Study On The Molecular Mechanism Of Bta-miR-677 Regulation Interferon Pathway Gene Affecting The Proliferation Of Caprine Parainfluenza Virus Type 3

The Caprine parainfluenza virus type 3?CPIV3?isolated a new strain of virus from the goats,which belongs to the single-stranded negative chain of the respiratory virus?Respirvirus?of Paramyxovirinea.CPIV3 can be transmitted through horizontal transmission of the healthy sheep around the enclosure,causing the diseased sheep to appear runny nose,high fever and mental depression and other clinical symptoms,the diseased sheep after a caesarean section found that their lungs and upper respiratory tract appear more serious lesions,and can continuously detect viremia and detoxification,which shows that CPIV3 has a strong infectious and pathogenic.Therefore,the threat posed by CPIV3 to the healthy development of sheep farming industry should be paid attention to.MicroRNAs?miRs?is a single-stranded RNA with a length of about 19-25 nt that can target binding certain protein molecules to form a silent complex?RISC?that inhibits the translation of m RNA or makes m RNA degrade,thus mediating gene expression.Studies have shown that viral infections can cause changes in the expression spectrum of host miRs,which can regulate natural immune response and viral infection.Therefore,miRs play an important role in antiviral infections and natural immune responses.To study the expression changes of miRs in CPIV3 infected MDBK cells,the previous study screened a large number of different expressions of miRs through high-throughput sequencing techniques,in which bta-miR-677 was significantly increased in the sample of infected cells of CPIV3.Bioinformatics software analysis such as GO,KEGG and Target Scan has found that bta-miR-677 may play an important role in type I interferon,inflammatory factors and apoptotic pathways.Therefore,this study will take bta-miR-677 in MDBK cells over/inhibited expression,and explore bta-miR-677 regulation of type I interferon pathway inhibited the proliferation of caprine parainfluenza virus molecular mechanism,for the study of bta-miR-677 based on the prevention and control of CPIV3 molecular drugs to provide an important scientific basis.1.Effect of Bta-miR-677 to CPIV3 replicationIn order to explore the effects of bta-miR-677 on CPIV3 proliferation,this study transfected bta-miR-677 mimics and inhibitor to MDBK cells,so that bta-miR-677 in MDBK cells was overexpressed or inhibited after inoclating CPIV3 virus fluid,and detected the virus load by q RT-PCR and TCID₅₀.the results showed that the transfection bta-miR-677mimics significantly inhibited CPIV3 proliferation,and the virus copy number in the transfection group was 8-16 times lower than that in the control group,and the virus copy titer(10^2.2 TCID₅₀-10^2.6TCID₅₀)in the transfection group was significantly lower than that in the control group(10^3.5TCID₅₀-10^4.3TCID₅₀).on the contrary,transfection bta-miR-677inhibitor significantly promoted CPIV3 proliferation.the virus copy number in the transfection group was 4-8 times higher than that in the control group,and the virus copy titer(10^3.5TCID₅₀-10^4.1TCID₅₀)was significantly higher than that in the control group(10^3.5TCID₅₀-10^4.2TCID₅₀).The results showed that bta-miR-677 could inhibit the proliferation of CPIV3.To investigate bta-miR-677 mechanisms that inhibit CPIV3 proliferation,bta-miR-677mimics/bta-miR-677inhibitor were transfected into MDBK cells to detect the transcript levels of interferon?Interferon,IFN?and interferon-stimulated genes?Interferon-stimulated genes,ISGs?.The results showed that the transcription level of type I interferon in the transfected bta-miR-677mimics group was significantly up-regulated,the IFN-?/IFN-?of the transfected group was up-regulated by about 2-4 times,and the IFI6?OAS1Y?OAS1Z?RSAD2?MX1?MX2 was up-regulated by 2-16 times.on the contrary,the transcription level of type I interferon in the transfection bta-miR-677 inhibitor group was significantly down-regulated,and the IFN-?/IFN-?of the transfection group was down-regulated by about 2-4 times,and the IFI6?OAS1Y?OAS1Z?RSAD2?MX1?MX2 compared with the control group down-regulated above 2 times.The results show that bta-miR-677 can promote the formation of I IFN and ISGs,and then inhibit the replication of CPIV3.2.Bta-miR-677 molecular mechanism to inhibit the replication of CPIV3To study the molecular mechanisms of bta-miR-677 to promote IFN-?or IFN-?,we learned from the analysis of miRs target gene prediction software such as Target Scan that bta-miR-677 can target MAVS by establishing a double-luciferase reporting system.Experiments such as q RT-PCR and Western blot are validated.The results showed that the luciferase activity of MAVS was lower than that of the control group by 2-3 times,there was no significant change in transcription levels,and the protein level of MAVS was significantly reduced.The results showed that bta-miR-677 inhibited MAVS expression at the protein level.Three si-RNAs are designed for the conservative region of MAVS,and the three si-MAVS are transfected to MDBK cells to detect the transcription levels of each gene by q RT-PCR.The results showed that transfection of si-MAVS was significantly reduced in MAVS transcription and expression?P<0.001?,improved the transcription level of type I IFN by 4-6 times,and IFI6,OAS1Y,OAS1Z,RSAD2,MX1,MX2 also increased by 4-17 times.Transfection of si-MAVS transfected to MDBK cells after inoculation of CPIV3,through q RT-PCR and TCID₅₀detection found that the number of copies of the transfection group CPIV3 was significantly lower than the control group 6 times,the virus replication effect(10^1.8TCID₅₀)significantly lower than the control group(10^3.5TCID₅₀).The results show edith of MAVS gene knock-down can raise the transcription levels of type I IFN and IFI6,OAS1Y,OAS1Z,RSAD2,MX1,MX2,and inhibit the replication of CPIV3.3.Establish stable MDBK cell lines that have been expressed over bta-miR-677In order to obtain a stable expression bta-miR-677 MDBK cell line,this study used PCR technology to amplify pri-bta-miR-677 fragments from the MDBK cell genome,amplify the enhanced green fluorescent p EGFP-N1 plasmids,and pre-clone p MD18-T vector,After the enzyme cut identification and sequencing are correct,they are cloned into the PCDH-CMV-MCS-EF1-Puro vector,respectively,to construct the slow virus recombinant plasmid PCDH-miR-677-EGFP containing the bta-miR-677 gene.The results show that PCDH-miR-677-EGFP over-expressed plasmid construction was successful,and the sequence ratio was up to100%.PCDH-miR-677-EGFP and two packaging plasmid PSP,PMD co-transfected 293FT cells,preparation of the bta-miR-677 gene recombinant slow virus VPCDH-miR-677.The harvested slow-virus VPCDH-miR-677 was inoculated with MDBK cells,screened by Puromycin for 10 generations,and the infection efficiency and q RT-PCR detection bta-miR-677 expression were detected with a fluorescence microscope.The results showed that a large amount of green fluorescence was observed,and bta-miR-677 was overexpressed 128 times in the MDBK-miR-677 cell line compared to normal MDBK.Inoculation CPIV3 in the MDBK-miR-677 cell line,while the establishment of CPIV3 infection normal MDBK as a control group,through q RT-PCR and TCID₅₀ detection.The results showed that the number of copies of CPIV3 was 6-64 times lower than that of the control group,and the viral replication effect(10^1.7TCID₅₀-10^2.1TCID₅₀)was significantly higher than that of the control group(10^3.3 TCID₅₀-10^4.7TCID₅₀).The results showed that bta-miR-677 was stable lysed over the MDBK-miR-677 cell line,which inhibited the replication of CPIV3.This lays the foundation for the next step in the study of the mechanism of the mechanism by which bta-miR-677 is infected with MDBK cells in CPIV3.4.Establishment of CPIV3 infected guinea pig animal modelTo study whether bta-miR-677 inhibited the proliferation of CPIV3 in vitro,the study injected the hyperexpression plasmid PCDH-miR-677-EGFP by 80 mg/kg muscle per guinea pig and extracted RNA from the guinea pig's lungs and trachea after 3d.By q RT-PCR test,injection OF PCDH-miR-677-EGFP can make miR-677 express168 times in the guinea pig body.After injecting the guinea pig with PCDH-miR-677-EGFP plasmid 3d,300?L per rat inoculated by a drip of the nose has been diluted to 10^-5.0/100?L CPIV3 virus solution A drug-taking group and a normal control group were set up to observe the symptoms of guinea pigs every day,and after 7d all guinea pigs were euthanized,the lungs and trachea were taken with one RNA extraction for q RT-PCR testing,and the other for pathological histological examination.A caesarean section observed the guinea pig and found that the 7d lung of the attack group after the attack appeared hyperemia,real change and swelling symptoms,while the immune attack group guinea pig's lungs only slightly swollen.QRT-PCR testing found that the number of copies of viral nucleic acid detected in the lungs of the experimental guinea pigs in the attack group was 6-64 times higher than that of the control group,while the number of copies of lung virus nucleic acid in the drug attack group was only 6-12 times higher than that of the control group.Pathological histology observation and identification found that the lung tissue of the experimental rat of the attack group showed more obvious changes such as pulmonary interval widening,alveoli fusion,inflammatory cell immersion and so on,while the drug attack group guinea pig had only slight lesions.The results showed that BTa-miR-677 can be expressed in guinea pigs by intramuscular injection PCDH-miR-677-EGFP,and the proliferation of CPIV3 can be effectively inhibited in guinea pigs.In summary,this study verifies the important role of bta-miR-677 in the natural immune antiviral process,identifies a target gene MAVS for bta-miR-677,and reveals bta-miR-677 regulation I The molecular mechanism of type I interferon and inflammatory factor expression confirms the molecular mechanism of bta-miR-677 inhibits the proliferation of CPIV3,and provides a scientific basis for the further development of bta-miR-677 as a drug for the treatment of CPIV3 infection.In summary,this study verifies the important role of bta-miR-677 in the natural immune antiviral process,identifies a target gene MAVS for bta-miR-677,and reveals bta-miR-677regulation the molecular mechanism of type I interferon and inflammatory factor expression confirms the molecular mechanism of bta-miR-677 inhibits the proliferation of CPIV3,and provides a scientific basis for the further development of bta-miR-677 as a drug for the treatment of CPIV3 infection.