Construction and Virus Rescue of an Infectious Full-length cDNA Clone of Porcine Reproductive and Respiratory Syndrome Virus(PRRSV) Expressing Foreign Gene
Abstract：A reverse genetic technology platform of Porcine reproductive and respiratory syndrome virus (PRRSV) with foreign gene is established, which can be used to develop a DIVA (differentiating infected from vaccinated animals) marker vaccine. In this study, a molecular marker of immunodominant B-cell epitope (49 amino acids) of Newcastle disease virus (NDV) nucleoprotein (NP) was inserted into nonessential region of nsp2 by the method of mutant PCR, basing on the infectious clone of HuN4-F112 vaccine strain. The marker full-length cDNA clone (psk-HuN4-F112-Δ52+NP49) was assembled by cloning and splicing of the gene fragments. The completely assembled full-length cDNA clone was confirmed by sequence and Swa Ι enzyme digestion. Capped RNA was transcribed in vitro from a full-length cDNA clone of the viral genome and transfected into baby hamster kidney cells(BHK-21) by liposome to acquire the rescued virus. The rescued recombinant virus was passaged on a highly permissive subclone of African green monkey kidney epithelial cell line(MARC-145). The successfully rescued virus was tested by RT-PCR, digestion, and genome sequence. The results showed that the rescued virus could be distinguished from the parental with the mutant genetic marker (MluⅠenzyme site of genome at 14667nt) and the inserted nsp2 gene region. The results of indirect immunofluorescence assay(IFA) showed that the inserted foreign marker could be expressed by the recombinant virus and the inserted foreign gene was stable during the virus serial passage. The results of plaque assay and viral growth curve showed that the recovery virus possessed similar characters of viral growth to those of the parental virus. The results suggest that this stable infectious clone can be used as an important tool for development of novel vaccine against PRRSV.
