MDBK Cells Research Articles

The chemokines CCL2 and CXCL10 produced by bovine endometrial epithelial cells induce migration of bovine B lymphocytes, contributing to transuterine transmission of BLV infection

Bovine leukemia virus (BLV) causes a lymphoproliferative disease in cattle and is transmitted horizontally and vertically via infected lymphocytes. Although transplacental infection is considered the predominant route of vertical transmission of BLV, the molecular mechanisms of this process remain to be elucidated. Notably, how BLV passes through the blood-placental barrier remains unclear, given that BLV is transmitted primarily by cell-to-cell contact. One hypothesis is that B cell migration to the placenta may be induced by certain endometrium-expressed chemokines. To test this hypothesis, we performed an in vitro cell migration assay using bovine B cell lines and endometrial epithelial cells. Cell migration assays showed that two bovine B cell lines, BL2M3 and BL3.1 cells, were attracted to the supernatant of bovine endometrial epithelial cells (BEnEpCs). Quantitative real-time RT-PCR showed that expression levels of mRNAs encoding the chemokines CCL2 and CXCL10 were higher in BEnEpCs than in MDBK cells. Additionally, an inhibition assay using immune serum against CCL2 and CXCL10 showed suppression of migration of bovine B cell lines. A syncytium assay showed that cells expressing BLV envelope (Env) protein fused with BEnEpCs. Here we found that bovine B cells are attracted by chemokines produced in the endometrium and that cells expressing BLV Env protein fused with endometrium epithelial cells. These results explain part of the molecular mechanism of transplacental transmission during BLV infection, although further analysis will be required. Advances in these areas are expected to contribute to controlling the spread of BLV.

Molecular and functional characterization of ovis aries IFN-epsilon

Interferon-epsilon (IFN-ε) is a type I IFN playing an essential role in innate and adaptive immunity against viral infection. Ovis aries IFN-ε (OvIFN-ε), consisting of 582 bp and which encodes a protein of 193 amino acids containing a signal peptide of 21 amino acids, was cloned and characterized. OvIFN-ε shares 51.6∼ 86.5% similarity to other species of IFN-ε, and evolves from the IFN-ε branch but not the other types of IFN. Additionally, OvIFN-ε gene is well conserved during evolution, and is highly transcribed in the liver, lung, brain, skin, ovary and uterus. Recombinant protein of OvIFN-ε was expressed in Escherichia coli and purified with nickel chelated column, which exhibited broad antiviral activity in vitro, sensitivity of trypsin, and stability of pH and temperature to some extent. Furthermore, OvIFN-ε could induce the transcription of ISG15, Mx1 and OAS in a time-dependent manner, as well as inhibit the VSV and BVDV replication in Ovis aries peripheral blood lymphocyte cells and MDBK cells. This study revealed OvIFN-ε has the typical characterization of type I IFNs and exerts antiviral activity against VSV and BVDV, and induces the expression of ISGs, which not only enriches the understanding of IFN-ε, but also facilitates further research on the antiviral defense responses of Ovis aries.

Evaluation of the Essential Oil of <i>Citrus paradisi</i> as an Alternative Treatment against <i>Candida albicans</i>

Introduction: The World Health Organization establishes that 80% of the world’s population uses traditional medicine for their primary care, because they contain compounds responsible for their properties. Objective: To evaluate the antifungal effect of the essential oil of Citrus paradisi against C. albicans and the cytotoxic effect in three cell lines in vitro. Methods: The phytochemical characterization of the oil was carried out by chemical methods and Gas chromatography (GC-MS) and the antifungal effect against C. albicans (ATCC 90029) was evaluated by the Kirby-Bauer method, which evaluated concentrations of 0.75 - 20 μg/mL and compared with nystatin 100,000 Ul/mL as a positive control. The percentage of the relative inhibitory effect was calculated. The minimum inhibitory concentration (MIC) was determined at 24 hours. Moreover, the cytotoxic effect on C. albicans and cell lines was determined by the colorimetric MTT tetrazolium assay. Finally, the antifungal effect against Candida strains isolated from clinical samples was evaluated at a concentration of 20 μg/mL. Results: The essential oil showed an antifungal effect with a percentage of inhibition of 123%. The MIC was 2.5 μg/mL, and the cytotoxicity index was 5.44 μg/mL for C. albicans. The IC50 values were 21.060, 9.482 and 4.176 μg/mL for Vero E6, J774A.1 and MDBK cells respectively. Conclusion: These results show the use of C. paradisi essential oil as an alternative treatment in oral antifungal therapy, it is beneficial due to its antifungal effect and its low toxicity on cell cultures.

Dewormer drug fenbendazole has antiviral effects on BoHV-1 productive infection in cell cultures.

BackgroundFenbendazole, a dewormer drug, is used widely in the clinical treatment of parasite infections in animals. Recent studies have shown that fenbendazole has substantial effects on tumor growth, immune responses, and inflammatory responses, suggesting that fenbendazole is a pluripotent drug. Nevertheless, the antiviral effects have not been reported. Fenbendazole can disrupt microtubules, which are essential for multiple viruses infections, suggesting that fenbendazole might have antiviral effects.ObjectivesThis study examined whether fenbendazole could inhibit bovine herpesvirus 1 (BoHV-1) productive infection in cell cultures.MethodsThe effects of fenbendazole on viral production, transcription of the immediate early (IE) genes, viron-associated protein expression, and the cellular signaling PLC-γ1/Akt pathway were assessed using distinct methods.ResultsFenbendazole could inhibit BoHV-1 productive infections significantly in MDBK cells in a dose-dependent manner. A time-of-addition assay indicated that fenbendazole affected both the early and late stages in the virus replication cycles. The transcription of IE genes, including BoHV-1 infected cell protein 0 (bICP0), bICP4, and bICP22, as well as the synthesis of viron-associated proteins, were disrupted differentially by the fenbendazole treatment. The treatment did not affect the cellular signaling pathway of PLC-γ1/Akt, a known cascade playing important roles in virus infection.ConclusionsOverall, fenbendazole has antiviral effects on BoHV-1 replication.

Development of pathological process and activity of etiotropic drugs in cell culture under condition of viral co-infection

The aim of the study: to investigate the peculiarities of the development of the pathological process in cells in conditions of mixed viral infection and to study the effectiveness of antiviral drugs in this model. Materials and methods of research. A model of simultaneous mixed infection of MDBK cells with human adenovirus serotype 5 (HAdV-5) and herpes simplex virus type 1 (HSV-1) was developed. Mitochondrial activity, ultrastructure and the state of the cell population were studied using MTT assay, transmission electron microscopy and flow cytometry with propidium iodide dye. The intensity of virus reproduction in cells and their infectious titer were studied by the cytomorphology method. The level of the synthesis of the major proteins of associate viruses was analyzed using flow cytometry and the corresponding monoclonal antibodies. Results of the research. Co-infected cells demonstrated a lower rate of development of pathomorphological changes compared to mono-infections, related to the inhibition of the reproduction of associate viruses. It was found that the co-infection of cells with HSV-1 and HAdV-5 results in a decrease in the number of cells with virus-induced intranuclear inclusions of both viruses by up to 40 % and viruses titer by 1.6 lg and 2.6 lg, respectively. Inhibition of synthesis of major capsid protein and glycoproteins of the herpes virus by 83 % and 64 %, respectively, and a less pronounced decrease in the amount of adenovirus hexon protein (by 17 %) were also noted. It is shown that the mitochondrial activity of co-infected cells increases to 64 % in comparison with herpetic mono-infection. An analysis of the influence of co-infection on cell cycle revealed that the number of cells in G1 phase remained unchanged compared with both mono-infections, while the number of apoptotic cells compared with herpes infection was reduced by 24 %. An analysis of the officinal drugs Acyclovir and Ribavirin effectiveness in conditions of mixed infection showed a reduction in their antiviral activity against associate viruses by 1 to 2.3 lg compared to mono-infections. Conclusions. The presence of a specific innovative cellular model of mixed infection with known aspects of the course of associated infections allows it to be used for preclinical study of antiviral activity of compounds and to obtain new data of the role of viral-viral interactions in the development of inefficient application of antiviral agents in medical practice. Key words: mixed viral infection, cytomorphological changes, cell cycle, reproduction of associate viruses, and antiviral activity. For citation: Biliavska LO, Povnitsa OY, Pankivska YB, Zagorodnya SD. Development of pathological process and activity of etiotropic drugs in cell culture under condition of viral co-infection. Journal of the National Academy of Medical Sciences of Ukraine. 2019;25(4):476–87

The Antiviral Effects of Sodium Phenylbutyrate Against BoHV-1 Infection In Vitro

Introduction: The alteration of histone acetylation is a known mechanism to regulate gene expression, and thereby affecting various cellular processes. Histone deacetylases (HDACs) are known to regulate histone acetylation by removal of the acetyl group from lysines. HDAC inhibitor such as Sodium Phenylbutyrate (PB) and Valproic Acid (VPA) have been reported to affect multiple virus infection while whether they affect BoHV-1 infection is unknown. Objective: The aim of the study is to investigate whether PB and VPA effects BoHV-1 infection and the virus induced inflammation related signaling including Erk1/2 and p38MAPK signaling. Methods: To assess the antiviral effects of PB and VPA on BoHV-1 infection, MDBK cells were treated with these inhibitors at different concentrations. Then time addition was performed to pinpoint which stages of virus infection was affected by the chemicals. In order to assess whether PB affect viral gene expression, we detected the viral IE genes such as bICP0, bICP4 and bICP22 using real-time PCR assay. The effects of PB had on the activation of inflammation related signaling including Erk1/2 and p38MAPK in response to the virus infection were also detected. Results: Here, for the first time we reveals that PB but not VPA affects BoHV-1 infection at late stages of infection. It affected the expression of IE genes such as bICP0, bICP4 and bICP22. Interestingly, PB enhanced the activation of both Erk1/2 and p38MAPK signaling stimulated by BoHV-1 infection. Conclusion: HDAC inhibitor PB significantly inhibited BoHV-1 infection partially through the interruption of certain viral IE gene expression. Though PB has been reported to have antiinflammatory effects, we found that it enhanced the activation of inflammation pertinent signaling of both Erk1/2 and p38MAPK stimulated by BoHV-1 infection.

RNA-Seq based transcriptome analysis during bovine viral diarrhoea virus (BVDV) infection

BackgroundBovine viral diarrhoea virus (BVDV) is the member of the genus Pestivirus within the Flaviviridae family and responsible for severe economic losses in the cattle industry. BVDV can employ ‘infect-and-persist’ strategy and ‘hit-and-run’ strategy to remain associated with hosts and thus contributes to BVDV circulation in cattle herds. BVDV have also evolved various strategies to evade the innate immunity of host. To further understand the mechanisms by which BVDV overcomes the host cell innate immune response and provide more clues for further understanding the BVDV-host interaction, in this descriptive study, we conducted a investigation of differentially expressed genes (DEGs) of the host during BVDV infection by RNA-Seq analysis.ResultsOur analysis identified 1297, 1732, 3072, and 1877 DEGs in the comparison groups mock vs. MDBK cells infected with BVDV post 2 h (MBV2h), mock vs. MBV6h, mock vs. MBV12h, and mock vs. MBV24h, respectively. The reproducibility and repeatability of the results were validated by RT-qPCR. Enrichment analyses of GO annotations and KEGG pathways revealed the host DEGs that are potentially induced by BVDV infection and may participate in BVDV-host interactions. Protein-protein interaction (PPI) network analyses identified the potential interactions among the DEGs. Our findings suggested that BVDV infection induced the upregulation of genes involved in lipid metabolism. The expression of genes that have antiviral roles, including ISG15, Mx1, OSA1Y, were found to be downregulated and are thus potentially associated with the inhibition of host innate immune system during BVDV infection. The expression levels of F3, C1R, KNG1, CLU, C3, FB, SERPINA5, SERPINE1, C1S, F2RL2, and C2, which belong to the complement and coagulation signalling cascades, were downregulated during BVDV infection, which suggested that the complement system might play a crucial role during BVDV infection.ConclusionIn this descriptive study, our findings revealed the changes in the host transcriptome expression profile during BVDV infection and suggested that BVDV-infection induced altering the host’s metabolic network, the inhibition of the expression of antiviral proteins and genes within the complement system might be contributed to BVDV proliferation. The above findings provided unique insights for further studies on the mechanisms underlying BVDV-host interactions.

COMPARISON OF THE EFFECTS OF FOETAL BOVINE CALF SERUM WITH THOSE OF BOTH CAMEL AND RABBIT SERUM ON SOME TYPES OF TISSUE CULTURE

We sought to investigate the feasibility of decreasing FBS or supplanting it with less expensive and more easily accessible sera, such as camel serum (CS) and rabbit serum (RS). Quality tests were performed for all three types of serum (commercial FBS, CS and RS). After MDBK and VERO cell lines were passaged three times for adaptation to CS and RS, the viability and damage levels in the cells cultured using the three types of serum were compared by3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) and comet assays. The results revealed that the best type of serum according to the MTT assays was (FBS), which improved cell viability from, 0.39 in the first passage to 0.69 in second passage, followed by FBS+ (CS), which improved viability from 0.40 in the first passage to 0.60 in the second passage and CS, which improved viability from 0.34 in the first passage to 0.46 in the second passage. The least suitable serum was RS; RS-cultured cells exhibited viabilities of 0.26 in the first passage and 0.34 in the second passage. The comet assays demonstrated similar outcomes; DNA damage was lowest in cells cultured with FBS, which demonstrated 7% damage in the first passage and 6% damage in the second passage, followed by cells cultured with CS, which demonstrated 19.33% damage in the first passage and 13% damage in the second passage. RS caused high levels of DNA damage compared with the other sera; 23.33% in the first passage and 16.13% in the second passage. Taken together, the results indicate that CS is superior to RS and can be used as a substitute for FBS or in an equal mixture with FBS for enhancement cell culture.

Cholesterol 25-Hydroxylase inhibits bovine parainfluenza virus type 3 replication through enzyme activity-dependent and -independent ways

Bovine parainfluenza virus type 3 (BPIV3) is one of the most important pathogens associated with bovine respiratory diseases in both young and adult cattle widespreadly around the world. The host factors which participate in the infection of BPIV3 are poorly understood. Here, we found the bovine protein Cholesterol 25-hydroxylase (CH25 H) plays an important role in the infection of BPIV3. CH25H is a multi-transmembrane and endoplasmic reticulum-related enzyme that catalyzes oxidation reaction of cholesterol to production of 25-hydroxycholesterol (25HC) and significantly inhibits the replication of several viruses. In this study, we found that CH25H is an interferon-stimulated gene (ISG), which taken part in the antiviral innate immunity. In addition, the overexpression of CH25H could inhibit the replication of BPIV3, and 25HC significantly inhibited BPIV3 infection by preventing the synthesis of both virus antigenomic RNA (cRNA) and genomic RNA (gRNA) in MDBK cells. Interestingly, CH25H-M, a mutant lacking hydroxylase activity, still had an antiviral effect against BPIV3. Taken together, our findings highlight the antiviral function of CH25H during BPIV3 infection, and suggest that CH25H inhibits viral infection through both enzyme activity-dependent and -independent ways.

A novel intracellularly expressed NS5B-specific nanobody suppresses bovine viral diarrhea virus replication

Bovine viral diarrhea virus (BVDV) infection causes significant economic losses to the cattle industry worldwide and still represents a huge pressure on agricultural production. Thus, the development of novel anti-BVDV strategies are urgently needed. The nonstructural protein 5 (NS5B) of BVDV is essential for viral replication. Further, the camel single-domain antibody (nanobody) represents a promising antiviral approach with the advantages of small size, stable structure, high specificity and solubility, and the recognition of specific epitopes. However, no NS5B-specific nanobodies against BVDV have been reported. In this study, NS5B-specific nanobodies were isolated from a phage display library of variable domains of Camellidae heavy chain-only antibodies (VHHs). Further, an MDBK cell line stably expressing Nb1 was established to explore antiviral activity. Results showed that Nb1 could markedly suppress BVDV replication and interact with the BVDV NS5B protein. This suggests that nanobodies have potential for the development of novel antiviral drugs against BVDV infection.

Eimeria tenella: specific EtAMA1-binding peptides inhibit sporozoite entry into host cells

Avian coccidiosis caused by Eimeria inflicts high economic losses to the poultry industry. Application of drugs and live vaccines presents particular challenges in pathogen resistance and cost, hence alternative anti-coccidial strategies are needed. In this study, peptides that specifically bind E. tenella AMA1 (EtAMA1) were screened from a phage display peptide library. The positive clones of target phages were characterized by ELISA after four rounds of biopanning. The binding capabilities with EtAMA1 and sporozoite proteins for the two selected peptides were detected by ELISA. The role of the two target peptides in inhibiting sporozoite invasion of MDBK cells was evaluated in vitro and the anti-coccidial effects of the two phages were assessed by an animal experiment. The three-dimensional (3D) structural model of EtAMA1 extracellular domain (EctoAMA1) protein was constructed based on the crystal template of TgAMA1 (PDB ID: 2 × 2Z), and the molecular docking between target peptides and EctoAMA1 model was analyzed. The results showed that two selected phages strongly interacted with EctoAMA1 and sporozoites protein. Two corresponding specific EtAMA1-binding peptide (named L and C) showed significant effects on inhibiting sporozoite invasion of MDBK cells. Chickens orally fed the two target phages showed partial protection against homologous challenge. Homology modeling analysis showed an apical hydrophobic groove was shaped on the top of the EctoAMA1 model. Molecular docking indicated the interaction between the EctoAMA1 protein and the two peptides, which was mainly reflected by the hydrophobic interaction and formation of intermolecular hydrogen bond. The above results suggest that the peptides L and C, especially L peptide, competed with E. tenella rhotry neck protein 2 (EtRON2) for binding to EtAMA1 located on the surface of sporozoites, and therefore inhibited the parasite invasion into cells.

Stress due to vaccination may induce re-activation of latent bovine herpesvirus-1 in cattle

This study evidences that, stress due to vaccination can cause re-activation and clinical outbreak of bovine herpesvirus-1 in latently infected cattle. Three to four days following mass-vaccination of cattle against Foot and Mouth Disease in Karnataka of India, symptoms of acute respiratory distress, conjunctivitis and vulvo-vaginitis were observed in vaccinated cattle in many villages of the state. Nasal and ocular swabs were collected from 25 ailing cattle in six villages of Hassan district of Karnataka. Upon bacteriological analysis, the samples were found negative for Pasteurella multocida. The serum samples from ailing animals were positive for bovine herpesvirus- 1 (BoHV-1) antibodies by Indirect-ELISA. The swab samples were found for BoHV-1 by PCR targeting glycoprotein- C gene of BoHV-1. The swab samples when subjected to virus isolation in MDBK cells yielded characteristic CPE of bunch of grape like clustering of cells by fifth passage. PCR targeting conserved region on glycoprotein-C gene on DNA extracted from cell-culture supernatants showing CPE confirmed the presence of BoHV-1. Nucleotide sequencing of the PCR amplicon showed that the BoHV-1 isolated during this study shared 100% sequence identity with BoHV-1 isolates from India, Switzerland and Brazil. This study emphasizes that stress due to vaccination can cause re-activation and clinical outbreak of bovine herpesvirus-1 in latently infected cattle.

Bovine Herpesvirus 1 Productive Infection Led to Inactivation of Nrf2 Signaling through Diverse Approaches.

Bovine herpesvirus type 1 (BoHV-1) is a significant cofactor for bovine respiratory disease complex (BRDC), the most important inflammatory disease in cattle. BoHV-1 infection in cell cultures induces overproduction of pathogenic reactive oxygen species (ROS) and the depletion of nuclear factor erythroid 2 p45-related factor 2 (Nrf2), a master transcriptional factor regulating a panel of antioxidant and cellular defense genes in response to oxidative stress. In this study, we reported that the virus productive infection in MDBK cells at the later stage significantly decreased the expression levels of heme oxygenase-1 (HO-1) and NAD(P)H quinone oxidoreductase-1 (NQO1) proteins, the canonical downstream targets regulated by Nrf2, inhibited Nrf2 acetylation, reduced the accumulation of Nrf2 proteins in the nucleus, and relocalized nuclear Nrf2 proteins to form dot-like staining patterns in confocal microscope assay. The differential expression of Kelch-like ECH associated protein 1 (KEAP1) and DJ-1 proteins as well as the decreased association between KEAP1 and DJ-1 promoted Nrf2 degradation through the ubiquitin proteasome pathway. These data indicated that the BoHV-1 infection may significantly suppress the Nrf2 signaling pathway. Moreover, we found that there was an association between Nrf2 and LaminA/C, H3K9ac, and H3K18ac, and the binding ratios were altered following the virus infection. Taken together, for the first time, we provided evidence showing that BoHV-1 infection inhibited the Nrf2 signaling pathway by complicated mechanisms including promoting Nrf2 degradation, relocalization of nuclear Nrf2, and inhibition of Nrf2 acetylation.

Antiviral effects of nisin, lysozyme, lactoferrin and their mixtures against bovine viral diarrhoea virus

BackgroundBovine viral diarrhoea virus (BVDV), an enveloped, single-stranded, positive-sense RNA virus from the Flaviviridae family, is a globally distributed bovine pathogen. BVDV infection in cattle, despite having a wide range of clinical manifestations, is invariably responsible for significant economic losses. To counteract these losses, various schemes to control and eradicate BVDV have been implemented, although safe drugs effectively inhibiting the replication of the virus are still lacking. The purpose of this study was to characterize the antiviral effect of naturally occurring proteins and peptide, such as bovine lactoferrin, chicken egg lysozyme, and nisin from Lactococcus lactis, used both individually and in combination, against the cytopathic NADL strain of BVDV in vitro. After determining the cytotoxicity level of each protein or peptide to MDBK cells, its antiviral effects were evaluated using virucidal, cytopathic effect inhibition and viral yield reduction assays. In addition, the influence of the tested compounds on the intracellular viral RNA level was determined.ResultsThe highest efficacy among the single treatments was achieved by bovine lactoferrin, which was effective both at the early stages of viral infection and during its entire course, although the effect weakened over time. Nisin and lysozyme were effective at later stages of infection, and the intensity of their effect did not diminish with time. Nisin+lactoferrin and lysozyme+lactoferrin combinations demonstrated stronger antiviral effects than did the single substances. The nisin+lactoferrin mixture present during the whole period of infection produced the strongest anti-BVDV effect in our entire research on both the extracellular viral titre (titre reduction up to 2.875 log ≈ 99.9%) and the intracellular viral RNA level (reduction up to 89%), and this effect intensified over the incubation time.ConclusionsThe tested substances could be applied in bovine viral diarrhoea prevention and therapy, especially when used in combination.

Molecular and antigenic characterization of bovine herpesvirus type 1 (BoHV-1) strains from cattle with diverse clinical cases in Turkey.

The present study reports the molecular and antigenic characterization of 13 bovine herpesvirus type 1 (BoHV-1) field viruses obtained from cattle with different clinical cases in Turkey between 1992 and 2017. We selected glycoprotein C (gC) of BoHV-1 as a target to detect and/or verify presence of the virus in suspect materials followed by virus isolation (VI) in MDBK cells. In seven out of 13 BoHV-1 positive samples, cytophatic effects (CPEs) were observed in MDBK cell cultures, although only four virus samples reached a sufficient titer to use in phylogenetic assay, restriction endonuclease analysis (REA), and virus neutralization test (VNT). According to the results of sequence analysis of the 13 BoHV-1 positive samples, nine BoHV-1 field viruses were determined as BoHV-1.1 and four as BoHV-1.2. Using REA, we demonstrated that two of our isolated viruses could be categorized as BoHV-1.1 while the other two isolates were BoHV-1.2 subtypes. Differences between the BoHV-1.1 and BoHV-1.2 isolates were also detected in the VNT results by assaying 125 suspected serum samples after testing with isolated (KY748023, KY748022, KY748020, and KY748021) and reference viruses (BoHV-1 Cooper and BoHV-5 Texas 89). These results are indicating the need to correctly identify BoHV-1 field isolates to better understand the epidemiology and pathogenesis of infection. In addition, it would be useful to identify the subtypes circulating in the specific geographical area while determining vaccination preferences.

An optimized aptamer-binding viral tegument protein VP8 inhibits the production of Bovine Herpesvirus-1 through blocking nucleocytoplasmic shuttling

Bovine herpesvirus 1 (BoHV-1) is a major pathogen of infectious bovine rhinotracheitis in bovine. Previously, we generated the aptamer IBRV A4 using systemic evolution of ligands by exponential enrichment. This aptamer inhibited infectivity of BoHV-1 by blocking viral particle absorption onto cell membranes. In this study, we found that the major tegument protein VP8 of BoHV-1 was involved in inhibition of infectious virus production by IBRV A4. We improved the affinity of IBRV A4 for VP8 by optimizing aptamer's structure and repeat conformation. An optimized aptamer, IBRV A4.7, was constructed with quadruple binding sites and a new stem-loop structure, which had a stronger binding affinity for VP8 or BoHV-1 than raw aptamer IBRV A4. IBRV A4.7 bound to VP8 with a dissociation constant (Kd) value of 0.2054 ± 0.03948 nM and bound to BoHV-1 with a Kd value of 0.3637 ± 0.05452 nM. Crucially, IBRV A4.7 had improved antiviral activity compared to IBRV A4, with a half-maximal inhibitory concentration of 1.16 ± 0.042 μM. Our results also revealed IBRV A4.7 inhibited BoHV-1 production in MDBK cells through blocking nucleocytoplasmic shuttling of viral VP8 in BoHV-1-infected MDBK cells. In conclusion, the aptamer IBRV A4.7 may have potency in preventing outbreaks in herds due to reactivation of latency.

Antiviral activity of PHA767491 on Caprine alphaherpesvirus 1 in vitro

Caprine alphaherpesvirus 1 (CpHV-1) induces genital lesions in its natural host similar to those caused by Human alphaherpesvirus 2 (HHV-2), commonly named herpes simplex virus 2 (HSV-2) in human patients. CpHV-1 infection in goats could represent a useful homologous animal model for the study of HSV-2 infection, chiefly for the assessment of antiviral drugs in in vivo studies. PHA767491 is a potent inhibitor of HSV-1 and HSV-2, being able to limit replication of HHVs both in vitro and in the mouse model. In the present study the antiviral efficacy of PHA767491 against CpHV-1 was evaluated in vitro in MDBK cells. PHA767491 inhibited significantly CpHV-1 replication in a dose-dependent fashion by up to 2.50 log10 TCID50/50 μl and was able to decrease viral DNA by nearly 8 log10. These findings confirm that PHA767491 is highly effective not only against simplexviruses (HSV-1 and HSV-2), but also against the varicellovirus CpHV-1. Experiments will be necessary to assess whether PHA767491 is suitable for treatment of vaginal lesions in CpHV-1-goat model. This could provide hints for the therapy of genital alphaherpesvirus infections in humans.

Comprehensive analysis of circRNAs expression profiles in different periods of MDBK cells infected with bovine viral diarrhea virus

CircRNAs play an important regulatory role in the regulation of disease. However, we have a limited understanding of the role of circRNAs in the host's complex protective and pathological mechanisms of BVDV infection. Transcriptome analysis of circRNAs in host cells after BVDV infection may allow us to understand the biological functions of circRNAs in the regulation of BVDV infection. Here, we identified a total of 19,118 circRNAs from the MBDK cells (at 12 h, 24 h, and 48 h post-infection) infected with BVDV by using RNA-seq technology. We confirmed several circRNAs using RT-PCR and DNA sequencing, and qRT-PCR analysis was performed to identify several circRNAs expression and circRNAs resistance to RNase R digestion. GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) enrichment analysis showed that the host genes of differentially expressed circRNAs were involved in the regulation of cell proliferation, apoptosis, cycle and viral infection related signaling pathways. These results indicate that circRNA in host cells plays a broad regulatory role after BVDV infection and provides a valuable resource for studying circRNA biology in host cells after BVDV infection.

Cellular microRNA bta-miR-2361 inhibits bovine herpesvirus 1 replication by directly targeting EGR1 gene

Bovine herpesvirus 1 (BHV-1) is an economically important pathogen of cattle and has led to significant consequences on the cattle industry worldwide. MicroRNAs (miRNAs) are a class of regulators that play critical roles in virus and host interaction. However, the roles of host miRNAs in BHV-1 infection remain largely unclear. In this study, a set of differentially expressed miRNAs by small RNA deep sequencing were analyzed in the Madin-Darby Bovine Kidney Cells (MDBK) infected with BHV-1 after 12 h, 24 h and 48 h post-infection compared to mock infection, and it was confirmed that bta-miR-2361 was significantly down-regulated. Moreover, bta-miR-2361 mimics transfection could inhibit BHV-1 replication. Combined with up-regulated genes from BHV-1-infected MDBK cells by deep RNA-sequencing and predicted by bioinformatics tools, early growth response 1 (EGR1) was putative target of bta-miR-2361. Furthermore, EGR1 was up-regulated during BHV-1 infection, and overexpression of EGR1 promoted BHV-1 replication whereas knockdown of EGR1 had the opposite effects. Subsequently, the target association between bta-miR-2361 and 3′UTR of EGR1 was further validated using a dual-luciferase reporter assay. In addition, overexpression of bta-miR-2361 resulted in decreased EGR1 mRNA and protein levels. Further mechanistic study showed that EGR1 stimulated BHV-1 UL46 promoter activity, but overexpression of bta-miR-2361 suppressed the production of UL46 gene. Collectively, this is the first study to reveal that bta-miR-2361 as a novel host factor regulates BHV-1 replication via directly targeting the EGR1 gene, which is a transcription factor that regulates viral UL46 gene of BHV-1. These results provide further insight into the study of BHV-1 pathogenesis.

Mycobacterium avium subspecies paratuberculosis (MAP) drives an innate Th17-like T cell response and upregulation of inhibitory signals

Abstract Johne’s disease (JD) is a chronic inflammatory gastrointestinal disorder of ruminants caused by Mycobacterium avium subspecies paratuberculosis (MAP). Our work suggested that MAP may drive a non-classical Th17-like response in PBMCs. Our objectives were to determine if Th17 cell activation by MAP required the presence of antigen presenting cells (APCs) and to determine how epithelial cells might influence Th17 activation. Monocytes were isolated by adhering and washing protocols and then cultured for 5 days to allow differentiation into macrophages. T cells and B cells were isolated using magnetic-activated cell sorting (MACS) by positive selection of CD3+ and sIgM+ cells, respectively. MACS cells were then cultured or cocultured with macrophages. MDBK cells were used as an epithelial cell model. qPCR was used to determine mRNA expression levels of MDBK, T cells alone, and T cells cultured with macrophages or B cells. All T cell cultures increased IL-17a and IL-23 mRNA expression with MAP-antigen stimulation when compared to untreated cells. APC containing cultures expressed significantly more IL-17a than T cells alone. IL-22 expression was only observed in T cell cultures stimulated with live MAP. T cell exhaustion and tolerance markers (PD-1, PD-L1, LAG-3) were increased in T cell only cultures when stimulated with MAP, but repressed in the presence of APCs. The presence of MDBK cells upregulated IL-23 following stimulation with MAP, suggesting that epithelial cells exposed to MAP may also help drive Th17 differentiation.