| Porcine Reproductive and Respiratory Syndrome(PRRS)is a serious contagious infectious disease caused by Porcine reproductive and respiratory syndrome virus(PRRSV),The epidemic disease can cause reproductive disorders in sows and respiratory symptoms in piglets,commonly known as "Blue-eared disease".PRRSV is a member of the family Arteriviridae that belongs to the order Nidovirales.The disease was first reported in Europe and the United States in the early 1990 s,while was first reported in China in 1996.In 2006,the highly pathogenic porcine reproductive and respiratory syndrome virus(HP-PRRSV)broke out.Since 2012,NADC30-like PRRSV and other strains had appeared one after another.At present,PRRS has become an important issue affecting swine industry all over the world.Subgenomic m RNA(sg m RNA)of PRRSV is synthesized by discontinuous transcription mechanism,and transcription regulatory sequence(TRS)plays an important role in this process.TRS can be divided into the Leader of virus genome(Leader-TRS)and the TRSs of downstream coding Body(Body-TRS).In order to deeply understand the role of TRSs in viral sg m RNA transcription and translation,the Leader-Body binding sites of HP-PRRSV attenuated vaccine strain Hu N4-F112 and European LV strain were firstly identified and analyzed in detail.The sequences of the Leader-TRS,each Body-TRS and their relative positions were also identified in the genome.It was found that there were great differences in the sequences and flanking sequences of the core oligonucleotides of the two strains,but the two bases of the core oligonucleotides in 3? terminal were identical.In this study,based on the full-length infectious clone platform of Hu N4-F112-EGFP expressing green fluorescent protein(EGFP),we constructed a series of full-length infectious clones against TRS6 mutations and constructed full-length infectious clones replacing TRS6 with other Body-TRSs.Through virus rescue and analysis of biological characteristics of virus,it was found that full-length mutant clones with two bases at 3? terminal of core oligonucleotide of TRS6 could not be rescued,the full-length mutant clones without flanking sequences at 5? or 3? terminals alone could also be rescued,however,full-length mutant clones with both flanking sequences deletion of 5? and 3? terminals could not be rescued.Compared with the parental virus,the titer of the virus rescued after flanking sequences deletion was lower.In addition,the viruses can be rescued by replacing TRS6 of Hu N4-F112-EGFP with TRS2,TRS3 and TRS7,which have basically similar growth characteristics with the parental virus,but the titer of recombinant virus containing TRS2 is relatively low.Nevertheless,the virus rescue of mutants with TRS6 substituted to TRS4 or TRS5 was failed.By predicting and analyzing the secondary structures of different inserted Body-TRS regions,it was found that the core oligonucleotides of TRS4 and TRS5 were located on the single chain structure and stem structure of stem-loop in the secondary structure formed by the insertion site region and flanking sequences.This relative position may affect the base-pairing process between TRS core oligonucleotides and the Leader-TRS,and then could affect the sg m RNA transcription of the virus,which can hinder the rescue of virus.The specific detailed mechanism needs more and further research.In conclusion,this study identified the sequences of Leader-TRS and a series of Body-TRS of PRRSV-1 and PRRSV-2 and their relative positions in the genome.It is proved that the primary sequence of TRS cassette has an influence on its regulation.At the same time,it is proved that four different Body-TRS cassettes in ORF1 b and ORF2 a gene regions can guide the stable and efficient expression of genes.This study laid a foundation for further analyzing the transcriptional regulatory elements of PRRSV and developing multivalent recombination vaccines using Hu N4-F112 vaccine strain as live viral vector in the future. |
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