| Porcine reproductive and respiratory syndrome virus (PRRSV) belongs to the family Arteriviridae,order Nidovirales,which includes the other viruses, equine arteritis virus (EAV), lactatedehydrogenase-elevating virus (LDV) and simian hemorrhagic fever virus (SHFV). Based on theserological and genetic difference, PRRSV is classified into two genotypes,the European genotype (type1) and the North American genotype (type2). ORF5is a newly dicovered ORF in all arteriviruses. The5a protein expressed from ORF5a has been proved as the eighth structural protein in EAV andPRRSV.Until now, the detail structure and function of5a protein is poorly understand. In this study,based on infectious full-length cDNA clones of PRRSV, the structure and function of5a protein aredissected. This will lay foundation to better understand of5a protein even arterivirus and to developnovel gene marker vaccines to prevent arterivirus infection.1.The structure and function of5a protein in PRRSV replicationORF5a has141or156nucleoides in type2,encoding5a protein ranging in size from46to51aminoacids. We have find several stop codons in frame of ORF5a behind the stop codon of ORF5a. Based onthe infectious cDNA clone pAJX25HB, we change the stop codon of ORF5a to elongate or shorten5aprotein by using PCR site-directed mutagenesis. The result shows that the size of5a protein can beelongated45residues at most, but the elongation of5a protein isn’t stable and PRRSV tends to use thefirst stop codon. The mutant viruses displayed reduced replication ability in MARC-145. Continuing toelongate5a protein results in inactivation of5a protein and failure to rescue viable virus.In addition,46residues is the minimum size of5a protein and no viable virus can be rescued with crippled5a protein.The results also indicate indirectly that5a protein is essential for PRRSV viability and it may play a rolein PRRSV package or budding.2. Separation of ORF5/ORF5a overlapping gene by developing intra-genotypic chimeric PRRSVMost sequences of ORF5a is overlapped with ORF5even ORF5a is fully embedded within ORF5,which makes it difficult to dissect the structure and function of5a protein. To study on the the structureand function of5a protein conveniently, we substitute ORF2a-5a of pAPRRSV with correspondingORFs of pSHE and construct three plasmid DNAs by using splicing overlapping extension polymerasechain reaction (SOE-PCR). Till now, ORF5a is separated from ORF5and5a protein can be encodedindependently. These plasmid DNAs were transfected into BHK-21and analyzed by molecular biologyand virology methods. The result shows that the chimeric viruses displayed robust genetic stability invitro, the corresponding proteins of type1PRRSV can play full function in type2virus and thebiological characteristics of the chimeric viruses were phenotypically indistinguishable from theparental virus. In this study, ORF5a is firstly separated from ORF5. This paved the way to furtherelucidate the structure and function of5a protein.3. Development and characterization of pAPRRS-LDV chimeric virus It has been proved that the chimeric virus, substitute ORF2-4of pAPRRSV with correspondingORFs of EAV, infected BHK-21cell which is the non-permissive cell of PRRSV. We substitutedORF2-4of pAPRRSV with corresponding ORFs of LDV and wanted to construct pARRS-LDV234chimeric virus which can infect mouse.Transfection BHK-21cell confirmed that pRRS-LDV234completed the transcription of subgenomic mRNA and the translation of GP5and N protein. Thisindicated that pARRS-LDV234is with biological activity.The pARRS-LDV234lost the infectivity toMarc-145(expected results), but can’t infect mouse and mouse peritoneal macrophages. We hasn’tfound any infectious passage cells for pARRS-LDV234yet. The study developed a new method forPRRSV propagation in mouse. |
