The MiRNA Expression Profile In NDV-infected Avain Macrophages And The Roles Of Gga-miRNA155 During NDV Infection

Newcastle disease is an avian highly contagious acute viral infectious disease caused by virulent strain of Newcastle disease virus(NDV),can affect most nonimmune brids,and spread mammals across species.The dominant genotype of NDV in China is genotype VII,brings enormous economic losses to the global poultry industry.At present,there are some limitations for the Newcastle disease mainly relying on vaccination in the prevention,which are capable of preventing clinical disease and death caused by infection with NDV strains to a certain extent,but do not completely prevent viral replication and shedding.microRNA(miRNA)are noncoding small RNAs of 18~25 nt in length,encoded animals,plants,virus,involved in tissue development,cell differentiation,proliferation and apoptosis.In addition,miRNA are crucial regulatory RNA molecules in gene expression,interference,assembly and release of infected virus.We performed a deep sequencing analysis of miRNA libraris of virulent NDV-infected and lentogen NDVinfected macrophages cells.The function and usable of a special miRNA-miRNA155 was exproed after the Q-PCR identification of the many variant miRNA in the highthroughput filter.The study is divided into the following parts:1.The miRNA expression profile of macrophages cells infected with strong and weakly toxic Newcastle disease virusInfected the macrophages cells with the strong(NA-1)and weakly(La Sota)toxic Newcastle disease virus.Mock infection cells were used as control.The infected and mock-infected cells were harvested at 24 h and 48 h post-infection(hpi)with Tripure regent(Roche)respectively for total RNA isolation and the qualified samples were conducted deep sequencing.For the identification of variant miRNA,miRNA expression profiles in the cells of macrophages cells infected by different strains at the same time point and the same strain at different time points was analysis(p value<0.01,singal value>100,diffrence value |Log2|>2).The results show that 2227 miRNA and 1954 express in the macrophages cells infected with NA-1 harvested at 24 hpi and 48 hpi respectively.Moreover,1950 miRNA and 1914 miRNA expressed in the macrophages cells infected with La Sota harvested at 24 hpi and 48 hpi respectively.2.Analysis of differential expression miRNAThe analysis of the variant miRNA indicated that 233 and 263 variant miRNA are present in HD11 cells with the infected of NA-1 at 24 hpi and 48 hpi compared with the miRNA in control cells.122 miRNA are co-expression in the cells by the infection of NA-1.There are 158 and 153 variant miRNA in the HD11 cells with the infection of La sota at 24 hpi and 48 hpi compared with the miRNA in control cells.35 miRNA are co-expression.Specially,18 miRNA were detected whether in macrophages cells infected with NA-1 or La Sota at 24 hpi or 48 hpi.Infected the HD11 cells with the strong(NA-1)and weakly(La Sota)toxic NDV.Mock infection cells were used as control.The infected and mock-infected cells were harvested at 24 h and 48 h postinfection(hpi)with Tripure regent(Roche)respectively for total RNA isolation.The reverse transcription(RT-PCR)was carried out with the total RNA.The cDNA was identified for the filter of differential expression miRNA by the fluorescent quantitative dye method.The results show that 2 miRNA performed significant difference in expression and show up-regulation expression.The biological function and pathways which the target genes involved in were perfomed by Go and KEGG.3.The effect of miRNA155 on the replication of NDV in the macrophages cellsInfected the HD11 cells with different dose of NA-1,mock infection cells were used as control.The infected and mock-infected cells were harvested at 24 h and 48 h post-infection(hpi)with Tripure regent(Roche)respectively for total RNA isolation.The RT-PCR was conducted for the cDNA following the miRNA reverse transcription kit.Detected the expression of miRNA155 and NDV in the HD11 cells with different dose of NDV at different time using the cDNA as template.For the effect of miRNA155 on the replication of NDV in the HD11 cells,the observation of cytopathic phenomenon was and the detection of the miRNA155,NDV and cytokine were carried out after the over-expression and inhibition of the function of the miRNA155 by the transfection of mimic,inhibitor.Finally,in the case of transfection of mimic and inhibitor,there were no cytopathic phenomenonwas and difference in content of NDV but the expression of certain cytokines.Moreover,miRNA155 can cause significant expression of TAB2 genes which is related to the p38 MAPK pathway.The above results show that the changes in the expression of miRNA155 are related to the inflammatory response caused by virus infection and the p38 MAPK pathway,inhibit the cytopathic phenomenon or the virus assembly and budding,but miRNA155 can not directly affect the replication of NDV in macrophages cells.