Madin-Darby Bovine Kidney Cells Research Articles

Lower disease activity but higher risk of severe COVID-19 and herpes zoster in patients with systemic lupus erythematosus with pre-existing autoantibodies neutralising IFN-α

ObjectivesType-I interferons (IFNs-I) have potent antiviral effects. IFNs-I are also overproduced in patients with systemic lupus erythematosus (SLE). Autoantibodies (AAbs) neutralising IFN-α, IFN-β and/or IFN-ω subtypes are strong determinants of...

The combination of high glucose and LPS induces autophagy in bovine kidney epithelial cells via the Notch3/mTOR signaling pathway

BackgroundAside respiratory diseases, beef cattle may also suffer from serious kidney diseases after transportation. Hyperglycemia and gram-negative bacterial infection may be the main reasons why bovine is prone to severe kidney disease during transportation stress, however, the precise mechanism is still unclear. The purpose of the current study is to explore whether the combined treatment of high glucose (HG) and lipopolysaccharide (LPS) could induce madin-darby bovine kidney (MDBK) cells injury and autophagy, as well as investigate the potential molecular mechanisms involved.ResultsAs we discovered, the combined effect of HG and LPS decreased MDBK cells viability. And, HG and LPS combination also induced autophagy in MDBK cells, which was characterized by increasing the expression of LC3-II/I and Beclin1 and decreasing p62 expression. LC3 fluorescence signal formation was also significantly increased by HG and LPS combination treatment. Furthermore, we measured whether the mammalian target of rapamycin (mTOR) and the Notch3 signaling pathways were involved in HG and LPS-induced autophagy. The results showed that the combination of HG and LPS significantly increased the protein expression of Notch3 and decreased protein expression of p-mTOR, indicating that Notch3 and mTOR signaling pathways were activated. However, co-treatment with the Notch3 inhibitor (DAPT) could reverse the induction of autophagy, and increased the protein expression of p-mTOR.ConclusionsThis study demonstrated that the combination effect of HG and LPS could induce autophagy in MDBK cells, and the Notch3/mTOR signaling pathway was involved in HG and LPS-induced autophagy.

Comparative transcriptome analysis of MDBK cells reveals that BoIFN-γ augmented host immune responses to bovine herpesvirus 1 infection.

Bovine herpesvirus 1 (BoHV-1) is an alphaherpesvirus that causes infectious bovine rhinotracheitis and infectious pustular vulvovaginitis in cattle. Ιnterferon-gamma (IFN-γ) is a pleiotropic cytokine with antiviral activity that modulates the innate and adaptive immune responses. In this study, we prepared high-purity bovine interferon gamma (BoIFN-γ) dimer protein using prokaryotic expression system and affinity chromatography. We subsequently investigated the effect of BoIFN-γ on BoHV-1 infection in Madin-Darby bovine kidney (MDBK) cells. The results showed that BoIFN-γ pre-treament not only decreased the production of BoHV-1 but also reduced the cytopathic effect of the virus. Differential gene expression profiles of BoHV-1 infected MDBK cells were then analysed through high-throughput RNA sequencing. The data showed that BoIFN-γ pre-treatment reduced lipid metabolism disorder and DNA damage caused by BoHV-1 infection. Furthermore, BoIFN-γ treatment upregulated the transcription of interferon regulatory transcription factors (IRF1 and GBP5) and interferon-stimulated genes (ISGs) of MDBK cells. Additionally, BoIFN-γ promotes expression of cellular protein involved in complement activation and coagulation cascades response as well as antigen processing and presentation process, while BoHV-1 infection dramatically downregulates transcription of these immune components including C3, C1r, C1s, PLAT, ITGB2, PROCR, BoLA, CD74, B2M, PA28, BoLA-DRA, and TAPBP. Collectively, our findings revealed that BoIFN-γ pre-treatment can improve host resistance to BoHV-1 infection and regulate transcription or expression of host protein associated with cellular metabolism and innate immune response. This provides insights into the development of prophylactic agents for prevention and control of BoHV-1 infection.

Flukicidal effects of abietane diterpenoid derived analogues against the food borne pathogen Fasciola hepatica.

Control of liver fluke infections remains a significant challenge in the livestock sector due to widespread distribution of drug resistant parasite populations. In particular, increasing prevalence and economic losses due to infection with Fasciola hepatica is a direct result of drug resistance to the gold standard flukicide, triclabendazole. Sustainable control of this significant zoonotic pathogen, therefore, urgently requires the identification of new anthelmintics. Plants represent a source of molecules with potential flukicidal effects and, amongst their secondary metabolites, the diterpenoid abietic acids can be isolated in large quantities. In this study, nineteen (19) chemically modified abietic acid analogues (MC_X) were first evaluated for their anthelmintic activities against F. hepatica newly excysted juveniles (NEJs, from the laboratory-derived Italian strain); from this, 6 analogues were secondly evaluated for their anthelmintic activities against adult wild strain flukes. One analogue, MC010, was progressed further against 8-week immature- and 12-week mature Italian strain flukes. Here, MC010 demonstrated moderate activity against both of these intra-mammalian fluke stages (with an adult fluke EC50 = 12.97 µM at 72 h post culture). Overt mammalian cell toxicity of MC010 was inferred from the Madin-Darby bovine kidney (MDBK) cell line (CC50 = 17.52 µM at 24 h post culture) and demonstrated that medicinal chemistry improvements are necessary before abietic acid analogues could be considered as potential anthelmintics against liver fluke pathogens

Lantana undulata Schrank – A New Source of Biological Potential in the Verbenaceae Family

Background : The presence of condensed tannins, flavonones, flavonols, flavones, xantones, catechins and alkaloids in the ethanolic extract of Lantana undulata leaves has been associated with cytotoxicity and virucidal effect on suid herpesvirus. Objective: To evaluate cellular toxicity, antimicrobial and antioxidant activities, and anti-phlogistic property of the ethanolic extract leaves of Lantana undulata, a plant from the Verbenaceae family. Method: Cytotoxicity was evaluated on Madin Darby bovine kidney (MDBK) cells and erythrocytes and through leukocyte DNA damage. Antibacterial and antifungal activities were performed using the agar diffusion technique and broth microdilution. The inhibition percentage and viral inhibition index were established against bovine herpesvirus. The antioxidant effect (EC50) was determined by the DPPH technique and the mouse ear edema model was used to determine the anti-phlogistic activity. Results: The maximum nontoxic concentration for MDBK cells was 650 μg/ml. The ethanolic extract of L. undulata was considered non-hemolytic from 2 to 16 μg/ml and not genotoxic at the concentration tested (<8 μg/ml). High concentrations (3500 μg/ml) were needed to inhibit bacteria and yeasts, and the action of the extract was bacteriostatic or fungistatic. The IIV was of 0.37 and the IP was of 57.34% against bovine herpesvirus. The antioxidant EC50 was 540.87 μg/ml and the reduction of oedema occurred at 10, 30 and 100 mg/kg in the mouse model used. Conclusion: Although the ethanolic extract of L. undulataleaves showed to be potentially toxic and to have a reduced spectrum of action on microorganisms, the outstanding antiphlogistic action puts the therapeutic potential of this plant into perspective.

Study of the dynamics of in vitro infection with bovine gammaherpesvirus type 4 and apoptosis markers in susceptible cells

Bovine gammaherpesvirus type 4 (BoHV-4) shows tropism for the endometrium, in which it causes the death of epithelial and stroma cells. Despite having anti-apoptotic genes in its genome, experiments based on immortalized cell lines have shown that BoHV-4 induces cell death by apoptosis. In the present study, we evaluated BoHV-4 replication, pro-apoptotic (Bax) and anti-apoptotic (Bcl-2) mitochondrial genes expression and chromatin condensation in bovine endometrium primary culture cells (BEC) and in the Madin Darby bovine kidney (MDBK) cell line. Results showed that BoHV-4 has a preference for replication in BEC cells over the MDBK cell line, demonstrated by the high viral titer that is consistent with the tropism of the virus. In BEC cells, chromatin condensation was consistent with the values of viral kinetics at the late stage of infection, accompanied with a balance in the mRNA levels of apoptotic mitochondrial proteins. As a consequence, in those cells viral transmission would be enhanced by inhibiting apoptosis in the early stage of virus proliferation, allowing the complete production of viral progeny, and then, the induction of apoptosis in late stages would allow neighboring cells infection. In MDBK cells replication kinetics was coincident with the up-regulation of Bcl-2, which suggests that the productive infection in MDBK is associated with a lytic phase of the virus or another cell death pathway (probably autophagy mechanism) at the late stage of infection. The results agree with the study of nuclear morphology, where a constant chromatin condensation was observed over time. It is clear that the documented BoHV-4 apoptotic responses observed in the cell lines studied above are not valid in cells from primary cultures. The data presented in this study suggest that BoHV-4 could induce apoptosis in BEC cells without a leading role of the mitochondria pathway. Further studies will be necessary to characterize in detail the programmed cell death pathways involved in BoHV-4 infection in the primary cell cultures evaluated.

Bta-miR-677 contribute to interferon pathway affecting the proliferation of caprine parainfluenza virus type 3

Caprine parainfluenza virus type 3 (CPIV3), a new strain of virus, was isolated from the goats in 2014 in China. Studies have shown that viral infection can induce changes in the expression profile of host miRNAs, which modulate natural immune responses and viral infection. In this study, we report that bta-miR-677 suppressed CPIV3 replication in Madin-Darby bovine kidney (MDBK) cells and guinea pigs. Bta-miR-677 overexpression promoted type I interferon (IFN–I) and IFN-stimulated genes (ISGs) production, thereby inhibiting CPIV3 replication, while bta-miR-677 inhibitor suppressed the antiviral innate immune response to promoted viral replication in MDBK cells. We showed that bta-miR-677 suppresses CPIV3 replication via directly targeted the 3′-untranslated region (3′-UTR) of mitochondrial antiviral signaling protein (MAVS) thus enhancing IFN pathway in MDBK cells. We also demonstrated that bta-miR-677 agomir could inhibit CPIV3 proliferation in guinea pigs, with much lower viral RNA levels in lung and trachea. Guinea pigs showed no obvious pathological changes and less severe lung lesions in bta-miR-677 agomir treated group at 7 dpi. This study contributes to our understanding of the molecular mechanisms underlying CPIV3 pathogenesis.

Molecular detection and characterization of lumpy skin disease viruses from outbreaks in Thailand in 2021.

Lumpy skin disease (LSD) is one of the most important transboundary and emerging diseases in cattle. The disease causes significant economic losses in animal production and trade worldwide. The first LSD outbreak was recorded in March 2021, at Roi-Et province in the northeastern region of Thailand. Thereafter, the disease had rapidly spread into neighbouring provinces and throughout the country. The aim of the present study was to provide information regarding to the molecular detection and characterization of LSD viruses from outbreaks in Thailand in 2021. There were 1,748,112 susceptible and 604,404 affected animals (n = 588,512 [36.30%], beef cattle; n = 12,367 [15.74%], dairy cattle and n = 3524 [7.35%], buffaloes). The morbidity and mortality rates were 34.57% and 3.47%, respectively, and the case fatality rate was 10.05% (60,713 deaths). Based on real-time polymerase chain reaction results, the p32 gene of LSD virus (LSDV) was detected more frequently in skin nodule samples (54/77, 70.13%) than in nasal swabs (26/55, 42.57%) and EDTA blood (16/77, 20.78%) samples. Moreover, the copy number of the p32 gene was higher in skin nodule samples than in nasal swab and EDTA blood samples (cycle threshold value = 21.94 ± 0.62 vs. 31.52 ± 0.66 and 34.27 ± 0.32, respectively). Furthermore, 29 (53.70%) of 54 capripoxvirus-positive skin nodule samples were successfully isolated from Madin-Darby bovine kidney cells, and the cytopathic effect was observed 72 h after inoculation. Based on the phylogenetic trees of the GPCR, ANK and RPO30 gene sequences, the LSDV isolates from Thailand were distinct from both the LSDV-field and LSDV-vaccine groups and were closely correlated with the LSDV strains isolated from mainland China, Hong Kong territory and Vietnam in 2020. Additionally, they could be a potential virulent vaccine-recombinant LSDV strain.

Development and assessment of a new bioassay for accurate quantification of Type I interferons induced by bovine respiratory viruses

Type I interferon (IFN-I) plays a major role in antiviral and inflammatory processes of the infected host. In the bovine industry, the bovine respiratory disease complex is a major cause of economic and health problems. This disease is caused by interactions of pathogens, together with environmental and host factors. Several pathogens have been identified as causal agents of respiratory diseases in cattle. To better understand how primary infections by viruses predispose animals to further infections by pathogenic bacteria, tools to accurately detect antiviral and immunoregulatory cytokines are needed. To facilitate the detection and quantification of bovine IFN-I, we have established a new specific and sensitive bioassay studies in the bovine host. This assay is based on a Madin–Darby Bovine Kidney (MDBK) cell line that carries a luciferase gene under the control of the IFN-I inducible bovine Mx1 promoter. Specific luciferase activity was measured after stimulation with serial dilutions of recombinant bovine alpha and beta IFNs and human IFN-α. With this novel bioassay we have successfully measured IFN-I production in supernatant from MDBK cells after stimulation of Toll-like receptors (TLR3, TLR7 and TLR8) and RIG-I-like receptors (RIG-I and MDA5), after viral infection with bovine respiratory pathogens, but also in samples from infected calves. Finally, this new bioassay is an easy-to-use and low cost tool to measure the production of bovine Type-I Interferon.

DDIT3 antagonizes innate immune response to promote bovine alphaherpesvirus 1 replication via the DDIT3-SQSTM1-STING pathway

ABSTRACT DNA damage-inducible transcript 3 (DDIT3), a transcription factor, is typically involved in virus replication control. We are the first to report that DDIT3 promotes the replication of bovine viral diarrhea virus, an RNA virus, by inhibiting innate immunity. However, whether the DDIT3 gene participates in DNA virus replication by regulating innate immunity remains unclear. This study reported that DDIT3 suppressed the innate immune response caused by DNA viruses to promote bovine herpesvirus 1 (BoHV-1) replication. After BoHV-1 infection of Madin-Darby bovine kidney (MDBK) cells, upregulated expression of DDIT3 induced SQSTM1-mediated autophagy and promoted STING degradation. Overexpression of the SQSTM1 protein effectively reduced STING protein levels, whereas SQSTM1 knockdown increased STING protein levels. Coimmunoprecipitation experiments and confocal laser scanning microscopy revealed that the SQSTM1 protein interacts with and colocalizes with STING. Knockdown of SQSTM1 expression in DDIT3-overexpressing cell lines restored STING protein levels. Moreover, a dual-luciferase reporter assay revealed that DDIT3 directly binds to the bovine SQSTM1 promoter and induces SQSTM1 transcription. Overexpression of SQSTM1 promoted BoHV-1 replication by inhibiting IFN-β and IFN-stimulated genes (ISGs) production; silencing of SQSTM1 promoted the expression of IFN-β and ISGs to inhibit BoHV-1 replication. In conclusion, DDIT3 targets STING via SQSTM1-mediated autophagy to promote BoHV-1 replication. These results suggest a novel mechanism by which DDIT3 regulates DNA virus replication by targeting innate immunity. DDIT3 antagonizes the innate immune response to promote bovine alphaherpesvirus 1 replication via the DDIT3-SQSTM1-STING pathway.

Comparison and efficacy of two different sheep pox vaccines prepared from the Bakırköy strain against lumpy skin disease in cattle.

PurposeLumpy skin disease (LSD) is a highly contagious and economically important viral infection of cattle, which leads to financial losses in the livestock industry of affected countries. Vaccination is the most effective control measure to prevent the disease. Heterologous sheep pox (SP) vaccine was used against LSD in Turkey. In this research, it was aimed to adapt SP Bakırköy vaccine strain attenuated in lamb kidney cells to Madin-Darby bovine kidney (MDBK) cells to provide better protection than commercial SP vaccine in cattle.Materials and MethodsTo evaluate safety and efficacy of vaccines, while animals were immunized with 10 doses (104.75 50% tissue culture infectious dose [TCID50]) and 5 doses of SP vaccine (104 TCID50) produced in MDBK cells, others were immunized with commercial Penpox-M vaccine (103.9 TCID50). Two cattle were kept as unvaccinated. At day 31 post-vaccination, all animals were challenged with the virulent LSD virus. Blood and swab samples were taken on certain days post-inoculation. Logarithmic differences challenge virus titers between vaccinated and unvaccinated animals were calculated.ResultsThe clinical sign was not observed in animals immunized with 10 doses of SP vaccine. The differences between the animals immunized with SP vaccine and control group was less than log 2.5 and the viremia occurred in immunized animals. The difference in titer was higher than log 2.5 in animals immunized with the Penpox-M, and viremia did not occur.ConclusionSP vaccine strain propagated in MDBK cells and can be used for immunization to prevent LSD infections. However, SP vaccine strain propagated in MDBK showed poor protection as compared to Penpox-M.

Design and synthesis of antivirals benzimidazoles and quinoxalines

Background Chronic hepatitis C can cause serious, even deadly, health problems like cirrhosis and liver cancer. There is no vaccine for hepatitis C. The hepatitis C virus (HCV) NS5B gene encodes RNA-dependent RNA polymerase, which is a key player in viral replication and is a promising target for the development of antiviral drugs. Drugs having benzimidazole and quinoxaline scaffolds were described to selectively block the activity of NS5B polymerase. New antiviral drugs have to be developed to overcome drug resistance.Objective The main goal of this work was to develop new effective anti-bovine viral diarrhea virus (BVDV) and anti-HCV agents by designing and synthesizing benzimidazole and quinoxaline derivatives.Materials and methods Synthesis of target compounds based on benzimidazole and quinoxaline scaffolds according to reported methods was done. Antiviral activity against BVDV was studied. BVDV and Madin-Darby bovine kidney cells were obtained from the American Type Culture Collection. Antiviral activity against HCV infectious system was evaluated. Huh7.5.1 cells were cultured and treated with different concentrations of studied compounds. GOLD molecular docking study was evaluated. The crystal structures of the HCV polymerases in complex with its co-crystalized native ligand were retrieved from the Protein Data Bank. Acute toxicity studies were carried out on animals. Results and conclusion A rational design based on the previous work was performed to indicate new promising benzimidazole and quinoxaline derivatives to be synthesized and tested as anti-HCV compounds. New benzimidazole and quinoxaline derivatives were synthesized and tested for anti-BVDV activity. All of the compounds showed strong activity against BVDV, except 17, which exhibited moderate antiviral activity. Compounds 12 and 13 were the most promising. The anti-HCV activity of 12 and 13 was investigated after infection of Huh 7.5.1 cells with HCV (JFH1). The IC50 values of 12 and 13 were found to be 19.1 and 49.4 μM, respectively; their CC50 values were 752.25 and 1480 μM, respectively; and their SI were calculated to be 39.3 for 12 and 30.03 for 13. The assigned compounds were docked into the hepatitis-C virus polymerase enzyme (pdb: 3FRZ) using GOLD 5.2.2 docking program. They revealed GoldScore fitness activities of 69.78–80.71, which is comparable to the native ‘PF-00868554’ ligand as a potent HCV polymerase inhibitor. They are bound by up to three hydrogen bonds, mainly with aminoacids R422 and S476, as well as they were embedded into the two small hydrophobic pockets formed by amino acid residues including L419, M423, L482,and L497. The acute toxicity of compound 12 on rats was tested. No signs of toxicity, no deaths, and no significant changes were observed in the biochemical parameters of liver and kidneys.

Microwave-Assisted Synthesis of Lactose Acetates with Antimicrobial, Cytotoxic, and Antiviral Properties

The task of this study was to perform the "green" synthesis of lactose octaacetate through microwave irradiation and to establish their biological activities. Lactose ester was prepared after microwave-assisted esterification of lactose with acetic anhydride (yield 85-90 %). Lactose octaacetate was characterized by a high degree of acetylation (DS 3.2-3.7). The lactose esters' structure was elucidated by infrared spectroscopy and nuclear magnetic resonance spectroscopy. Lactose octaacetate showed better antifungal activities than antibacterial activities. It possessed slight to moderate antifungal activities against Aspergillus niger ATCC 1015, Penicillium sp., Rhizopus sp., Fusarium moniliforme ATCC 38932. Lactose acetates demonstrated low cytotoxicity against three cell lines: Madin-Darby bovine kidney (MDBK) cells, human epithelial type 2 (HEp-2), and Madin-Darby canine kidney (MDCK) cells. This is the first report for antiviral activity of lactose acetates against herpes simplex virus type 1 (HSV-1), influenza virus A/Panama/2007/99/H3N2 - (IAV/H3N2), PV-1 and Coxsackievirus B1. It was found that this compound showed activity with SI = 2.4 only against PV-1, but against HSV-1, IAV/H3N2, and Coxsackievirus B1 was inactive. The current study demonstrated the applications of lactose acetates as antimicrobial and antiviral substances in food, pharmaceutical, agricultural and cosmetic preparations.

Establishment of a cell culture-qPCR system to quantify early developmental stages of Eimeria zuernii

Bovine coccidiosis is caused by apicomplexans of the genus Eimeria and results in significant economic losses in the cattle industry worldwide. Numerous anticoccidial drugs are available for the treatment of bovine Eimeria infections. However, many compounds have been on the market for decades, and multidrug resistance is commonly observed in avian Eimeria. Recent reports of anticoccidial resistance in ovine Eimeria indicate the need for a rapid and inexpensive in vitro method to assess drug efficacy against ruminant Eimeria. Currently, no such assay exists for bovine Eimeria. The aim of this study was to develop a Madin-Darby bovine kidney (MDBK) cell culture-qPCR model to support the development of Eimeria (E.) zuernii in laboratory settings. The established in vitro assay was applied on three field strains of E. zuernii from the western United States to identify its general suitability for a variety of field strains. Infected cells were observed microscopically and analyzed by quantitative PCR (qPCR) at 48 and 192 h post infection (hpi). Light microscopy observations demonstrated E. zuernii sporozoite invasion as early as 24 hpi, while confocal laser scanning microscopy revealed early meront formation by 48 hpi. Gene copy numbers displayed variations in parasite copy numbers directly after infection and over the observation period over 192 h. Based on these findings, this assay is suitable for detecting E. zuernii gene copies in MDBK cells over an experimental period of 192 h. Though total gene copy numbers did not increase over time, we conclude that this assay is a suitable for sustaining the growth and development of E. zuernii stages in vitro. This testing system will allow for further investigations of bovine Eimeria while reducing the use of animal experiments.

Evaluating Large Spontaneous Deletions in a Bovine Cell Line Selected for Bovine Viral Diarrhea Virus Resistance.

Bovine viral diarrhea virus’s (BVDV) entry into bovine cells involves attachment of virions to cellular receptors, internalization, and pH-dependent fusion with endosomal membranes. The primary host receptor for BVDV is CD46; however, the complete set of host factors required for virus entry is unknown. The Madin-Darby bovine kidney (MDBK) cell line is susceptible to BVDV infection, while a derivative cell line (CRIB) is resistant at the level of virus entry. We performed complete genome sequencing of each to identify genomic variation underlying the resistant phenotype with the aim of identifying host factors essential for BVDV entry. Three large compound deletions in the BVDV-resistant CRIB cell line were identified and predicted to disrupt the function or expression of the genes PTPN12, GRID2, and RABGAP1L. However, CRISPR/Cas9 mediated knockout of these genes, individually or in combination, in the parental MDBK cell line did not impact virus entry or replication. Therefore, resistance to BVDV in the CRIB cell line is not due to the apparent spontaneous loss of PTPN12, GRID2, or RABGAP1L gene function. Identifying the functional cause of BVDV resistance in the CRIB cell line may require more detailed comparisons of the genomes and epigenomes.

Advancements in the Growth and Construction of Recombinant Lumpy Skin Disease Virus (LSDV) for Use as a Vaccine Vector.

Attenuated vaccine strains of lumpy skin disease virus (LSDV) have become increasingly popular as recombinant vaccine vectors, to target both LSDV, as well as other pathogens, including human infectious agents. Historically, these vaccine strains and recombinants were generated in primary (lamb) testis (LT) cells, Madin–Darby bovine kidney (MDBK) cells or in eggs. Growth in eggs is a laborious process, the use of primary cells has the potential to introduce pathogens and MDBK cells are known to harbor bovine viral diarrhea virus (BVDV). In this study, data is presented to show the growth of an attenuated LSDV strain in baby hamster kidney (BHK-21) cells. Subsequently, a recombinant LSDV vaccine was generated in BHK-21 cells. Partial growth was also observed in rabbit kidney cells (RK13), but only when the vaccinia virus host range gene K1L was expressed. Despite the limited growth, the expression of K1L was enough to serve as a positive selection marker for the generation of recombinant LSDV vaccines in RK13 cells. The simplification of generating (recombinant) LSDV vaccines shown here should increase the interest for this platform for future livestock vaccine development and, with BHK-21 cells approved for current good manufacturing practice, this can be expanded to human vaccines as well.

Novel Preparation Technique of Hyperimmune Globulins against Bovine Coronavirus as Surrogate of Beta Coronavirus

Consuming time and effort to prepare hyperimmune globulins using Freund’s adjuvant is a sophisticated and harsh technique. In this work, a novel, safe, and rabid method was proposed using monolaurin as an immune-stimulating agent in hyperimmune globulins production against Bovine coronavirus (BCoV). The mentioned virus was used as a surrogate to family Betacoronavirus. Bovine coronavirus (Mabus strain) with titer log10 5.8 tissue culture infective dose infectivity (TCID50)/ml was used in this experiment. The inactivation of the virus was done using 1% ascorbic acid for 24h. Monolaurin emulsion (10% w/v) of was prepared by sonication using tween 20 and water. The inactivated bovine coronavirus was added to the emulsion by 20% of the final volume. The immunoglobulins were prepared by inoculating the inactivated virus with the adjuvant in rabbits and evaluated on the Madin-Darby bovine kidney (MDBK) cell line by virus neutralization test (VNT). The effect of the adjuvant was assessed by histopathological examination of vital organs such as the kidney and liver. The antibody titer against the BCoV was reached its peak, log2 1024 TCID50/ml, at the 3rd-week post-inoculation in the rabbits. The level of the globulin reached a high level and its peak (14.3g/dL) at the end of the experiment. No abnormalities were seen in the livers and kidneys of the negative control group of rabbits. Monolaurin showed a new level of safety and efficacy when used as an adjuvant during the preparation of the immunoglobulins against BCoV.

Establishment of a Suspension MDBK Cell Line in Serum-Free Medium for Production of Bovine Alphaherpesvirus-1.

The Madin–Darby bovine kidney (MDBK) cell line is currently used for the production of bovine alphaherpesvirus-1 (BoHV-1) vaccine. For the purpose of vaccine manufacturing, suspension cells are preferred over adherent ones due to simplified sub-cultivation and an easier scale-up process, both of which could significantly reduce production cost. This study aimed to establish a procedure for the culture of BoHV-1 in the suspended MDBK cell line in serum-free medium. We screened several commercially available serum-free media and chose ST503 for subsequent experiments. We successfully adapted the adherent MDBK cells to suspended growth in ST503 in the absence of serum. The maximum density of suspension-adapted MDBK cells could reach 2.5 × 107 cells/mL in ST503 medium with optimal conditions. The average size of suspension-adapted cells increased to 18 ± 1 µm from 16 ± 1 µm. Moreover, we examined tumorigenicity of the suspended cells and found no sign of tumorigenicity post adaptation. Next, we developed a protocol for the culture of BoHV-1 in the cell line described above and found that ultrasonic treatment could facilitate virus release and enhance virus yield by 11-fold, with the virus titer reaching 8.0 ± 0.2 log10TCID50/mL. Most importantly, the prototype inactivated BoHV-1 vaccine we generated using the suspension cultures of MDBK cells induced neutralizing antibodies to a titer comparable to that of the commercial inactivated BoHV-1 vaccine. Overall, we established and optimized a protocol for the production of inactivated BoHV-1 vaccine in MDBK cells adapted for suspension culture, which provides insights for future large-scale manufacturing of BoHV-1 vaccine.

The in vitro antiviral activity of Lacticaseibacillus casei MCJ protein-based metabolites on bovine viral diarrhea virus

Bovine viral diarrhea virus (BVDV) is a ubiquitous immunosuppressive etiological agent which is economically important for a wide host range in the livestock industry. Lactobacillus spp. has widely been using in the field of management and treatment of gastro-enteric disease for both humans and animals. The ability of Lacticaseibacillus casei MCJ protein-based metabolized to suppress BVDV infection in Madin–Darby Bovine Kidney cell line was demonstrated in this study. The protein-based metabolites were extracted from the cultured L. casei to obtain the safest and beneficial form of the probiotic bacteria. It is revealed that LPM have no cytotoxic effect and the cell viability remain more than 80% even after the cells are treated with 3000 µg/mL of LPM. The results of the plaque formation assay showed that LPM can reduce the viral infection rate. To know the mechanism of LPM for anti-BVDV activity, MDBK cells were exposed to LPM before, after and co-incubation of virus infection. The co-treatment of LPM with BVDV revealed the best results. The results suggest that the LPM has a potential anti-BVDV activity which could be a prospective candidate for the prevention and control of BVDV infection in an animal.

Bovine endometrium-derived cultured cells are suitable for lipofection

Bovine-derived cultured cells, including Madin-Darby bovine kidney cells, are used worldwide; however, lipofection tend to result in low transfection efficiency, which has impeded the progress of veterinary research. We performed experiments to confirm the lipofection efficiency of bovine-derived cultured cells, to identify cells that suitable for lipofection. Several bovine tissues (endometrium, testis, ear tissue and foetal muscle) were collected, and primary cultured cells were prepared. Lipofection assay showed that only bovine endometrium (BE)-derived cells could be transfected efficiently (50‒70%). BE cells can be divided into at least two types of cell populations (BE-1 and BE-2). The BE-1 cells, which were suitable for lipofection, were obtained by passages at short intervals and were negative for cytokeratin- and positive for vimentin-expression; the BE-2 cells did not have these characteristics and were not suitable for lipofection. Furthermore, the BE-1 cells and artificially immortalised cells of BE-1, iBE-1 cells, were utilised in a reporter assay requiring the introduction of multiple DNAs. Endometrial tissues can be collected from living cows, and BE-1 cells can be obtained easily by controlling passaging timing. The production of BE-1 cells and sharing the methods required to prepare them will contribute to the development of veterinary research.