The Construction And Application Of Chimeric Full-length CDNA Clone Of Arteriviruses

Equine arteritis virus (EAV), porcine reproductive and respiratory syndrome virus (PRRSV), lactate dehydrogenase elevating virus (LDV) and simian haemorrhagic fever virus (SHFV) were united in the family of enveloped positive-stranded RNA viruses, the Arteriviridae. The host range of the currently known arteriviruses is restricted to horses and donkeys (EAV), pigs (PRRSV), mice (LDV), and several genera of African and Asian monkeys (SHFV).The outcome of arterivirus infection can range from an asymptomatic, persistent carrier state to abortion or lethal haemorrhagic fever. There are still lots of mysteries about arterivirus genome replication, transcription and protein translation. The development of infectious cDNA clones made it possible to construct chimeric arteriviruses to investigate the functional complementation of a protein in two inter- or intra- species viruse strains. Chimeric viruses can also provide a powerful tool for molecular dissection of the pathogenesis of the increaseing virulence of arteriviruses.1: Construction of full-length cDNA clone of equine arteritis virus and analysis of genome sequenceTo develop a reverse genetics system of EAV, six pairs of oligonucleotides were designed based on the full-length genomic sequence of EAV Bucyrus strain. By RT-PCR, six overlapping cDNA fragments, designated as PE124,PE631,PE1854, PE5191, PE61107 and PE97Q respectively, were amplified, followed by being cloned into pCR BluntⅡ-TOPO vector. A NotI enzyme site and a T7 promoter sequence were introduced immediately upstream of 5′-end, with a XhoI cutting site in downstream of poly(A) tail in 3′-end. Full-length cDNA clone pWEAV was obtained by connecting the six cDNA fragments utilizing single restriction endonuclease site into the pBluescriptⅡKS(+) vector. The construction of a full-length genomic cDNA clone of EAV is a crucial step to obtain the infectious clone,which may facilitate further dissecting of structure and function relationship of EAV genome.2: Investation of the regulator of arterivirus replicationIn arterivirus, the regulating mechanisms of the virus life circle, such as genomic RNA replication, mRNA transcription, translation and virus particle packaging were mysterious. Furthermore, the deeper investigations need to be done to determine the roles virus coding proteins taking in these regulating mechanisms.The in vitro transcript of full length cDNA clone pWEAV was transfected in BHK-21 cell, and then IFA and RT-PCR were used to detect the virus protein expression and RNA replication. No RNA synthesis and protein expression were observed during the passages, so lethal mutations existed in the cDNA clone pWEAV. In order to identify the lethal mutations, based on the backbone of infectious clone pEAV030, we constructed a series of chimeric pEAV030/pWEAV cDNA clones. The in vitro transcript of the chimeric cDNA clones were transfected in the BHK-21.The results showed that two nucleotides variants in 5‵UTR and 10 amino acids variants in nsp1-nsp12 and 11 amino acids varants in GP2-GP4 also did not effect t the infectity of chimeric clone. On the other hand, we found that 6 animo acids variants in nsp2-nsp3 of chimeric clone are lethal. There animo acids effect genomic RNA replication and subgenomic RNA transcription. We also found one single amino acids mutation (A→S ) in GP5 and H→Y mutation in N protein, respectively, did not effect the genomic RNA replication and subgenomic RNA transcription. But both amino acids were responsible for the lethal of the pWEAV. It indicated that the both two amino acids may have a function in virus particle packaging and budding. Roles of these lethal mutations involving in the EAV life cycle will be studied in the future.3: The function of nsp6 in the arteriviruses replicationArterivirus non-structural protein (nsp1-nsp13) can assemble a Replicase/Transcriptiase Complex (RTC) for genomic RNA replication and subgenomic RNA transcription. The function and mechanism of each nsp assosiated for RTC is unclear. Nsp6, only 13-22 amino acids,.is the smallest protein in the arterivirus. From now on, the structure and function of nsp6 is unknown. To study the role of the nsp6 in replication of arterivirus, in this study, we construct a series of clones with nsp6 deletion and substitution with other arterivirus nsp6 by SOE-PCR. RT–PCR and IFA were used to detect the genomic RNA replication and subgenomic RNA transcription of the nsp6 mutants. The results showed that the mutants with nsp6 completely (16aa) or partially (3aa, 6aa) deletions can not be rescued, but all mutants were detected subgenomic RNA synthesis. The pAPRRS mutant whose nsp6 was substituted with LV, 3 amino acids changed, was viable. When pAPRRS nsp6 was substituted with LDV nsp6, ,the chimeric clone presented replication, transcription but not CPE, although 6 amino acids changed. Then pAPRRS nsp6 was replaced with the EAV nsp6, resulting in the lethal chimeric clone which can not tolerate the 22aa substitution. In conclusion, nsp6 is essential for virus infectivity, but the mechanism of nsp6 action will be studied in next step.We also found that 3 amino acid mutations were nonessential for virus replication and virus infectivity; however more mutations of nsp6 effected the process of virus replication in a way. 4:The construction of chimeric clone of high pathogencity PRRSV and attenuated PRRSV strain and identification of chimeric viruses rescuedPRRSV, the causative agent of the ongoing"porcine high fever syndrome"in China, is capable of genetic and antigenic mutations at high frequency. How to design vaccine rationally to keep up with the ever-changing prevalent PRRSV variant is of great interest. In this study, based on an infectious cDNA clone of an attenuated TypeⅡPRRSV strain pCSA, which was further manipulated by inserting polylinker (PacI,SwaI,AscI)between ORF1 and ORF2, and the high pathogencity PRRSV cDNA clone pJX143, we replaced the coding sequence of pCSA structural protein(s) and/or non-structual protein with those of the HP PRRSV to develop a series of chimeric clones. Upon transfection of chimeric clones cDNAs into Marc-145 cells, typical PRRSV cytopathic effects were observed. This study provided a valuable tool to develop the chimeric PRRSV as vaccine candidate offering cross-protection to HP PRRSV strains. Furthermore the infectious chimeric cDNA clone provides a powerful tool to molecular dissection of the mechanism of pathogenesis of the increasing- virulence of the on-going prevalent PRRSV in China.