MiRNAs And MRNAs Expression Profiles Analysis Of Newcastle Disease Virus Infected Chicken Embryos And Screening Of Antiviral Genes

Newcastle disease(ND)caused by the virulent Newcastle disease virus(NDV),which could lead serious damages in the poultry industry.The main clinical symptoms are dyspnea,high fever,diarrhea,nervous disorders,mucous membranes and pulp blooding with high mortality and contagiousness.Since the disease was discovered in 1926,it has already experienced four major pandemics in the world and brought huge economic losses to the poultry industry.Due to multiple genotypes and rapid mutations of NDV and immune failure,traditional vaccines have insufficient protection and cannot effectively control ND.Therefore,it is urgent to explore new strategies for the prevention of this disease.Transcription of gene mRNAs and microRNAs(miRNAs)plays an important regulatory role in virus-infected hosts.The study of miRNAs and mRNAs transcription profiles in post infection is helpful to elucidate the regulation relationship between NDV infection and host,which may provide new ideas to control ND.The research contents and results of this project are as follows:1.Sequencing analysis of miRNAs expression profile of chicken embryonic visceral tissues infected with NDVThe miRNAs expression profiles of chicken embryos infected with NDV velogenic strain(F48E9)and lentogenic strain(La Sota)were obtained by small RNA sequencing.Screening miRNAs expression profiles showed that F48E9 infection caused 33 up-regulated and 31 down-regulated miRNAs,while La Sota infection induced 36 up-regulated and 25 down-regulated miRNAs.Among those miRNAs,there were 27 up-regulated and 22 down-regulated miRNAs between La Sota and F48E9 infections.The differential miRNAs,gga-miR-34a-5p,gga-miR-375,gga-miR-122-3p,gga-miR-187-3p,gga-miR-124a-3p,gga-miR-203 a,gga-miR-205 a,gga-miR-183,gga-miR-9-3p and gga-miR-451,were selected for verification by real-time quantitative PCR(RT-qPCR),the expressed levels of those miRNAs were similar to the results from small RNA sequencing.The miRNA target genes were predicted and analyzed by bioinformatics,which suggested that differentially expressed miRNAs might involve in regulating cell growth,metabolism,cell cycle,immune and inflammatory responses.Intensive study of gga-miR-375 with better antiviral effect revealed that gga-miR-375 overexpression could significantly inhibit the proliferation of NDV.Both RT-qPCR,dual luciferase repoters and western blot assays showed that gga-miR-375 could target the NDV M gene to inhibit the proliferation of the virus.In addition,gga-miR-375 was also found to be secreted into the cell supernatant after overexpression,and it was found that exogenous gga-miR-375 still inhibited viral proliferation after the NDV-infected cell treated with the collected supernatant.These results indicated that gga-miR-375 has a post-transcriptional regulation of NDV M gene and can be secreted to the extracellular to regulate the surrounding cells for antiviral action.2.Transcriptomic sequencing analysis of visceral tissues of NDV-infected chicken embryos and antiviral gene screeningTo reduce the range of target genes of differentially expressed miRNAs,RNA-Seq sequencing technique was used to analyze mRNAs transcriptome of NDV-infected chicken embryonic visceral tissues.The matching rates of chicken genome in the control,F48E9 infection,and La Sota infection groups were 70.05%,68.55%,and 71.76%,respectively.Compared with the control group,F48E9 and La Sota infection caused changes at mRNA expression levels of 2035 and 1604 genes,respectively.Compared with F48E9 infection,La Sota infection caused 360 up-regulated and 668 down-regulated genes.The differentially expressed genes(DEGs),ISG12(1),ISG12(2),IFI35,Mov10,DHX58,Mx,OASL,TGM2,C8ORF4 and RUNX2,were selected for verification by RT-qPCR,and the expressed levels of those mRNAs were consistent with the results from RNA-Seq.The GO and KEGG enrichment analysis of DEGs,such as IFI35,NMI,Mx,OAS*A,STAT1 and IFN?,revealed multiple key regulatory factors involved in the regulation of viral replication via different signaling pathways.Further studies on IFI35 revealed that the gene was poorly conserved in different species,but it was well conserved in birds.The tissue expressed profiling showed that IFI35 presented the highest expression in the lungs of healthy chickens,following by brain and bursa of fabricius,and its distribution in the tissue after virus infection was similar to the viral tissue distribution.Overexpression of IFI35 could significantly inhibit NDV proliferation.The transcriptional changes of viral genes were detected by RT-qPCR,which revealed that NP,P,F and HN genes were significantly decreased after overecpression of IFI35.Western blots results showed that the expression of V protein was significantly inhibited when IFI35 was co-expressed with viral genes.In addition,the expressed trends between IFI35 and IFN?/?/? were similar based on RT-qPCR,and IFI35 overexpression could promote the expression of multiple genes in the signaling pathway,inducing IFNs production.Those results preliminarily proved that IFI35 played an antiviral effect by inhibiting the expression of V proterin and subsequently increasing the production of IFNs.3.miRNAs-mRNAs association analysis and antiviral gene screeningIn order to obtain the correlation and universality of the regulations between different types of genes during NDV infection,we further analyzed the association between differentially expressed miRNAs and mRNAs in visceral tissues of chicken embryos infected with virulent or attenuated NDVs respectively.The total of 1069 miRNA-mRNA pairs were obtained.Bioinformatics analysis of these DEGs revealed that most of them involved in the regulation of metabolism and growth,but several genes were involved in immune response,inflammatory response,apoptosis,cytokine expression and etc.Further refining the genes involved in the host immune or inflammatory response pathways and 130 immune-or inflammation-related miRNA-mRNA pairs were screened,such as gga-miR-375-RUNX2,gga-miR-122-3p-MMP13,gga-miR-122-5p-IL17 RD and etc.The predicted regulatory relationship between gga-miR-203 a and TGM2 was further verified.Both RNA-Seq and RT-qPCR results showed that NDV infection can significantly reduce gga-miR-203 a expression,but promote the expression of TGM2.Overexpression of gga-miR-203 a significantly promoted the proliferation of NDV,while overexpression of TGM2 inhibited the replication of NDV.Both RT-qPCR and dual luciferase reporter assays further confirmed that gga-miR-203 a overexpression inhibited the expression of TGM2,conversely,interference with endogenous gga-miR-203 a could promote the TGM2 expression,without effect on the expression of TGM2 with binding site mutations.Those results demonstrated that the host could control NDV by inhibiting the gga-miR-203 a and promoting the expression of the antiviral gene TGM2.4.High-throughput sequencing of viral gene transcriptionBased on the high-throughput sequencing technology,transcripts of viral genes,NP,P,M,F,HN,L,V,and W,were obtained from chicken embryonic tissues infected with different virulent NDV strains.The transcriptional ratios of NP,P,M,F,HN,L,V,and W genes were 24.94%,22%,10.14%,12.65%,8.27%,3.37%,13.37% and 8.21% for F48E9 infection,and 26.76%,8.14%,19.43%,9.42%,15.10%,4.16%,5.22% and 3.85% for La Sota infection.The transcripts of P,V and W were analyzed separately and showed that the ratio of P:V:W was 50.48%: 30.68%: 18.84% for F48E9 infection and 47.3%: 30.33%: 22.37% for La Sota infection,which indicated that the difference in P/V/W transcripts between F48E9 and La Sota was not significant.Further transcriptional changes of viral genes from F48E9 infected bursa of fabricius under different infection time were analyzed.The results showed that the transcriptional ratios of NP,P,M,F,HN,L,V and W genes in the bursa of fabricius were 23.89%,15.21%,9.21%,17.93%,13.51%,4.46%,9.95% and 11.73% for 72 h post infection(hpi),and 25.58%,16.35%,10.15%,15.86%,12.54%,4.14%,9.84% and 5.57% for 96 hpi.The transcriptional ratio of P:V:W was 48.84-49.2%:31.92-32.23%:18.87-18.94% and 48.6-54.54%:29.24-30.63%:16.22-18.77% for 72 and 96 hpi,without significant difference.In order to verify the experimental results,we constructed a method for detecting the transcriptional changes of P/V/W after ND V infection based on high-throughput technology.The changes of P/V/W transcripts at different time point after F48E9 or La Sota infected DF-1 cells were detected using the above method.It was found that the transcriptional ratios of P/V/W were stable under different infection time points,which was similar in the same virus strain but different in NDV strains.And the maximum insertion of G during NDV transcription was detected up to 10.Those results indicated that the editing frequencies of P gene were relatively stable,but differences in different virulent viruses.Compared with attenuated NDV,transcript of virulent NDV V gene was relatively high,with a relatively low transcript of W gene,which suggested that the transcriptional level of V gene may be related to the viral virulence.In summary,on the one hand,miRNA and mRNA expression profiles of NDV-infected SPF chicken embryonic visceral tissues were obtained based on high-throughput sequencing,and the differences in host responses to F48E9 or La Sota infection were analyzed.Differentially expressed miRNAs or mRNAs were analyzed and the DEGs of the host against NDV infection were screened.Further studies confirmed that gga-miR-375 could regulate the expression of NDV M gene to achieve anti-NDV infection.It was also found that IFI35 could promote IFN production by affecting the expression of NDV V protein,and negatively regulate the NDV infection.In addition,correlation analysis of miRNA and mRNA expression profiles in NDV infection identified 130 immune-related miRNA-mRNA pairs.Further experiments confirmed that gga-miR-203 a had post-transcriptional regulation on the expression of TGM2,and the host could inhibit the replication of NDV by promoting TGM2 expression.On the other hand,high-throughput sequencing was used to study the editing frequency of NDV P gene,and established a method for detecting the transcriptional ratio of P/V/W genes.It was confirmed that the editing frequency of P gene was relatively stable under different infection time points,but the transcription of virulent NDV V gene was higher than that of attenuated NDV,which may be one of the reasons why virulent NDV has higher ability to antagonize interferon.In this thesis,we studied the miRNA and mRNA-mediated host-virus interactions in NDV infection,laying the foundation for understanding mechanisms of NDV pathogenicity and host antiviral responses.