MDBK Cells Research Articles

Bovine A20 gene overexpression during bovine viral diarrhea virus-1 infection blocks NF-κB pathway in MDBK cells

Viruses have developed cellular strategies to ensure progeny survival. One of the most interesting is immune camouflage, where the virus triggers a controlled-intensity immune response that prevents total destruction of the infected cell, thus “winning time” for the virus. This study explored the regulatory contexts of the bovine A20 gene during bovine viral diarrhea virus (BVDV)-1 infection, using IL-8 as an immune-response sentinel molecule. Assessments were conducted through RT-qPCR, Western blotting, gene silencing/overexpression, luciferase assays, and the use of pharmacological inhibitors, among other approaches. The results demonstrated that a) BVDV-1 increased A20 levels in Madin-Darby bovine kidney cells, b) increased A20 led to decreased IL-8 expression, and c) the virus affected the NF-κB signaling pathway. Collectively, these data identify bovine A20 as a strong regulator of immune marker expression. In conclusion, this is the first report on BVDV-1 modulating bovine IL-8 activation through the NF-κB/A20 pathway.

Autophagy induced by bovine viral diarrhea virus infection counteracts apoptosis and innate immune activation.

Bovine viral diarrhea virus (BVDV) is an important pathogen of cattle that plays a complex role in disease. There are two biotypes of BVDV: non-cytopathic (NCP) and cytopathic (CP). One strategy that has been used to treat or prevent virus-associated diseases is the modulation of autophagy, which is used by the innate immune system to defend against viral infection; however, at present, the interplay between autophagy and BVDV remains unclear. Madin-Darby bovine kidney cells stably expressing microtubule-associated protein 1 light chain 3B (LC3B) with green fluorescent protein (GFP) (GFP-LC3-MDBK cells) and autophagy-deficient MDBKs (shBCN1-MDBK cells) were constructed. Then MDBK, GFP-LC3-MDBK and shBCN1-MDBK cells were infected with CP or NCP BVDV strains. The LC3-II turnover rate was estimated by western blot, autophagosomes were visualized by confocal microscopy, and ultrastructural analysis was performed using electron microscopy. Autophagy flux was observed using chloroquine as an inhibitor of the autophagic process. The influence of autophagy on BVDV replication and release was investigated using virus titration, and its effect on cell viability was also studied. The effect of BVDV-induced autophagy on the survival of BVDV-infected host cell, cell apoptosis, and interferon (IFN) signalling was studied by flow cytometric analysis and quantitative RT-(q)PCR using shBCN1-MDBK cells. we found that infection with either CP or NCP BVDV strains induced steady-state autophagy in MDBK cells, as evident by the increased number of double- or single-membrane vesicles, the accumulation of GFP- microtubule-associated protein 1 light chain 3 (LC3) dots, and the conversion of LC3-I (cytosolic) to LC3-II (membrane-bound) forms. The complete autophagic process was verified by monitoring the LC3-II turnover ratio, lysosomal delivery, and proteolysis. In addition, we found that CP and NCP BVDV growth was inhibited in MDBK cells treated with high levels of an autophagy inducer or inhibitor, or in autophagy deficient-MDBK cells. Furthermore, our studies also suggested that CP and NCP BVDV infection in autophagy-knockdown MDBK cells increased apoptotic cell death and enhanced the expression of the mRNAs for IFN-α, Mx1, IFN-β, and OAS-1 as compared with control MDBK cells. Our study provides strong evidence that BVDV infection induces autophagy, which facilitates BVDV replication in MDBK cells and impairs the innate immune response. These findings might help to illustrate the pathogenesis of persistent infection caused by BVDV.

Cyclophilin B facilitates the replication of Orf virus

BackgroundViruses interact with host cellular factors to construct a more favourable environment for their efficient replication. Expression of cyclophilin B (CypB), a cellular peptidyl-prolyl cis-trans isomerase (PPIase), was found to be significantly up-regulated. Recently, a number of studies have shown that CypB is important in the replication of several viruses, including Japanese encephalitis virus (JEV), hepatitis C virus (HCV) and human papillomavirus type 16 (HPV 16). However, the function of cellular CypB in ORFV replication has not yet been explored.MethodsSuppression subtractive hybridization (SSH) technique was applied to identify genes differentially expressed in the ORFV-infected MDBK cells at an early phase of infection. Cellular CypB was confirmed to be significantly up-regulated by quantitative reverse transcription-PCR (qRT-PCR) analysis and Western blotting. The role of CypB in ORFV infection was further determined using Cyclosporin A (CsA) and RNA interference (RNAi). Effect of CypB gene silencing on ORFV replication by 50% tissue culture infectious dose (TCID50) assay and qRT-PCR detection.ResultsIn the present study, CypB was found to be significantly up-regulated in the ORFV-infected MDBK cells at an early phase of infection. Cyclosporin A (CsA) exhibited suppressive effects on ORFV replication through the inhibition of CypB. Silencing of CypB gene inhibited the replication of ORFV in MDBK cells. In conclusion, these data suggest that CypB is critical for the efficient replication of the ORFV genome.ConclusionsCellular CypB was confirmed to be significantly up-regulated in the ORFV-infected MDBK cells at an early phase of infection, which could effectively facilitate the replication of ORFV.

Selection and characterization of specific nanobody against bovine virus diarrhea virus (BVDV) E2 protein.

Bovine viral diarrhea-mucosal disease (BVD-MD) is caused by bovine viral diarrhea virus (BVDV), and results in abortion, stillbirth, and fetal malformation in cows. Here, we constructed the phage display vector pCANTAB 5E-VHH and then transformed it into Escherichia coli TG1-competent cells, to construct an initial anti-BVDV nanobody gene library. We obtained a BVDV-E2 antigen epitope bait protein by prokaryotic expression using the nucleotide sequence of the E2 gene of the BVDV-NADL strain published in GenBank. Phage display was used to screen the anti-BVDV nanobody gene library. We successfully constructed a high quality phage display nanobody library, with an initial library capacity of 4.32×105. After the rescue of helper phage, the titer of the phage display nanobody library was 1.3×1011. The BVDV-E2 protein was then expressed in Escherichia coli (DE3), and a 49.5 kDa band was observed with SDS-PAGE analysis that was consistent with the expected nanobody size. Thus, we were able to isolate one nanobody that exhibits high affinity and specificity against BVDV using phage display techniques. This isolated nanobody was then used in Enzyme Linked Immunosorbent Assay and qRT-PCR, and ELISA analyses of BVDV infection of MDBK cells indicated that the nanobodies exhibited good antiviral effect.

Recombinant Swinepox Virus Expressing Glycoprotein E2 of Classical Swine Fever Virus Confers Complete Protection in Pigs upon Viral Challenge.

Classical swine fever (CSF) is a highly contagious and serious viral disease that affects the pig industry worldwide. The glycoprotein E2 of the classical swine fever virus (CSFV) can induce neutralizing antibodies, and it is widely used for novel vaccine development. To explore the development of a vaccine against CSFV infections, the gene of glycoprotein E2 was inserted into the swinepox virus (SPV) genome by homologous recombination. The culture titers of rSPV-E2 remained at about 4.3 × 106 TCID50 for more than 60 passages in PK15 and swine testis cell lines. The rSPV-E2 could not be replicated in Vero, MDBK or other non-porcine cell lines. After two to three passages, the SPV specific gene of rSPV-E2 could not been detected in the non-porcine cell culture. To evaluate the immunogenicity of rSPV-E2, 20 CSFV seronegative minipigs were immunized with rSPV-E2, a commercial C-strain vaccine, wild-type SPV (wtSPV; negative control), or PBS (a no-challenge control). After challenge with CSFV, pigs in the rSPV-E2-immunized group showed significantly shorter fever duration compared with the wtSPV-treated group (P < 0.05). E2-specific antibodies in the rSPV-E2-immunized group increased dramatically after vaccination and increased continuously over time. CSFV genomic copies in the serum of rSPV-E2-immunized pigs were significantly less compared with the wtSPV-treated group at all time points after challenge (P < 0.01). Significant reduction in gross lung lesion scores, histopathological liver, spleen, lung, and kidney lesion scores were noted in the rSPV-E2-immunized group compared with the wtSPV-treated group (P < 0.01). The results suggested that the recombinant rSPV-E2 provided pigs with significant protection from CSFV infections; thus, rSPV-E2 offers proof of principle for the development of a vaccine for the prevention of CSFV infections in pigs.

Cholesterol-rich lipid rafts play a critical role in bovine parainfluenza virus type 3 (BPIV3) infection

Lipid rafts are specialized lipid domains enriched in cholesterol and sphingolipid, which can be utilized in the lifecycle of numerous enveloped viruses. Bovine parainfluenza virustype3 (BPIV3) entry to cell is mediated by receptor binding and membrane fusion, but how lipid rafts in host cell membrane and BPIV3 envelope affect virus infection remains unclear. In this study, we investigated the role of lipid rafts in the different stages of BPIV3 infection. The MDBK cells were treated by methyl-β-cyclodextrin (MβCD) to disrupt cellular lipid raft, and the virus infection was determined. The results showed that MβCD significantly inhibited BPIV3 infection in a dose-dependent manner, but didn't block the binding of virus to the cell membrane. Whereas, the MDBK cells treated by MβCD after virus-entry had no effects on the virus infection, to suggest that BPIV3 infection was associated with lipid rafts in cell membrane during viral entry stage. To further confirm lipid rafts in viral envelope also affected BPIV3 infection, we treated BPIV3 with MβCD to determine the virus titer. We found that disruption of the viral lipid raft caused a significant reduction of viral yield. Cholesterol reconstitution experiment showed that BPIV3 infection was successfully restored by cholesterol supplementation both in cellular membrane and viral envelope, which demonstrated that cholesterol-rich lipid rafts played a critical role in BPIV3 infection. These findings provide insights on our understanding of the mechanism of BPIV3 infection and imply that lipid raft might be a good potential therapeutic target to prevent virus infection.

Non-cytopathic bovine viral diarrhea virus 2 induce autophagy in MDBK cell

Abstract Objective Bovine viral diarrhea virus (BVDV) is classified into two distinct species, BVDV1 and BVDV2. Both species consist of two biotypes, cytopathic (cp) and non-cytopathic (ncp). Autophagy is an essential control process by which cells degrade unnecessary or dysfunctional cellular components or organelles in the cytoplasm. In this study, we explored autophagy activity in ncp BVDV2-infected MDBK cells. Materials and methods Ncp BVDV 2 (12F001) strain was cultured in MDBK cell and titration of the 50% tissue culture infective dose (TCID50) was conducted using the Kärber method. Transfection with GFP-LC3 was performed using the LipofectamineTM 2000 reagent. MDBK cells (8′105) were seeded in 6-well plates, infected with ncp BVDV2 or treated with rapamycin or DMSO, and then incubated. Subsequently, cells were used for western blot for LC3B, p62/SQSTM1, ATG14, and β-Actin. Results Our results showed that LC3 protein expression levels were increased in ncp BVDV2-infected MDBK cells by western blot and confocal microscopy. This expression was high in rapamycin-treated cells, followed by ncp BVDV2 infection, and DMSO treatment. We determined autophagy activity in ncp BVDV2-infected MDBK cells by assessing the protein levels of SQSTM/p62, LC3 and ATG14 by western blot. The decreased levels of SQSTM/p62 were accompanied by an enhanced expression of LC3 and ATG14 in ncp BVDV2-infected MDBK cells. Conclusion These results suggest that ncp BVDV 2 infection activate autophagy in MDBK cells. We conclude that autophagy may be linked to the establishment of persistent infection through virus survival. Thus, further studies should be needed to determine the association between autophagy and virus replication.

Identification and characterization of GRA6/GRA7 of Neospora caninum in MDBK cells.

Neospora caninum, an apicomplexan parasite, is recognized as a major bovine abortifacient. Dense granule antigens (GRAs) play important roles in the formation and modification of parasitophorous vacuoles (PVs) in Toxoplasma gondii. However, a few studies investigating GRAs have been reported in N. caninum. The aim of the present study was to characterize the dense GRA6/GRA7 of N. caninum in PVs using MDBK cells as a host cell model. Neospora caninum was inoculated into MDBK cells, and changes were observed using a transmission electron microscope (TEM). Neospora caninum GRA6/GRA7 were identified and characterized using bioinformatics, cell fractionation, and immunofluorescence. The TEM results revealed that integrated PVs were present in MDBK cells after N. caninum infection. Bioinformatics analysis showed that NcGRA6/NcGRA7 shared 28.76% and 29.66% homology with T. gondii GRA6/GRA7 (TgGRA6/TgGRA7) but had similar signal peptides, transmembrane domains, and motifs. Cell fractionation and subcellular localization analyses both showed that NcGRA6 was distributed in the lumen and intravacuolar network in soluble and transmembrane forms. The transmembrane form of NcGRA7 was observed in the PV membrane. These data lay a foundation for further study on bovine neosporosis and NcGRA6/NcGRA7 function during PV formation.

Chemical and cytotoxic analyses of brown Brazilian propolis (Apismellifera) and its invitro activity against itraconazole-resistantSporothrix brasiliensis.

This study aimed to evaluate the chemical composition and cytotoxic activity of brown Brazilian propolis and its invitro activity against itraconazole-resistant Sporothrix brasiliensis from animal sporotrichosis. Propolis was acquired commercially and prepared as a hydroalcoholic extract. Chemical analysis was evaluated by liquid chromatography coupled to mass spectrometry of ultra-efficiency. The cell viability was evaluated by MTT test in MDBK cells of 50 to 0.09μg/mL. For antifungal tests, twenty isolates of Sporothrix brasiliensis from dogs (n=11) and cats (n=9) with sporotrichosis were tested to itraconazole (16-0.0313μg/mL) and to propolis (3.125-0.09mg/mL) by broth microdilution technique (CLSI M38-A2), adapted to natural products. The results were expressed in minimal inhibitory concentration (MIC) and minimal fungicidal concentration (MFC). Itraconazole showed activity between MIC values of 0.25 to greater than 16μg/mL, and 88.9% (08/09) and 72.7% (08/11) of S.brasiliensis from cats and dogs, respectively, were considered itraconazole-resistant. All Sporothrix brasiliensis were sensitive to brown propolis between MIC values of 0.19-1.56mg/mL, including the itraconazole-resistant isolates, whereas the MFC values of propolis were from 0.78 to greater than 3.125mg/mL. Propolis maintained a medium to high cell viability between concentration of 0.78 to 0.09μg/mL, and p-coumaric acid was the major compound. Brown Brazilian propolis is a promising antifungal candidate against sporotrichosis and more studies need to be undertaken to evaluate its safe use to understand its efficacy.

Molecular characterization and biological activity of bovine interferon-omega3

Bovine interferon-omega3 (BoIFN-ω3) gene was amplified from bovine liver genomic DNA, which encodes a 195-amino acid protein containing a 23-amino acid signal peptide. Analysis of the molecular characteristics revealed that BoIFN-ω3 evolving from IFN-ω, contained four cysteine residues and five alpha helices, showing that BoIFN-ω3 presented the typical molecular characteristics of type I interferon. BoIFN-ω3 exhibited antiviral and antiproliferative activities, which exerted a protective effect against VSV in several mammalian cell lines, as well as against BEV, IBRV, and BVDV in MDBK cell. Moreover, BoIFN-ω3 was shown to be highly sensitive to trypsin, but remaining stable despite changes in pH and temperature. Additionally, BoIFN-ω3 induced the transcription of Mx1, ISG15, and ISG56 genes, as well as the expression of Mx1 protein in a time-dependent manner. These findings will be useful to further study BoIFN-ω in host's defence against infectious diseases, particularly viral infections. Furthermore, results will facilitate further research on the bovine interferon family.

Genome sequence of bubaline alphaherpesvirus 1 (BuHV1) isolated in Australia in 1972.

Bubaline alphaherpesvirus 1 (BuHV1) is a member of the family Herpesviridae, subfamily Alphaherpesvirinae, genus Varicellovirus. To date, no full genome sequence of BuHV has been published. Here, we report the complete genome sequence of bubaline alphaherpesvirus 1 (BuHV1) strain b6 (BuHV1-b6), isolated from a water buffalo (Bubalus bubalis) in 1972 in Australia. The virus was multiplied in MDBK cells, and the DNA was extracted and subjected to high-throughput sequencing. The reads were aligned and combined into a single genome sequence, with bovine alphaherpesvirus 5 (BoHV5) strain SV507/99 (accession number NC005261) as a reference. The BuHV1-b6 genome is a linear double-stranded DNA molecule, 137,452 bp long, with a GC content of 76.8%. The genome consists of two unique sequences: a long, or UL, sequence (103,818 bp) and a short, or US, sequence (9,586 bp), with the latter being flanked by inverted IR and TR elements of 12,024 bp each. The arrangement is typical of herpesvirus genomes of the D-type. The overall sequence has a 92.2% similarity at the nucleotide level to the reference BoHV5 strain. Our report provides a significant landmark in the history of herpesviruses, represented by the genome sequence of this 44-year-old virus isolate.

Activity of the antimicrobial peptide P34 against bovine alphaherpesvirus type 1

ABSTRACT: Previous studies have demonstrated the antimicrobial activity of the peptide P34. In this study, the antiviral potential of P34 and the in vitro mechanism of action were investigated against bovine alphaherpesvirus type 1 (BoHV1). P34 exhibited low toxicity, a high selectivity index (22.9) and a percentage of inhibition of up to 100% in MDBK cells. Results from antiviral assays indicated that P34 did not interact with cell receptors, but it was able to inhibit the viral penetration immediately after pre-adsorption. In addition, BoHV1 growth curve in MDBK cells in the presence of P34 revealed a significant reduction in virus titer only 8h post-infection, also suggesting an important role at late stages of the replicative cycle. Virucidal effect was observed only in cytotoxic concentrations of the peptide. These findings showed that the antimicrobial peptide P34 may be considered as a potential novel inhibitor of in vitro herpesviruses and must encourage further investigation of its antiherpetic activity in animal models as well as against a wide spectrum of viruses.

Calcium signaling involved in bovine herpesvirus 1 replication in MDBK cells.

Calcium is one of the most prominent second messengers in eukaryotic cells. The involvement of calcium signaling in bovine herpesvirus 1 (BoHV-1) replication was not yet reported. In this study, we revealed that the L-type Ca2+ calcium channel blocker, Verapamil and store-operated calcium channel blocker, 2-aminoethyl diphenylborinate (2-APB) inhibited BoHV-1 replication in MDBK cells at the post-entry stages, and the Na+/Ca²+ exchanger inhibitor, N-arachidonoyl glycine exchanger (NAGly) interfered with the viral entry process. NAGly also effected the phosphorylation of PLCγ-1 at Ser1248, which corroborated our previous findings, that PLCγ-1 is important for BoHV-1 entry. Collectively, these results suggest that diverse calcium channels are employed by BoHV-1 for efficient replication.

First report of bovine herpesvirus 1 isolation from bull semen samples in China.

Bovine herpesvirus 1 (BoHV-1) infection causes substantial economic losses to the cattle industry worldwide. So far, the isolation of BoHV-1 field virus has not been reported in China. Here, for the first time we report that two isolates of BoHV-1 designated as NJ16-1 and NJ16-2 were obtained from semen samples from breeding bulls in China. Typical cytopathic effect in MDBK cells, detection of viral protein VP16 in western blot analysis, PCR detection of BoHV-1 gB gene proved BoHV-1 infection and subsequent nucleotide sequence analysis showed a 99% identity with BoHV-1 Cooper strain. These results suggest that these isolated viruses are BoHV-1.

Lentivirus-mediated bta-miR-193a Overexpression Promotes Apoptosis of MDBK Cells by Targeting BAX and Inhibits BVDV Replication

Lentivirus-mediated bta-miR-193a Overexpression Promotes Apoptosis of MDBK Cells by Targeting BAX and Inhibits BVDV Replication

The p38 MAPK inhibitor, SB203580, inhibits cell invasion by Neospora caninum

The Apicomplexan parasite Neospora caninum is an obligate intracellular parasitic protozoan. It can cause severe diseases in a number of animals throughout the world. Infection with N. caninum leads to abortions in pregnant animals and neuromuscular disorders of newborns which cause great economic losses to animal husbandry. However, the mechanism of cell invasion by N. caninum is still unclear. This paper aims to investigate the impact of SB203580, a p38 MAPK inhibitor, on host cell invasion by N. caninum. The results suggested the presence of putative p38 MAPK homologues in N. caninum, and incubation of N. caninum with SB203580 markedly reduced the tachyzoite motility and microneme exocytosis (NcMIC2, 3, and 6). Furthermore, treatment or pretreatment of MDBK cells with SB203580 effectively reduced cell invasion by N. caninum. Therefore, SB203580 affected both, parasites and host cells, resulting in inhibition of cell invasion by N. caninum.

Infectious Bovine Rhinotracheitis in a Cattle Farm at Sharkia Governorate with Special Reference to its Effect on T lymphocytes

This study was applied on a cattle farm of Holstein cows at Sharkia Governorate for the isolation and identification of Bovine herpesvirus type-1 (BoHV-1) and to examine its effect on T lymphocytes. The results of clinical examination revealed that there were respiratory disorders in 30 out of 150 (20%) of cattle including elevated body temperature (40 to 42°C), nasal and ocular discharges, some animals developed severe rhinitis, conjunctivitis, corneal opacity, cough and diarrhea. Out of 30 nasal swabs, 15 swabs were positive for virus isolation as indicated by cytopothic effect (CPE) on MDBK cells. Only 11 of the 15 isolates were confirmed by virus neutralization test (VNT) as BoHV-1 isolates. In addition, only 3 out of 4 BoHV-1 isolates were detected by PCR. Peripheral blood T lymphocytes (PBTL) were analyzed using electron microscopy and comet assay to examine the effect of BoHV-1 on lymphocytes. Electron micrographs of T lymphocytes revealed peripheral condensation of chromatin and fragmentation of the nucleus end of the cell leading to the formation of apoptoic bodies. Comet assay denoted fragmentation of cellular DNA. It could be concluded that BoHV-1 can infect T lymphocytes of cattle, causing directly and indirectly apoptosis which subsequently lead to suppression of cell-mediated immunity, enhancing establishment of latency and increasing the probability for secondary bacterial infection.

Isolation and Molecular Detection of Bovine Parainfluenza Virus Type 3 in Cattle in Serbia

Abstract The presence of bovine parainfluenza virus type 3 (BPIV3) was examined in 119 nasal swabs collected from cattle with severe respiratory infection. All samples were conducted for virus isolation on the MDBK cell line. The cytopathic effect was observed after 48h to 72h in cells inoculated with eight samples (8/119; 6.7%). The confirmation of isolated strains of BPIV3 was done by the virus-neutralization test. In addition, all samples of bovine nasal swabs were also examined for the presence of BPIV3 virus using RT-PCR with primers specific for the part of HN gene. The presence of BPIV3 was detected in eight samples (8/119; 6.7%) that were also positive upon virus isolation. The molecular characterization based on nucleotide sequencing of the part of the HN gene showed that all BPIV3 isolates belonged to genotype C of BPIV3. They branched in one distinct cluster with three different branches, but these branches were very similar to each other (98.1% to 99.8%). Serbian BPIV3c isolates were most similar to the Chinese BPIV3c isolates SD0805, SD0809 and SD0835 (from 97.92% to 99.7%), and to South Korean (12Q061), Japanese (HS9) and American (TVMDL16 and TVMDL20) BPIV3c strains (from 97.1% to 98.8%), and distinct from American (TVMDL15and TVMDL17) and Australian (Q5592) BPI3V genotype B strains (only 79.9% to 82.3% similarity), as well as from the genotype A BPIV3 strains from different countries published in GenBank.

Flow Cytometry Analysis of Cell Cycle and Specific Cell Synchronization with Butyrate.

Synchronized cells have been invaluable in many kinds of cell cycle and cell proliferation studies. Butyrate induces cell cycle arrest and apoptosis in MDBK cells. We explore the possibility of using butyrate-blocked cells to obtain synchronized cells and we characterize the properties of butyrate-induced cell cycle arrest. The site of growth inhibition and cell cycle arrest was analyzed using 5-bromo-2'-deoxyuridine (BrdU) incorporation and flow cytometry analyses. Exposure of MDBK cells to 10 mM butyrate caused growth inhibition and cell cycle arrest in a reversible manner. Butyrate affected the cell cycle at a specific point both immediately after mitosis and at a very early stage of the G1 phase. After release from butyrate arrest, MDBK cells underwent synchronous cycles of DNA synthesis and transited through the S phase. It takes at least 8 h for butyrate-induced G1-synchronized cells to begin the progression into the S phase. One cycle of cell division for MDBK cells is about 20 h. By combining BrdU incorporation and DNA content analysis, not only can the overlapping of different cell populations be eliminated, but the frequency and nature of individual cells that have synthesized DNA can be determined.

Roles of bta-miR-29b promoter regions DNA methylation in regulating miR-29b expression and bovine viral diarrhea virus NADL replication in MDBK cells.

MicroRNAs (miRNAs) are an important class of small, non-coding RNAs that control target genes expression by degradation of target mRNAs or by inhibiting protein translation in many biological processes and cellular pathways. In a previous study, we found that miR-29b interfered with bovine viral diarrhea virus (BVDV) replication. However, the mechanisms of regulation of miR-29b expression are not well known. DNA methylation is an important epigenetic mechanism for silencing gene transcription, and plays an important role in promoter choice, protein expression, and regulation of miRNAs expression. In this study, we focused on the roles of DNA methylation of miR-29b promoter in regulating miR-29b expression and investigated the effects of DNA (cytosine-5) methyltransferase 1 (DNMT1) knockdown on miR-29b expression and BVDV (strain NADL) replication. Our results showed that methylation levels of miR-29b promoter were significantly decreased in BVDV NADL-infected MDBK cells. Furthermore, DNMT1 silencing significantly decreased the methylation levels of miR-29b promoter, up-regulated miR-29b expression and inhibited BVDV NADL replication, which supports the important roles of DNA methylation in regulating miRNA expression and further proves an evidence for our previous views.