Construction Of The HP-PRRSV Infectious Clone And Its Application In Viral Visualization

Porcine reproductive and respiratory syndrome virus(PRRSV) is the causative agent of porcine reproductive and respiratory syndrome(PRRS) that can result in reproductive failure in pregnant sows and respiratory complications in piglets. PRRS is still a severe problem of swine industry today, especially the highly pathogenic PRRS(HP-PRRS) caused by highly pathogenic PRRSV(HP-PRRSV) outbreak in 2006 gave the swine industry with significant losses in China. For a more convenient procedure to study molecular mechanisms and pathogenicity of HP-PRRSV and the vaccine design, an infectious clone of HP-PRRSV SD16 strain based on bacterial artificial chromosome(BAC) system was constructed. Fluorescent protein genes(EGFP or DsRed) were inserted into SD16 genome to achieve viral visualization, and the expression level of fluorescent protein can serve as the indicator in the validation of antiviral drugs. 1. Construction of the HP-PRRSV infectious clone of SD16 strain based on BAC system.Modification of the genome give a quick procedure for the verification of particular site functions in the genome, and construction of viral infectious clone is a powerful tool to achieve this goal in the virus. The BAC system, derived from the plasmid of E.coli Fertility factor, have the advantages of large capacity, high fidelity and convenient of operation, have been applied to a variety of viruses rescue.According to the complete sequence of HP-PRRSV SD16 strain(GenBank No. JX087437) genome, specific primers were synthesized and be used in the PCR amplification. All PCR-amplified fragments were gel purified and ligated for the construction of pEasy-blunt plasmid. The cytosine(C) at the position 9893 was mutated to thymine(T) by PCR to silence a restriction enzyme site Bmt I as the genetic marker. The nucleic acid sequence containing SD16 genome 5’(fused with part of the CMV promoter sequence in upstream) and 3’ end(flanked by the HDV ribozyme and the bovine growth hormone terminator sequence after the poly A) were synthesized, and then be inserted into pBAC plasmid by Sfi I and Rsr II restriction sites, and then got the backbone plasmid pBAC-SD16-5’-3’. Fragments of SD16 genome were inserted into the pBAC-SD16-5’-3’ with the proper restriction sites. The completely assembled full-length cDNA clone pBAC-SD16 FL was confirmed by sequencing. pBAC-SD16 FL was transfected into MARC-145 cells. The virus was rescued and identified by IFA, genetic marker detection and western blot. In vitro studies demonstrated that the rescue virus maintained growth properties similar to parental virus. These results suggest that infectious clone of HP-PRRSV SD16 strain was successfully constructed, and which can be used as a powerful tool to study the mechanism and biological characteristics of PRRSV. 2. Application of HP-PRRSV infectious clone of SD16 strain in viral visualization.Record of "live" state of organisms reflects what is the real situation of life, can give us a directly understanding of the real world. Visualization labeling includes organisms(such as viruses) component and the host proteins, and then for the study the real-time interaction between them. The molecular markers were widely used in visualization labeling, including the genetically encoded fluorescent protein and the peptides which have the binding properties to fluorescent substance.Based on infectious clone of HP-PRRSV SD16 strain, green fluorescent protein(EGFP) or red fluorescent protein(DsRed) were introduced into SD16 genome to achieve PRRSV visualization. EGFP or DsRed gene were inserted into the position between N gene and 3’UTR of SD16 genome, serve as a separate transcription unit to initiate its transcription by PRRSV transcriptional regulatory sequences TRS6 or TRS2, respectively. Recombinant virus were rescued in MARC-145 cells. In vitro studies on MARC-145 cells demonstrated that the rescue virus maintained growth properties similar to parental virus. Fluorescent protein expression in cells maintain high efficiency and intensity, flow cytometry results shows an increased proportion of fluorescent positive cells along with the prolonged culture time. To some extent, as the live cell imaging showing, the appearance of fluorescence could be serve as a early sign of PRRSV infection. In the function verification of antiviral drugs, the fluorescent signal generated by the fluorescent virus in host cells can be used as a positive correlation indicator of PRRSV infectivity.This recombinant HP-PRRSV containing fluorescence genes not only serve as a powerful tool to study the mechanism and biological characteristics of PRRSV, but also to provide an expression vector system for other exogenous tags expression.