| Porcine Reproductive and Respiratory Syndrome(PRRS)is an infectious disease of pigs caused by Porcine Reproductive and Respiratory Syndrome Virus(PRRSV).PRRSV belongs to the family arterygidae,arterygidae and arterygitis viruses.It is a single-stranded plus-stranded RNA virus with a gene length of about 15 kb,which can be divided into European type and north American type according to the genotype.The disease broke out in North America and Europe in the late 1980 s and early 1990 s,then spread to Asia and other regions,causing huge economic losses to the world pig industry.During transcription,PRRSV forms a series of subgenomes(sgRNA)with a common 5 'UTR and 3' UTR.The intermediate structure of these sgRNA is an open reading frame(ORF)of one or more proteins at the 3'end of the viral genome.That is,L-TRS and TRS(B-TRS)located in the genome play an important role in the transcription process of sgRNA,but the variable PRRSV B-TRS site has not been fully revealed.In this paper,the distribution,type and function of PRRSV b-trs were analyzed using high-throughput sequencing technology and virus reverse genetic technology,and the results were as follows:(1)PAM cells infected with PRRSV HuN4 strain were collected 48 h after infection for high-throughput sequencing.The results showed that there were 168 effective B-TRS transcription sites in PRRSV HuN4 genome.The analysis of the base composition of B-TRS showed that B-TRS in PRRSV HuN4 had a complex diversity.Although the 6 sites of B-TRS could be any type of base,they tended to be TTAACC combinations.The more similar the base composition of B-TRS was to L-TRS(TTAACC),the higher the transcription abundance.(2)PRRSV HuN4 strain was infected with PAM cells and MARC-145 cells,respectively,and samples were collected for high-throughput sequencing at 6 h,12 h,24 h,36 h and 48 h after infection.The results showed that the transcripts of PRRSV HuN4 strain(6 classical sgmRNAs)changed with time;6 h after infection,the abundance of virus sgmRNA was lower than the detection threshold;at 12 h after infection,the ratio of the abundance of each transcriber was 2.25 : 1 : 1 : 2.5 : 2 : 16;at 24 h after infection,the ratio of the abundance of each transcriber was 1.2 : 1 : 3.4 : 10.8 : 8 : 35;at 36 h after infection,the ratio of the abundance of each transcriber was 1.06 : 1 : 1.24 : 3.38 : 3.46 : 15.36;at 48 h after infection,the ratio of the abundance of each transcriber was 1 : 1 : 1.35 : 3.45 : 3.69 : 15.27.PRRSV HuN4 strain has the same ratio of transcriptons except ORF6 in different cells.The ratio of virus transcriptional daughter abundance in PAM cells was 1 : 1.09 : 1.46 : 3.28 : 5.03 : 17.91.In addition,sgmRNA structure analysis showed that PRRSV HuN4 strain sgmRNA encoded at least 13 new ORF.(3)Using reverse genetic manipulation,RFP gene and RFP gene with TRS6 were inserted between ORF7 and 3' UTR in PRRSV HuN4 genome by reverse genetic manipulation.Both infectious clones could rescue the recombinant virus,but HuN4-RFP strain did not express red fluorescent protein,The same sgmRNA was used for RFP and ORF7.Hun4-TRS6-RFP strain can express red fluorescent protein and transcribe a single sgmRNA,and the expression level of RNA is higher than that of ORF7.However,the red fluorescent protein expression of Hun4-TRS6-RFP strain was not stable during passage,and the base 1 to 517 of the 4th generation RFP gene was deleted.To sum up,the number of B-TRS of PRRSV HuN4 strain is far more than the 12 known strains,and the sequence is diverse;sgmRNA transcriptional ratio fluctuated at different time points after virus infection,but there was little difference among different cells;sgmRNA of PRRSV HuN4 strain encodes at least 13 new ORF.By using reverse genetic manipulation,the role of B-TRS in sgmRNA transcription was analyzed,and it was found that the presence of B-TRS plays an important role in the formation of PRRSV sgmRNA. |
PDF Full Text Request