| Bovine viral diarrhea virus(BVDV),an infectious agent that can infect a variety of cloven-hoofed animals,causes bovine viral diarrhea/mucosal disease(BVD/MD)which results in significant economic losses.Innate immunity plays an important role during viral infection,and its molecular mechanisms of interaction with virus can provide new ideas for the prevention and control of the animal diseases.Interferon(IFN)system is an important contributor of innate immunity with IFN inducible proteins which are the effector of interferon system and have a direct antiviral role.Mx protein is the key component of interferon induced antiviral state.Understanding the mechanisms of Mx proteins against viral infection will greatly elaborate on the interaction between virus and host.From the perspective of the interaction between BVDV and the host,our research focuses on the transcriptome changes of MDBK cells infected with BVDV and the impact of bovine Mx1 protein on the replication of BVDV.The results were shown as follows.1.Isolation and identification of two BVDV isolates,BVDV BJ-2013 and BVDV BJ-2016(1)In this study,the two BVDV isolates,one isolated from clinical bovine serum and second from fetal bovine serum,were identified by RT-PCR,cell culture and indirect immunofluorescence assay(IFA).The two isolates,named BVDV BJ-2013 and BVDV BJ-2016,belong to 1a and 1b genotype according to the genotyping analysis based on BVDV 5'UTR,respectively.(2)The alignment of amino acid sequences showed that 12 amino acids(LGEGNWLVNADR)insertion was found in the NS2 protein of BVDV BJ-2013 isolate.The homology analyses suggested that BVDV BJ-2013 isolate shared the highest nucleotide and amino acid homology with BVDV 08GB44-1 strain isolated from South Korea,whereas BVDV BJ-2016 isolate had the highest nucleotide and amino acid homology with BVDV HJ-1 strain isolated from China.The phylogenetic analysis based on the nucleotide sequences of BVDV polyproein showed that BVDV BJ-2013 shared the same evolutionary branch with BVDV 08GB44-1 and Oregon strain which suggested that BVDV BJ-2013 was closely related to 08GB44-1.The simultaneous clustering of BVDV BJ-2016 with BVDV HJ-1,PJ and 08GB45-2 in same evolutionary branch indicated that all these isolates were closely related to one another.2.The comparative analyses of transcriptome changes in MDBK cells infected with BVDV(1)The comparative analyses of transcriptome changes in MDBK cells infected with BVDV BJ-2016 was performed.The transcriptome analyses showed that 53929 transcripts were obtained and a total of 24616 reference genes were annotated.The prediction of new transcripts and new genes suggested that 26651 new transcripts and 2359 new genes were obtained.(2)The analyses of differential gene expression showed that 1297,1732,3072 and 1877 differentially expressed genes(DEGs)were screened in the comparative group of Mock vs MBV2h?Mock vs MBV6h?Mock vs MBV12h?Mock vs MBV24 h,respectively.The enrichment analyses of DEGs showed that BVDV infection induced the up-regulation of lipid metabolism related genes,which indicated that BVDV infection might change the metabolic network of cells infected with BVDV;BVDV infection also led to the down-regulation of F3?C3?C1R and other genes of the complement and coagulation cascade pathway and the significant changes of the expression of some antiviral genes(such as ISG15,IFITM1 and Mx1),which suggested that the complement system may play an important role in BVDV infection and the inhibition of innate immunity after BVDV infection may contribute to the formation of BVDV infection.3.Bovine Mx1 protein inhibited BVDV replication(1)Through lentivirus infection,the MDBK cell lines of the overexpression and knockdown of Mx1 gene were established.The identification results showed that the expression of Mx1 gene was up-regulated 5.15 times in the cells of overexpressed Mx1 gene and the gene knockdown efficiency in the knockdown cell line of Mx1 gene was up to 93.03%.The effect of bovine Mx1 protein on BVDV replication was verified by detecting the virus loading in the culture supernatant of Mx1 gene overexpression and knockdown cells.The results showed that the overexpression of Mx1 gene significantly inhibited BVDV replication(P<0.01),while the knockdown of Mx1 gene significantly promoted BVDV replication(P<0.01).These results suggested that bovine Mx1 protein inhibited the replification of BVDV.(2)The strand-specific real-time fluorescence quantitative RT-PCR was established and used for detecting the changes of BVDV positive and negative-stranded RNA in MDBK cells with Mx1 gene overexpressed and knockdown after BVDV infection.The results showed that the overexpression of Mx1 gene caused the reduction of BVDV positive and negative-stranded RNA in the cells.The significant reduction of the positive-stranded RNA was found between 12 h and 24 h after BVDV infection(P<0.05 or P<0.01)and that of the negative stranded RNA was observed between 6h and 36 h after BVDV infection(P<0.05 or P<0.01).The knockdown of Mx1 gene resulted in the increase of BVDV positive and negative-stranded RNA in the cells.Significant increase in BVDV negative-stranded RNA was observed between 6h and 36 h after BVDV infection(P<0.01),whereas BVDV positive-stranded RNA only increased significantly at 24 h and 36 h after BVDV infection(P<0.05).These findings indicated that Mx1 protein had a significant inhibitory effect on the replication of BVDV and its positive and negative-stranded RNA.In this study,two BVDV strains,named BVDV BJ-2013 and BVDV BJ-2016,were isolated and identified;the comparative analyses of transcriptome changes of MDBK cells before and after infection with BVDV and the role of some DEGs were identified;strand-specific real-time fluorescence quantitative RT-PCR of BVDV was established;It was found that bovine Mx1 protein significantly inhibited the replication of BVDV and its positive and negative RNA.The results provide a scientific basis for the further study of the interaction between BVDV and its host,and for the elucidation of the anti-BVDV mechanisms of bovine Mx1 protein. |
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