| Porcine reproductive and respiratory syndrome (PRRS) caused by PRRS virus (PRRSV), is aneconomically important disease characterized by reproductive failure in sows and respiratory disease inpigs of all ages. In2006, highly pathogenic porcine reproductive and respiratory syndrome virus(HP-PRRSV) caused great economic losses emerged in China and continues to be a threat for the pigindustry. Evolutions and mutations of the PRRSV easily caused the changes of antigenicities, tropismsand pathogenicities, therefore, the traditional vaccines usually did not provide enough immunologicpotency to swine herds, which brought great challenges to prevention and control of PRRSV in clinicalpractice. In order to further understand the molecular basis of phenotypic variation of PRRSV, and todevelop more effective vaccines, the present study describes the establishment of a full-length cDNAcloneofa live-attenuatedHP-PRRSVvaccinestrainbyutilizingreversegenetic manipulationtechnology,which was driven by RNApolymerase (pol) I and (pol) II, and the development of Marc-145-T7cell linestably and efficiently expressing T7RNAPby using retroviral gene transfer technology, finally we testedinfectivity of the cDNAclone. Tremendous progresses having been made in understanding the molecularbiology and pathogenesis of HP-PRRSV by the reverse genetics system would provided a perspective onpotential novel strategies and approaches that may be applicable to the development of safe and effectiveHP-PRRSV vaccine.To obtain genome sequence and full length cDNA clone of a live-attenuated highly pathogenicporcinereproductiveandrespiratorysyndrome virus (HP-PRRSV) strain, thePCR primers were designedmainly according to the sequences of HP-PRRSV HuN4and classical CH-1a. Six overlapped fragmentsspanning the full-length genome were subsequently generated by RT-PCR amplification. Each PCRproduct was cloned into PMDT-20vector and then sequenced. The subclone products were cloned intothe modified pOK12vector in proper order by suitable enzyme sites to obtain viral full length genomiccDNA clone, named pAPRRSVOK12. In addition, polymerase II, I and the T7RNA polymerasepromoter and hammerhead ribozyme (HamRz) fragments were inserted into upstream of the viralgenomic5’-terminal sequences by introduced restriction enzyme Mlu I and EcoR I, and the terminatorfragments and hepatitis delta virusribozyme (HdvRz) wereinsertedinto downstreamofthe viralgenomic3’-terminal sequences by introduced restriction enzyme Not I and Fse I. Sequencing and enzymaticdigestion results showed that the viral full length genomic cDNAclone was constructed successfully. Theplasmid not only was the in vivo rescue system based on RNApolymerase I/Ⅱ system, but also it was invivo transcription system based on T7RNA polymerase. When Marc-145cell was directly transfectedwith the full-length cDNAof PRRSV, but infectious PRRSV was not successfully recovered.To develop an efficient and vaccinia virus-free system, the T7RNAP gene was inserted into thechromosome of Marc-145cells by using retroviral gene transfer technology and then Marc-145-T7cellline stably and efficiently expressing T7RNAPwere established under selection pressure. The T7RNAPproducedintheMarc-145-T7cell line was abletoefficiently drivein vivotranscription ofenhanced greenfluorescent protein(EGFP) reporter gene controlledby the T7promoter. These data showed the T7RNAPin Marc-145-T7cells have good transcriptional activity and can be used to rescue virus from full-length cDNA. We attempted at obtaining an infectious full-length cDNA clone of a live-attenuated HP-PRRSVvaccine strain by utilizing stable Marc-145-T7cell line constitutively expressing T7RNA polymerase,but we did not succeed. Although failed to save infectious HP-PRRSV vaccine strain, correct andobjective analysis of the causes of failure would provided a perspective on potential novel strategies andapproaches that may be used to rescue virus from cloned cDNAof some RNA viruses. |
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