HP-PRRSV-attenuated vaccine as a vector and embedding immune genes of two other pathogens to construct a recombinant strain may be an effective attempt to achieve cross-protection against three pathogens, providing an important research basis for the design of trivalent vaccines. KEY POINTS: • A recombinant PRRSV that expressed two foreign proteins was constructed. • The recombinant PRRSV could induce antibodies against PRRSV, EGFP, and mCherry. • PRRSV MLV strain had the potential to be used as a viral vector for triple vaccines.