Rescue and in vitro growth characterization of NSP2 multisite-deleted strain of porcine reproductive and respiratory syndrome virus GS15

Abstract:

[Objective] A genetically engineered virus strain rGS15-△2 with deletion of the residues at the positions 519-565 and 628-747 of the non-structural protein 2 (NSP2) had been rescued based on the PRRSV/GSWW/2015 infectious clone. This study aims to construct and rescue an engineered virus strain with the deletion of three sites in NSP2 based on rGS15-△2. [Methods] Based on the infectious clones of rGS15-△2, two recombinant plasmids with the deletion of three sites were constructed by fusion PCR. Specifically, the dominant epitope at the amino acid site 323-364 or 372-433 of NSP2 was further deleted on the basis of rGS15-△2. The recombinant plasmids were linearized and mixed with liposome, which were transfected into Marc-145 cells for virus rescue. The growth characteristics of the engineered virus strains were analyzed by electron microscopy, immunofluorescence assay (IFA), virus titer determination, and growth curve establishment. [Results] The engineered virus strains rGS15-△3-1 and rGS15-△3-2 were rescued successfully. Virions with the diameter from 50 nm to 80 nm can be observed under an electron microscope. The results of IFA confirmed the expression of PRRSV N protein by the rescued virus strains and the parent strain GS15. Furthermore, the rescued viruses were cultured with Marc-145 cells for 40 passages, and the deletion regions were confirmed to be stable by RT-PCR and sequencing. The titers of rGS15-△3-1 and rGS15-△3-2 were 2.00×10^6.0 TCID₅₀/mL and 2.25×10^5.8 TCID50/mL, respectively, which had differences from that of the parent strain (P<0.05). The growth curves showed that the rescued viruses had lower replication levels than the parent strain, and they reached the peak titers 24 h later than the parent strain. [Conclusion] We characterized the growth of the viruses with the deletion of multiple sites in NSP2 of PRRSV. The findings laid a foundation for the development of novel PRRSV-labeled vaccines and provided a new strategy for the prevention and control of porcine reproductive and respiratory syndrome.