Reverse Genetic Manipulation Of Coding Regions Of Porcine Reproductive And Respiratory Syndrome Virus

Porcine Reproductive and Respiratory Syndrome(PRRS) is a severe infectious disease.PRRS is a economically significant infectious disease in the swine industry worldwide,from 2006,"Porcine High Fever Syndrome" has spreaded all over the Chinese swine industry,the highly pathogenic(HP) PRRSV is one of the major cause pathogen.As the virus genome recombinating and mutating,the biological type,antigen type and genotype of PRRSV variated quickly,current control for PRRS is insufficient. The available vaccines are lack of antigencity and cross protection.It is urgent that more efficient PRRS vaccine should be developed.At the same time,the virulence factor of PRRSV is not known clearly.PRRSV uses subgenomic mRNA to express its genetic information,a uique RNA discontinuous minus-strand transcription.In our study,we constructed a series of mutantion inserting foreign sequence in the structural protein coding regions of PRRSV,aiming at dissecting the cis-acting element for transcription regulation and translation mechanism,the detailed summarized as follows.1 Reverse genetic manipulation of structural protein coding regions of highly pathogenic(HP) porcine reproductive and respiratory syndrome virusManipulation of the genome of porcine reproductive and respiratory syndrome virus(PRRSV) is complicated for their condensed genomic structure.To investigate the importance of overlapping genes arrangement and to facilitate the genetic manipulation of the viral genome,seven mutant full-length cDNA clones were constructed in which one restriction site were inserted in between HP PRRSV ORF1/2,ORF4/5,ORF5/6,ORF 6/7,Our results demonstrated only the plasmid pJXM-12 with Nde I in between ORFland 2 was infectious,the mutant viruse stably maintained the mutations that were introduced restriction sites during five serial passages,the biological characteristics of the mutant viruses were phenotypically indistinguishable from the parental virus.But for other plasmid no viable virus was resued,and also the triscription and replication.That may be related to the limited length of packaged genomal RNA of PRRSV,the position of insertion,or the inserted sequence causes a hidden signal,via RNA secondary structure or protein binding sites,that affects viral transcription, replication,and/or translation.This research may provide support for a mechanism of discontinuous minus-strand transcription and translation.2 Reverse genetic manipulation of ORF6/7 and 3′UTR of porcine reproductive and respiratory syndrome virus(PRRSV)In recent years,mass outbreaks of highly pathogenic(HP) porcine reproductive and respiratory syndrome virus(PRRSV) have spread all over the Chinese swine industry.There is no highly efficient PRRS vaccine to control it.Based on the first infectious cDNA clone of HP PRRSV strain pJX143 and that of an attenuated PRRSV(pAPRRS),we constructed several chimeric clones with various substitutions of structural protein gene(ORF7) and 3′UTR between attenuated pAPRRS and virulent pJX143,.and also 32nt including two restriction sites was inserted in between ORF6 and ORF7,ORF7 and 3′UTR Upon transfection on Marc-145 cell lines,all chimeric constructs pBJ327,pBJ732,pBJD32,pBJD32R were rescued.The rescued viruses maintained the similar virological properties, based on the results of the plaque morphologies of the rescued viruses.For the chimeric infectious clones are constructed successfully,the chimeric viruses can be passaged stably on Marc-145 cell.In this study,we construct the direct repeat sequence of 32nt,that could provide the significant platform for searching the direct repeated-mediated deletions in RNA virus and this may be a method for developing PRRS vaccine.3 Mapping the transcriptional promoter of mRNA5 of PRRSVAfter the discontinuous minus-strand transcription,a series of proteins were translated,GP5 is by ORF5.GP5 is one of the major factor of virus to which the host response well with protective antibody production.In this study a series of mutagensis of an infectious clone of pAPRRS were conducted, different nucleotides have been inserted into the different position of the spacer of 10nt between ORF4 and ORF 5.Our result showed that the spacer can suffer 13nt if the insertion is behind the stop codon of ORF4,otherwise only 3nt was permitted before the start codon of ORF5,distinguished sequence with 18nt can be inserted between the spacer,this study could provide the significant platform for further dissecting the mechanism of transcription and translation,and also exploring for PRRSV as foreign gene expression vectors.