BHK-21 Cell Cultures Research Articles

Optimization of Virus Yield as a Strategy to Increase the Efficiency of Rabies Vaccine by BHK-21 Cells in a Bioreactor

Background: Rabies is a usually fatal viral zoonotic and preventable disease. The efficacy and safety of animal rabies vaccination made in permanent BHK-21 cell culture have been proven over a long period of use. By increasing the yield of cells and viruses, the efficacy of the vaccine can be increased. Objectives: The objective of this study was to optimize and maximize the output of a rabies vaccine made on BHK cells in a bioreactor. Methods: This study examined the impacts of independent parameters, such as pH, temperature, cell density, and dissolved oxygen (DO), on rabies virus strain PV-PARIS yield for a central composite design. To achieve high viral production, this study used the central composite approach to optimize cell development. Results: The findings showed that BHK-21 cells were grown under the ideal conditions of pH 7.21, temperature 35.05ºC, 68.75% for DO, and 2.30 × 106 cell/mL of cell density to produce high titers of rabies virus (4.7 × 107 plaque-forming unit [PFU]/mL). High correlation coefficients (0.927) validated that the predicted model was well-fitted with the data, and the statistical analysis of the collected data indicated that the experimental data and predicted model were well-matched. The accuracy of this model’s predictions was correlated with values of adjusted R-squared (R2Adj) and predicted R-squared (R2Pred). Conclusions: These upgrades lead to a more reliable and economical procedure that makes industrialization and commercialization easier.

FORMATION OF SILVER-CONTAINING FILMS BASED ON POLYELECTROLYTE COMPLEXES BY SPUTTERING DEPOSITION AND THEIR ANTIMICROBIAL AND ANTIVIRAL ACTIVITY

Silver-containing film materials are formed by vacuum sputtering of silver nanoparticles on the surface of polyelectrolyte complexes based on chitosan and sodium salt of carboxymethylcellulose (pectin). The obtained samples were characterized by the methods of wide-angle X-ray scattering and transmission electron microscopy, and their antimicrobial, antiviral and cytotoxic properties were also investigated. The presence of metallic silver on the surface of polyelectrolyte complexes was confirmed by the method of wide-angle X-ray diffraction. It was established that upon sputtering of silver, a ~200 nm thick layer is formed within 5 minutes. It was shown that Na-CMC–Ag–chitosan and pectin–Ag–chitosan samples, formed by silver sputtering, exhibit antimicrobial activity against test cultures of S. aureus, E. coli, P. aeruginosa, and C. albicans. Antiviral activity of samples against influenza A virus and herpes simplex virus type 1 was also established. The obtained samples were not cytotoxic, did not inhibit the viability of MDCK and BHK cell cultures.

Genetic characterization and phylogenetic analysis of foot and mouth disease virus vaccine strains and recent field isolate

Foot and Mouth Disease (FMD) control is a national and regional responsibility. FMD outbreaks were detected in three farms in Port-Said Governorate in 2020, although animals in these farms were vaccinated with the local polyvalent inactivated vaccine. Samples from tongue epithelium and vesicular fluid were collected from these farms, where FMDV isolation was performed on BHK-21 cell culture. Viral RNA was extracted from the virus isolates, and vaccine strains were then screened for FMDV using conventional Reverse transcriptase polymerase chain reaction. The substitution rates and phylogenetic relationship between the field isolate and the vaccine strains were determined using DNA sequence and bioinformatics analyses. The results illustrate that the sequence analysis for the 1D region of FMDV field isolate was serotype A‑Africa topotype, Genotype IV, and closely related to isolated A‑Africa topotype Genotype IV by Animal Health Research Institute, 2020 with nucleotide similarity ranging from 97.1% to 99.74%, and revealing genetic variation of 5.63-7.88% from previously Egyptian isolated A‑Africa topotype, Genotype IV in 2016 and 2018. High genetic variations were determined from the locally used vaccine strain serotype A of the Asia topotype, lineage Iran‑05, in the major antigenic sites of the VP1 region with a nucleotide difference of 26.34%. Depending on these findings, the Veterinary Serum and Vaccine Research Institute produced an emergency inactivated monovalent vaccine using the newly isolated strain, and we recommended adding it to the subsequent prepared vaccine batches.

Preparation, Characterization, and Antimicrobial and Antiviral Properties of Silver-Containing Nanocomposites Based on Polylactic Acid-Chitosan.

Antimicrobial and antiviral nanocomposites based on polylactic acid (PLA) and chitosan were synthesized by a thermochemical reduction method of Ag+ ions in the PLA-Ag+-chitosan polymer films. Features of the structural, morphological, thermophysical, antimicrobial, antiviral, and cytotoxic properties of PLA-Ag-chitosan nanocomposites were studied by X-ray diffraction (XRD), transmission electron microscopy (TEM), differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), and antiviral, antimicrobial, and cytotoxic studies. The effects of temperature and the duration of reduction of Ag+ ions on the structure of PLA-Ag-chitosan nanocomposites were established. During the thermochemical reduction (T = 160 °C, t = 5 min) of silver palmitate ions in PLA-Ag+-chitosan polymer films, Ag nanoparticles with an average size of 4.2 nm were formed. PLA-Ag-chitosan polymer nanocomposites have strong antimicrobial activity against S.aureus and E.coli strains. In particular, for PLA-chitosan samples containing 4% Ag, the diameters of the S.aureus and E.coli growth inhibition zones were 25.8 and 25.0 mm, respectively. The antiviral activity of the nanocomposites against influenza A virus, herpes simplex virus type 1, and adenovirus serotype 2 was also revealed. The PLA-4%Ag-chitosan nanocomposites completely inhibited the cytopathic effect (CPE) of herpes virus type 1 by 5.12 log10TCID50/mL (high antiviral activity) and the development of the CPE of influenza virus and adenovirus by 0.60 and 1.07 log10TCID50/mL (relative antiviral activity). The obtained nanocomposites were not cytotoxic; they did not inhibit the viability of MDCK, BHK-21, and Hep-2 cell cultures.

Cytotoxicity of 5% ethanol extracts of moringa oleifera leaf as an alternative of root canal irrigant to fibroblast BHK-21 cell culture

Cytotoxicity of 5% ethanol extracts of moringa oleifera leaf as an alternative of root canal irrigant to fibroblast BHK-21 cell culture

Anticoronavirus Activity of Water-Soluble Pristine C60 Fullerenes: In Vitro and In Silico Screenings.

The emergence of a new member of the Coronaviridae family, which caused the 2020 pandemic, requires detailed research on the evolution of coronaviruses, their structure and properties, and interaction with cells. Modern nanobiotechnologies can address the many clinical challenges posed by the COVID-19 pandemic. In particular, they offer new therapeutic approaches using biocompatible nanostructures with "specific" antiviral activity. Therefore, the nanosized spherical-like molecule (0.72 nm in diameter) composed of 60 carbon atoms, C60 fullerene, is of interest in terms of fighting coronaviruses due to its high biological activity. In here, we aim to evaluate the effectiveness of anticoronavirus action of water-soluble pristine C60 fullerene in the model and in vitro systems. As a model, apathogenic for human coronavirus, we used transmissible gastroenteritis virus of swine (TGEV), which we adapted to the BHK-21 cell culture (kidney cells of a newborn Syrian hamster). The shape and size of the particles present in C60 fullerene aqueous colloidal solution (C60FAS) of given concentration, as well as C60FAS stability (value of zeta potential) were studied using microscopic (STM, scanning tunneling microscopy, and AFM, atomic force microscopy) and spectroscopic (DLS, dynamic light scattering) methods. The cytopathic effect of TGEV was determined with the help of a Leica DM 750 microscope and the degree of monolayer changes in cells was assessed. The microscopy of the viral suspension was performed using a high resolution transmission electron microscope (HRTEM; JEM-1230, Japan). Finally, the search for and design of optimal possible complexes between C60 fullerene and target proteins in the structure of SARS-CoV-2 coronavirus, evaluation of their stability in the simulated cellular environment were performed using molecular dynamics and docking methods. It was found that the maximum allowable cytotoxic concentration of C60 fullerene is 37.5±3.0μg/ml. The investigated C60FAS reduces the titer of coronavirus infectious activity by the value of 2.00±0.08TCID50/ml. It was shown that C60 fullerene interacts directly with SARS-CoV-2 proteins, such as RdRp (RNA-dependent RNA polymerase) and 3CLpro (3-chymotrypsin-like protease), which is critical for the life cycle of the coronavirus and, thus, inhibits its functional activity. In both cases, C60 fullerene fills the binding pocket and gets stuck there through stacking and steric interactions. Pioneer in vitro study to identify the anticoronavirus activity of water-soluble pristine C60 fullerenes indicates that they are highly promising for further preclinical studies, since a significant inhibition of the infectious activity of swine coronavirus of transmissible gastroenteritis in BHK-21 cell culture was found. According to molecular modeling results, it was shown that C60 fullerene can create the stable complexes with 3CLpro and RdRp proteins of SARS-CoV-2 coronavirus and, thus, suppress its functional activity.

ПРОТИВІРУСНА АКТИВНІСТЬ АМІНОКАПРОНОВОЇ КИСЛОТИ ПО ВІДНОШЕННЮ ДО КОРОНАВІРУСУ ІНФЕКЦІЙНОГО БРОНХІТУ В УМОВАХ IN VITRO

I. V. Dziublyk, O. P. Trokhimenko, S. O. Soloviov, G. L. Gumeniuk, O. Ya. Dziublyk, N. I. Gumeniuk, O. K. Yakovenko Abstract The aim of the study is a preclinical evaluation of the antiviral activity of aminocaproic acid (ACA) against the prototype strain IBV (Infectious bronchitis virus) of Coronavirus family in vitro. Material and methods. During the research, modern methods were used to determine the cytotoxic effect of the evaluation on a monolayer of BHK-21 cell culture in vitro; cultivation, accumulation and determination of the infectious titer of IBV by cytopathic action on a monolayer of cell cultures; assessment of the antiviral effect of the drug — the establishment of the inhibitory concentration and the chemotherapeutic index (CTI) of ACA in various modes of drug administration: 2 hours before infection, simultaneously with infection and 2 hours after infection. Results. With the introduction of ACA 2 hours before infection, a decrease in the infectious titer of the IBV virus was not established. The antiviral activity of ACA was detected when the drug was added in 2 modes: simultaneously and 2 hours after infection. The introduction of ACА into the medium for cell cultivation at non-toxic concentrations of 7.91–15.82 mg / ml led to a decrease in the infectious titer of the virus by 1.4–2.0 lg TCD50 / 0.1 ml. The CTI of the ACA was 6 in the indicated concentrations and modes, which is an indicator of its promising potential for further studies of antiviral activity in vivo, including clinical studies. Conclusions. The direct antiviral effect of ACA against the prototype H-120 virus strain from the Coronaviridae family in vitro was revealed. The suppression of viral reproduction with an established low toxicity of the drug, a decrease in the infectious titer of IBV by 1.4–2.0 lg TCD50 / 0.1 ml and with a CTD equal to 6.0, indicate the prospects for further study of the antiviral properties of ACA in clinical trials. Key words: aminocaproic acid, coronavirus, antiviral activity.

THE CULTURAL PROPERTIES ALTERATIONS OF PORCINE ENTEROVIRUS DURING LONG-TERM STORAGE

Enterovirus infections remain one of the urgent problems in modern infectious pathology and are represented in numerous publications of domestic and foreign researchers, including publications in the field of veterinary virology. The causative agents of enterovirus infections of viral etiology (enteroviruses) are characterized by relative resistance to adverse environmental conditions, including thermal stability, acid resistance, resistance to proteolytic enzymes, which allows them to survive in the environment and facilitates their transmission by various ecological routes (water, food, aerosols, contaminated objects, etc.). The purpose of this study was to elucidate the changes in the infectious properties of porcine enteroviruses in vitro under conditions of long-term storage at a temperature of minus 32 °C. In the course of this study, a re- cultivation process was carried out with the subsequent adaptation of two variants of viruses: the porcine teschovirus of the first serotype (Teschovirus A), the “Dniprovsky 34” strain and Porcine sapelovirus 1 (porcine enterovirus of serogroup 8), the reference V-13 strain. The re-cultivation was performed on BHK-21 cell cultures / clone 13 and on SPEV, in which they were previously cultivated, in order to determine the infectious activity after storage under negative temperatures (minus 32 °C) for two and twenty years. On the example of porcine enterovirus of serogroup 8 (the causative agent of viral gastroenteritis), it was proved that during long-term storage (20 years) at a temperature of – 32 °C, the virus did not lose its infectious properties, although a change in the cytopathogenic effect in vitro during re-cultivation was found. The infectious properties of the porcine teshovirus of the first serotype are also capable of long-term storage (2 years) under conditions of minus 32 °C temperature.

Determination of the effects of Enrofloxacin, Linco-Spectin and Florphenicol antibiotics on BHK-21 cell culture and FMD 146S Virus particles-infective titers

Abstract: Enrofloxacin, linko-spectin and florphenicol antibiotics were intended to be used in the BHK-21 An30 cells and the foot and mouth disease virus (FMDV) culture during the vaccine production process. Methylthiazolyldiphenyl-tetrazolium bromide (MTT) and cell cultures assays were performed in the BHK-21 An30 cells treated with the antibiotics that took place in this study to determine the concentration that inhibits cell proliferation and adverse effects on cell morphology. Virus cultures were performed by inoculating of FMDV serotypes (A/TUR/11, O/TUR/07, Asia-1/TUR/15) to the treated cell cultures with the antibiotics. 146S and infective tites of the obtained virus suspensions were determined. The non-toxic upper limit was determined to be 15μg /ml for enrofloxacin and 300μg/ml for linco-spectin in terms of cell morphology and cell numbers versus positive control (gentamycin, penicillin-streptomycin) and negative control (antibiotic-free medium) as a result of MTT and cell culture tests on BHK cells. It was observed that Florfenicol also inhibited cell proliferation and induced cell degeneration, even at a concentration of 5μg/ml. The mean 146S values of FMD virus cultures containing enrofloxacin and linco-spectin were determined to be 0.49, 0.46, ; 0.53, 0.47 and 0.30, 0.28µg/ml for serotype A/TUR/11, O/TUR/07 and Asia-1/TUR/15 respectively. The mean values of the infective titres were 107,04, 107,25, 106,04, 106,59 and 107,26, 107,6 pfu/ml. for serotype A/TUR/11, O/TUR/07, and Asia-1/TUR/15 respectively. In the control group used gentamycin, penicillin-streptomycin and antibiotic-free medium, the mean 146S FMD virus particles were obtained as 0.51, 0.50, 0.50; 0.52, 0.55, 0.52 and 0.36, 0.33, 0.30 µg/ml for A/TUR/11, O/TUR/07 and Asia-1/TUR/15 respectively. The mean values of the FMD infective virus titres were 107,35, 107,40, 107,11; 106,24, 106,46, 106,62, and 107,70, 107,75, 107,77 pfu/ml for A/TUR/11, O/TUR/07 and Asia-1/TUR/15 respectively. As a result, the infective FMDV titer and 146S results obtained in the control group (gentamicin, penicillin-streptomycin) and FMD virus cultures using Enrofloxacin (15µg / ml) and linco-spectin (300µg / ml) were very close to each other. According to these data, it was concluded that enrofloxacin and linco-spectin can be used up to the upper limit in the BHK-21 An30 cell and FMD virus cultures. However, florfenicol should not be used in cell and virus cultures.

A vaccine-matching assessment of different genetic variants of serotype O foot-and-mouth disease virus isolated in Ethiopia between 2011 and 2014.

The aim of this study was to assess the vaccine-matching and antigenic properties of foot-and-mouth disease virus (FMDV) isolates collected from Ethiopia between 2011 and 2014. Samples (n = 51) were collected from cattle and pigs with clinical signs consistent with foot-and-mouth disease (FMD) on farms in Debre-Berhan, Debre-Zeit/Bishoftu, Sidamo, Mekelle, and Addis Ababa. Infectious FMDV was isolated using BHK-21 cell cultures from 38 of the 51 field samples (74.5%). All of these FMDV-positive samples were characterized as serotype O, belonging to two East Africa topotypes (EA-3 and EA-4), and their VP1-encoding sequences demonstrated amino acid sequence variability encompassing 27 positions in comparison to the vaccine strain (O/ETH/38/2005) currently provided by the National Veterinary Institute of Ethiopia. One-dimensional virus neutralization test (1dm VNT) results showed that O/ETH/38/2005 was antigenically matched to 10 of the 16 serotype O viruses. These findings indicate that the O/ETH/38/2005 vaccine strain can provide protection against outbreaks caused by the O/EA-3 topotype, although poorer vaccine-matching results for the O/EA-4 topotype reinforce the importance of using a good-quality vaccine with high coverage in the susceptible herds with supporting post-vaccination serosurveillance to ensure that sufficient antibody titers are generated in the vaccinated animals.

Miramistin effect on BHK-21 and PSGK-30 cell line proliferation and FMD virus reproduction in these cells

At present, Miramistin drug (benzyldimethyl [3-(myristoylamino) propyl] ammonium chloride monohydrate), which has a broad bactericidal effect, is used in veterinary practice. This antiseptic is active against most viruses, mycoplasmas, bacteria, fungi and protozoa. According to toxicometric parameters, Miramistin is classified as a low-hazard substance. Use of cell structures as test systems for assessing the toxicity of pharmacological substances instead of conventional tests on experimental animals allows us to better clarify the possible mechanism of the effect of the investigated substance. Since many types of mycoplasmas affect the genitourinary system organs, to assess the cytotoxicity of Miramistin kidney mammalian cells of various mammals can be used as test systems, in particular, the newborn Syrian hamster (VNK-21), Siberian mountain ibex (PSGK-30), and others. The possibility of using BHK-21 and PSGK-30 cell monolayer as test systems for assessing the baseline cytotoxicity of Miramistin was shown. When studying toxicity of the drug, the effect of its various concentrations on the cell morphology was studied, the number of viable cells and the total protein content were determined as an indicator of the cell mass increase. The results of a cytomorphological study indicate that the Miramistin maximum permissible concentration of for BHK-21 and PSGK-30 cell monolayer is 25 μg/cm3. The use of this antibacterial drug in higher concentrations caused the appearance and increased signs of endogenous intoxication and degeneration. When evaluating the proliferative activity of cells under the influence of the Miramistin antibiotic in different concentrations, it was found that increasing the dose to 50, 75, 100, 125, 150 μg/cm3 leads to decrease in the rate of cell growth compared to the control. The Miramistin content in the growth medium in an amount of up to 25 μg/cm3 did not affect the intensity of protein synthesis. Presence of Miramistin in the culture medium at the maximum permissible concentration causes a slight decrease in the reproduction of foot and mouth disease virus in BHK-21 and PSGK-30 cell cultures by 4.5 and 4.0%, respectively, compared with the control without antibiotic. Since mycoplasmas are the most common contaminants of cell cultures, further studies will be aimed at exploring the possibility of using Miramistin for decontamination of BHK-21 and PSGK-30 cell monolayers.

Senecavirus A (SVA) in finishing swine: diagnosis and viral isolation

ABSTRACT: Senecavirus A (SVA) has been a problem in Brazil since the end of 2014. The infections caused by SVA have disrupted the productive chain in Brazil, as it can be confused with foot-and-mouth disease. Although, the virus has remained endemic in the country, an increase in the number of cases of the disease was observed in 2018. The aim of the present study was to conduct the differential diagnosis of foot-and-mouth disease in an outbreak of vesicular disease in finishing swine. Animals (160-170 days old) were kept on a farm with 6000 pigs in Minas Gerais State, Brazil. The morbidity and mortality rates were 20% and 2.2%, respectively. The diagnosis was performed by RT-PCR, using primers that determine the amplification of an internal region of the 3D gene. Furthermore, samples were inoculated into BHK-21 cell culture for viral isolation. In the first passage under cultivation, a cytopathogenic effect compatible with SVA replication (rounding and detachment of the cell monolayer) was observed. The viral identity was confirmed using two additional assays: indirect immunofluorescence assay (IFA) and nucleotide sequencing. Both tests confirmed that the infection was caused by SVA. In summary, we described a method for the diagnosis and viral isolation of SVA, a virus that arrived in Brazil in 2014 and has become endemic in the country.

The Difference of The Effects of Conventional Flowable Composite and Self-Adhering Flowable Composite on BHK-21 Fibroblast Cells

Background: One type of composite resin material on the market is flowable composites (FC) which has low viscosity, can be applied to areas with low stress or require good penetration such as pit and fissure sealants, restoration of class II, class III, and class V. Along with technology development, self-adhering flowable composite (SAFC) material has been developed which shorten the applications time because it combines etch, priming, and bonding in one system. The incomplete composite polymerization process can release residual monomers which affect pulp and gingiva The effects of composite materials can be seen from the viability of BHK-21 fibroblast cells after being exposed by these materials. Aims: Determine the viability of BHK-21 cells after being exposed to conventional flowable composite (CFC) and SAFC. Method: The research was in-vitro experimental laboratory with post-test only control group design. BHK-21 cell cultures were included in a 96-well microplate and divided into control group (N=16) and two treated groups (N=16). The treated group was given CFC and SAFC in a disk form with 5mm in diameter and 2,5mm in thickness, then incubated for 24 hours. MTT was given, the optical density value was read by ELISA reader and cell viability was calculated. Optical density data were analyzed using Tukey HSD to compare between groups. Results: The BHK-21 cells viabitlity of SAFC group is greater than the CFC, , indicated by the optical density SAFC (value=0.1233) and CFC (value=0.0936). Conclusion: The viability of BHK-21 cells exposed to SAFC is higher than that of CFC.

Semliki Forest Virus replicon particles production in serum-free medium BHK-21 cell cultures and their use to express different proteins.

The production of biopharmaceuticals as vaccines in serum-free media results in reduced risk of contamination and simpler downstream processing. The production of enveloped viruses and viral vectors such as Semliki Forest Virus (SFV) typically requires lipids that are provided by supplementation with animal serum, so production under serum-free conditions is challenging. In this work, the capacity to deliver genetic material of SFV-viral replicon particles (SFV-VRPs) produced in BHK-21 cells adapted to serum-free medium (BHK/SFM) was evaluated. Three transgenes were evaluated: GFP used as a model protein, while hepatitis C virus nonstructural protein 3 protease domain (HCV-NS3p) and rabies virus glycoprotein (RVGP) were selected based on their distinct nature (enzyme and glycoprotein, respectively). BHK/SFM cells produced a sevenfold higher number of SFV-VRPs, as determined by qRT-PCR. These particles showed similar capacities of infecting BHK/FBS or BHK/SFM cells. GFP expression was evaluated by flow cytometry, HCV-NS3p activity by enzymatic assay, and RVGP expression by ELISA and Western Blot. Expression analysis revealed higher levels of GFP and HCV-NS3p in BHK/SFM, while the levels of RVGP were similar for BHK/SFM and BHK/FBS. In conclusion, the BHK/SFM cells showed increased SFV-VRP production yields, without affecting vector infectivity or heterologous gene expression, hence validating the use of BHK/SFM for industrial applications.

POSTMORTEM DETECTION OF BLUETONGUE AND EPIZOOTIC HEMORRHAGIC DISEASE VIRUSES IN THE BONE MARROW OF WHITE-TAILED DEER (ODOCOILEUS VIRGINIANUS)

We determined the temporal aspects of detecting bluetongue virus (BTV) and epizootic hemorrhagic disease virus (EHDV) in postmortem bone marrow samples of white-tailed deer (Odocoileus virginianus) using molecular and in vitro cell culture techniques. Bone marrow samples from carcasses were collected and assayed on the day of death and at intervals up to 16 wk after death. We recovered BTV and EHDV from fresh bone marrow collected at day 0 by isolation in Vero and BHK-21 cell cultures. However, attempts to replicate the viruses from aged bone marrow in Vero and BHK-21 cell cultures failed. The real-time quantitative reverse transcriptase PCR (qRT-PCR) results confirmed that EHDV and BTV can be detected in aged bone marrow for up to 12 and 16 wk, respectively, after death. The RNA of BTV and EHDV could be detected by qRT-PCR in white-tailed deer bone marrow for extended periods of time postmortem. This technique will provide a useful tool for retrospective determination of BTV or EHDV infection of white-tailed deer at the time of death.

Design, Synthesis, Antiviral Evaluation, and SAR Studies of New 1-(Phenylsulfonyl)-1H-Pyrazol-4-yl-Methylaniline Derivatives.

A series of N-((3-phenyl-1-(phenylsulfonyl)-1H-pyrazol-4-yl)methyl)anilines 7a-p and 8a-l, structurally related to previously synthesized and tested (N-(1,3-diphenyl-1H-pyrazol-4-yl)methyl)anilines (1a-v), were designed and synthesized. The new derivatives were evaluated in cell-based assays for their cytotoxicity and antiviral activity against a large panel of RNA and DNA viruses of public health significance. Generally, the tested compounds did not display cytotoxicity toward the cell lines used. The majority of derivatives 7a-p were able to interfered with YFV and RSV replication in the micromolar range showing a marked improvement in potency and selectivity with respect to the reference inhibitors 6-azauridine and ribavirin, respectively. The introduction of a p-methoxy substituent on the phenylsulfonyl group (compounds 8a-l) completely abolished the anti-RSV activity and reduced or eliminated the potency against YFV. On the contrary, several p-methoxy analogs were able to interfere with BVDV replication with a comparable (8b, 8c, 8g, and 8k) or better (8a and 8f) potency than the reference inhibitor, ribavirin. Compound 7e, selected for time of addition experiments on BHK-21 cell cultures infected with YFV, achieved the highest reduction of virus titer when added 2 h post infection and maintained up to 4 h post infection.

Optimization of Vaccine Virus Accumulation in the Development of Smallpox Drugs Based on Cell Cultures

Objective. Optimization of vaccine virus cultivation in the suspended cell culture BHK-21 for infectious activity increment of virus-containing suspension as the base material for smallpox vaccine preparations. Materials and methods. We used suspended culture line of the cells BHK-21 of 72-hour age and nutrient medium of the MEM type in accordance with the guidelines on preparation in our studies. For challenging of the cells, vaccine virus (strain B-51) was used. The virus was adapted through three consequent passages on horion-allantois shell of developing chicken embryos of commercial dermovaccine series 449а at the premises of the Federal State Budgetary Institution “the 48th Central Research Institute” of the Ministry of Defense of the Russian Federation. Information on its genetic features is absent. Cultivation and precipitation of infected cells BHK-21 was carried out in bioreactor with priming volume of 1 liter at (36.5±0.5) °C and aeration with air mixture with varying content of CO2. Results and conclusions. Gas massexchange intensity was enhanced alongside simultaneous maintaining of sparing hydrodynamic conditions for mixing suspended cell cultures in bioreactor. Two-fold increase (up to (4.48±0.63)·109 cell/l) in suspended BHK-21 cell culture concentration at the end of reproduction cycle was achieved. Concentration of the vaccine virus was 3–5 times raised, from (8.1±0.3) lg PFU (plaque forming unit)/ml up to the level of infectious activity – (8.8±0.3) lg PFU/ml. Specific multiplicity of cell infection in recalculation per a cell was 1–5 PFU/cell and by virus yield – 20–100 PFU/cell. Enhanced infectious activity of the virus in concentrated suspension of infected BHK-21 cells substantiates the perspectives of the proposed method for improvement of vaccine virus accumulation phase in the development of anti-smallpox preparations based on cell cultures.

Inherent biophysical stability of foot-and-mouth disease SAT1, SAT2 and SAT3 viruses

Foot-and-mouth disease (FMD) virus (FMDV) isolates show variation in their ability to withstand an increase in temperature. The FMDV is surprisingly thermolabile, even though this virus is probably subjected to a strong extracellular selective pressure by heat in hot climate regions where FMD is prevalent. The three SAT serotypes, with their particularly low biophysical stability also only yield vaccines of low protective capacity, even with multiple booster vaccinations. The aim of the study was to determine the inherent biophysical stability of field SAT isolates. To characterise the biophysical stability of 20 SAT viruses from Southern Africa, the thermofluor assay was used to monitor capsid dissociation by the release of the RNA genome under a range of temperature, pH and ionic conditions.The SAT2 and SAT3 viruses had a similar range of thermostability of 48–54 °C. However, the SAT1 viruses had a wider range of thermostability with an 8 °C difference but with many viruses being unstable at 43–46 °C. The thermostable A-serotype A24 control virus had the highest thermostability of 55 °C with some SAT2 and SAT3 viruses of similar thermostability. There was a 10 °C difference between the most unstable SAT virus (SAT1/TAN/2/99) and the highly stable A24 control virus. SAT1 viruses were generally more stable compared to SAT2 and SAT3 viruses at the pH range of 6.7–9.1.The effect of ionic buffers on capsid stability showed that SAT1 and SAT2 viruses had an increased stability of 2–9 °C and 2–6 °C, respectively, with the addition of 1 M NaCl. This is in contrast to the SAT3 viruses, which did not show improved stabilisation after addition of 1 M or 0.5 M NaCl buffers. Some buffers showed differing results dependent on the virus tested, highlighting the need to test SAT viruses with different solutions to establish the most stabilising option for storage of each virus. This study confirms for the first time that more stable SAT field viruses are present in the southern Africa region. This could facilitate the selection of the most stable circulating field strains, for adaptation to cultured BHK-21 cells or manipulation by reverse genetics and targeted mutation to produce improved vaccine master seed viruses.

Cytotoxicity of Combination Chitosan with Different Molecular Weight and Ethanol Extracted Aloe vera using MTT Assay

The use of combination chitosan and ethanol extracted Aloe vera is considered to have activity to promote bone healing. The requirement dental material used in oral cavity include good biocompatibility, non toxic, non carcinogenic and nor allergenic response. The aim of this study was to examine the cytotoxicity combination chitosan with different molecular weight and ethanol extracted Aloe vera using MTT assay. 1 % chitosan gel (w/v) was made from chitosan powder with high (ChH) and low (ChL) molecular weight. 1 % chitosan gel was combined with ethanol extracted Aloe vera with concentration 50 % (Av1 and 75% (Av2). It divided five groups: control group, ChH-Av1, ChH-Av2, ChL-Av1 and ChL-Av2. Each of sample was immersed in eppendorf microtubes consist of media culture. After 24 hours, the immersion of media culture were used to investigate the cytotoxic effect to BHK-21 cell lines by MTT assay method. The density of optic formazan was indicated the number of living cells. All data were statistically analyzed by one-way Anova. The result showed that percentage of the living cells of all groups was uper 50 % using parameter CD50. There was no significant difference among treatment groups. It can be concluded that combination chitosan with different molecular weight and ethanol extracted Aloe vera were non toxic for BHK-21 cell culture.

Biological Assay of Foot and Mouth Disease Virus (FMDV) Serotypes for Titrating BLRI Developed Trivalent FMD Vaccines Seed

Foot and Mouth Disease (FMD) is an important viral as well as transboundary disease affecting almost all cloven-hoofed animals. The aim of the present study is the focused on determination of biological titer by tissue culture infective dose50 (TCID50) assay of currently available FMD virus serotype in Bangladesh. For adaptation of FMD virus (FMDV), BHK-21 cell line was used. BHK-21 cell subculture was done from preserved cultured bottle of Foot and Mouth Disease (FMD) Research Laboratory of Bangladesh Livestock Research Institute (BLRI), Savar, Dhaka. RT-PCR confirmed selected three different positive serotypes (O, A and Asia 1) of FMD virus were inoculated into BHK-21 cell culture and cytopathic effects (CPE) were observed after adaptation into BHK-21 cell from 3rd to 5th passages. Clear infectious BHK-21 cell culture fluid was collected and preserved at -80℃ temperature. The TCID50 assay was performed to determine the biological titer of the three positive serotype of FMDV. The biological titer of this study was found 106.5/ml viral titer for O type, 106.75/ml viral titer for A type, 106.66/ml viral titer for Asia-1 type. These three specific serotypes can be used as vaccine seed against FMD virus. The effective vaccination of susceptible animals is considered to be the corner stone to the disease in our country Bangladesh. The findings of this study can be helpful for the trivalent vaccine development in Bangladesh and it may effective in limiting the spread of FMD.