| Porcine reproductive and respiratory syndrome (PRRS) first reported in the late 1980s, is recognized as a serious swine disease and is characterized with either reproductive failure in pregnant sows, or respiratory tract distress particularly in sucking pigs. PRRS has brought great economic losses to the swine industry every year. Vaccine is the most effective measure to counteract PRRS, but there are still lots of problems with available commercial vaccines, for example, the low protection efficiency to heterologous strain, and safety. In this study, based on the reverse genetic system of low virulent PRRSV which was attenuated by serial passage in MARC-145, we constructed an improved DNA-launched reverse genetic system. Then, based on sequence alignment analysis, we got the consensus amino acid sequence of GP5 and codon optimized the coding sequence using codon preferred in pigs. Codon optimized ORF5 was swapped into the improved reverse genetic system, through which we dissected the function of virus genome coding sequence in virus replication, viral protein expression, and laid a theoretical foundation for the development of PRRSV vaccine.Firstly, a DNA-launched reverse genetics system (RGS) was constructed and optimized. As far as I know, most PRRSV RGS are based on RNA transfection. There were some disadvantages with RNA based RGS, such as stability, low virus rescue efficiency, complicated in vitro transcription and high cost. In this study, in order to improve the virus rescue efficiency and stability of cDNA clone, T7 promoter was replaced with hCMV promoter, the optimal transcriptional initiation site of hCMV was analyzed, and a ribozyme element of delta hepatitis virus and BGH terminal signal at the 3'end of ployA. Five cDNA clones were constructed by site-direct mutation and splicing overlapping extension polymerase chain reaction. And the distances between the TATA box of hCMV and 5'viral genome were 21nt, 23nt, 25nt and 27nt respectively. The virus rescue efficiency was determined by the time when CPE can be observed, the level of N protein detected by indirect fluorescence assay and virus supernatant titers at different time phases. When the distances were 23nt and 25nt, CPE could be observed at 84 hours post transfection (hpt), and virus supernatant could be titrated by TCID50. However, when the distances were 21 and 27, typical CPE could be observed by passaging virus supernatant in fresh MARC-145 cell, and virus supernatant could not be titrated by TCID50. So, it was indicated that the optimal transcriptional initiation site was between 23 nt and 25 nt. Furthermore, pAJX25HB was constructed by adding a delta hepatitis virus ribozyme element based on pAJX25. In comparison with parental clone, typical CPE could be observed 12h earlier, and the virus titers at different time phase were 2 logs higher. To sum up, it indicated that modifications of 5'and 3'of viral genome could improved virus rescue efficiency remarkably.Subsequently, we tried to re-code the GP5 consensus sequence using more preferred codon in pigs for the purpose to raise the level of GP5 expression and improve cross protection efficiency. Also, we wanted to investigate the function of GP5 coding sequence in the process of virus replication by codon optimization. Fifty northern American PRRSV strains, including 5 typical strains of PRRSV abroad, 5 typical strains of domestic classical PRRSV and 5 typical strains of high pathogenic PRRSV, were selected as samples, and the genomic sequences were obtained from Genbank. Based on the sequence analysis of GP5, we found that the viruses belonged to different subgroup and could represent heterologous strains. GP5 consensus sequence was maintained by alignment of the GP5 amino acid sequences of 15 typical PRRSV strains, and codon optimized by online software DyNAVcaS(http://miracle.igib.res.in/dynavac/). Finally, the coding sequence of codon optimized GP5 consensus sequence was synthesized by Genscript Co.. pAJX25HB was used as backbone, pAJXM5, pAJXM5S1, pAJXM5S2, pAJXM5S3 and pAJXM5S4 were constructed by swapping the ORF5, and the regions of 1-555 nt,1-180 nt,181-555 nt,132-555 nt and 104-555 nt were codon optimized respectively. Then, viruses were rescued by DNA transfecting MARC-145 cell and BHK-21 cell. The results of DNA transfection showed that PRRSV could be rescued when the codon optimized region was between 104 nt and 555 nt of ORF5. Then, virological characteristic vM5S2 were identified by virus multi-step growth curve and RT-PCR. The results of RT-PCR and sequencing indicated that no reversal mutation emerged from P1 to P3 virus. And the growth characteristic of vM5S2 was similar to parental virus, which indicated that codon optimization didn't change the characteristic of virus replication. PRRSV could not be rescued from pAJXM5 and pAJXM5S1. However, subgenomic mRNA5, 6, and 7, and major structural proteins GP5 and N were detected by RT-PCR and indirect fluorescence assay,Which showed that the transcription and translation were not destroyed by codon optimization. Therefore, we could get a conclusion that, not only GP5 amino acid sequence played significant role in virus life cycle, but also N terminal 103 nt was essential for virus replication. On the other hand, it needs further investigation to dissect the mechanism.
|
PDF Full Text Request