BHK-21 Cell Line Research Articles

Synthesis and Cytotoxicity of Some Thieno[2,3-d]pyrimidine Derivatives

A series of compounds 5-amino-2-ethylmercapto-4-phenyl-6-subistitutedthieno[2,3-d]pyrimidines (8a–d), 4-chloro-7-ethylmercapto-9-phenylpyrimido[5′,4′:4,5]thieno[3,2-d]triazine (9), and 2-ethylmercapto-8-oxo-4-phenyl-7-(4-chlorophenyl)pyrimido [4′,5′:4,5]thieno[2,3-d]pyrimidine (10) were synthesized and their structures were confirmed by 1H NMR, 13C NMR, and MS. All compounds were evaluated for their IC50 values against three cancer cell lines (MCF-7, HUH-7 and BHK) and WISH cells. The IC50 of compound (8d) was calculated for each cell line. Interestingly, the IC50 for the normal human amnion WISH cell line was much higher (723 µg/mL) than those found for the tumor cell lines BHK (17 µg/mL), HUH-7 (5.8 µg/mL), and MCF-7 (8.3 µg/mL). The proliferation inhibition of normal (WISH) and tumor (BHK, HUH-7, and MCF-7) cells by compound (8d) was investigated using MTT assay, and the IC50 was calculated after 48 h of treatment for each cell line.

Development of an Immunoperoxidase Monolayer Assay for the Detection of Antibodies against Peste des Petits Ruminants Virus Based on BHK-21 Cell Line Stably Expressing the Goat Signaling Lymphocyte Activation Molecule.

From 2013 to 2015, peste des petits ruminants (PPR) broke out in more than half of the provinces of China; thus, the application and development of diagnostic methods are very important for the control of PPR. Here, an immunoperoxidase monolayer assay (IPMA) was developed to detect antibodies against PPR. However, during IPMA development, we found that Vero cells were not the appropriate choice because staining results were not easily observed. Therefore, we first established a baby hamster kidney-goat signaling lymphocyte activation molecule (BHK-SLAM) cell line that could stably express goat SLAM for at least 20 generations. Compared with Vero cells, the PPR-mediated cytopathic effect occurred earlier in BHK-SLAM cells, and large syncytia appeared after virus infection. Based on this cell line and recombinant PPR virus expressing the green fluorescent protein (GFP) (rPPRV-GFP), an IPMA for PPR diagnosis was developed. One hundred and ninety-eight PPR serum samples from goats or sheep were tested by the IPMA and virus neutralization test (VNT). Compared with the VNT, the sensitivity and specificity of the IPMA were 91% and 100%, respectively, and the coincidence rate of the two methods was 95.5%. The IPMA assay could be completed in 4 h, compared with more than 6 d for the VNT using rPPRV-GFP, and it is easily performed, as the staining results can be observed under a microscope. Additionally, unlike the VNT, the IPMA does not require antigen purification, which will reduce its cost. In conclusion, the established IPMA will be an alternative method that replaces the VNT for detecting antibodies against PPRV in the field.

Expression System Based on an MTIIa Promoter to Produce hPSA in Mammalian Cell Cultures

Because of the limitations of standard culture techniques, the development of new recombinant protein expression systems with biotechnological potential is a key challenge. Ideally, such systems should be able to effectively and accurately synthesize a protein of interest with intrinsic metabolic capacity. Here, we describe such a system that was designed based on a plasmid vector containing promoter elements derived from the metallothionein MTIIa promoter, as well as processing and purification elements. This promoter can be induced by heavy metals in a culture medium to induce the synthesis of human prostate-specific antigen (hPSA), which has been modified to insert elements for purification, proteolysis, and secretion. We optimized hPSA production in this system by comparing the effects and contributions of ZnCl2, CdCl2, and CuSO4 in HEK293FT, HeLa, BHK-21, and CHO-K1 cells. We also compared the effectiveness of three different transfection agents: multi-walled carbon nanotubes, Lipofectamine 2000, and X-tremeGENE HP Reagent. hPSA production was confirmed via the detection of enhanced green fluorescent protein fluorescence, and cell viability was determined. The expression of hPSA was compared with that of the native protein produced by LNCaP cells, using enzyme-linked immunosorbent assay and sodium dodecyl sulfate polyacrylamide gel electrophoresis. X-tremeGENE reagent, the BHK-21 cell line, and CuSO4 showed the highest hPSA production rates. Furthermore, BHK-21 cells were more resistant to the oxidative stress caused by 100 μM CuSO4. These results suggest that the proposed optimized inducible expression system can effectively produce recombinant proteins with desired characteristics for a wide range of applications in molecular biology.

Genetic and biological characterization of Zika virus from human cases imported through Shenzhen Port

During February to March 2016, a total of 13 Zika virus (ZIKV) cases were imported into China by travelers. Infection with ZIKV in humans results in fever, rash and has been associated with microcephaly as well as Guillain-Barre syndrome. ZIKV is a mosquito-borne flavivirus and Aedes mosquitoes are the main transmission vector species. Since Aede s are prevalent throughout China, ZIKV has the potential to become widely disseminated if not properly controlled. The genetic and biological characteristics of ZIKV should be thoroughly studied in order to enact effective countermeasures. A novel ZIKV strain (SZ_SMGC-1) was successfully isolated from the sera of an imported case returning to China from Fiji and Samoa at the Shenzhen Port. The entire viral genome was sequenced and compared to whole ZIKV genomes downloaded from GenBank. Results showed that SZ_SMGC-1 possesses the highest nucleotide identity (99.9%) to ZIKV from Fiji and Samoa, and also has high nucleotide identity (99.0%–99.4%) to ZIKV from Venezuela. Additionally, ZIKV is know to have evolved into distinct Asian and African lineages, and our phylogenetic analyses showed that the ZIKVs isolated from all imported cases into China clustered into the Asian lineage. To study the biological characteristics of ZIKV, SZ_SMGC-1 was used to infect different cell lines including the mosquito- derived C6/36, monkey-derived Vero and hamster-derived BHK-21 cells. Infection with SZ_SMGC-1 in vitro resulted in considerable cytopathic effects (CPE) to mammalian cells and supported replication of the virus to high titers, but did not cause readily observable CPE in C6/36 cells despite virus replication to comparable titers with mammalian cell lines. Interestingly, two clones of Vero and BHK-21 cell lines were used in the present study, respectively, but ZIKV SZ_SMGC-1 replicated much quicker to high virus titers in one clone than the other. Furthermore, there are no effective vaccine or drug for ZIKV until now. The results of the genetic and biological characteristics and the sensitive cell lines form the basis for further investigation into the pathogenesis and development of an effective medical countermeasure against ZIKV.

Characterization of Recent Isolates of Newcastle Disease virus

Fifty five tissue samples were collected aseptically from 5 flocks suspected to be naturally infected with NDV in EL- Ismalia, Al-sharkia, El- Gharbia, kafr-Elshiekh & El-Geza and subjected to trials of virus isolation. Nine field samples from 2 flocks (Kafr-Elshiekh and Giza) were positive for virus isolation. Inoculation of SPF chicken eggs induced death of the embryo within 48-72 hours post inoculation. HA test applied on the obtained allantoic fluids showed positive results suggesting that inoculated samples contained NDV. It was noticed that highest infectivity titer of the virus was 9.5 log10 EID50/ml by the 5th passage in SPF chicken eggs. Cell culture passage of the obtained virus isolate from the allantoic fluid through ten consecutive passages in BHK cell line. the CPE developed at the 3rd passage within 24-48 hours post cell infection where polykaryocytosis, cell syncitia, rounding of cells. Tissue cultured fluids (TCF) were found to be direct HA possitive. The virus titer was 3 log10 TCID50 /ml at the 3rd passage and reach to 7 log10 TCID50 /ml by the 6th passage. At the 9th passage, the virus titer was reached to 8.5 log10 TCID50 /ml. Identification of the obtained virus isolate was confirmed by VNT using specific anti-NDV in BHK cell. The isolate of NDV with HA-positive allantoic fluids of El-Giza flockes were virulent NDVand the F gene give expected size of 724 bp. The isolate of this study also compared against the reference and vaccinal strains from gene bank and the results revealed that the isolate NDV/chicken/Egypt/Giza/2015 was velogenic type resembling the genotype VIId strains. F gene analysis of the NDV/chicken/Egypt/Giza/2015 revealed that the most nucleotides identity from gene bank with this isolate were NDV/Chicken/GC/IS/2010/1224 and NDV/buzzard/Israel/714/2011 with a nucleotide identity of 92.7%. It was also closely related to Chicken/China/SDWF07/2011; Chicken/China/Shandong/01/2012 and NDV/Turkey/Israel/111/2011 with a nucleotide identity of 92.5%. Otherwise nucleotides identity between the selected NDV field isolate in this study and NDV/Lasota was 72.4%. Phylogenetic analysis of NDV/chicken/Egypt/Giza/2015 with other reference and vaccinal strains of NDV revealed that this isolate was related to NDV/B7/RLQP/CH/EG/12 and present in the same group with NDV/F388/RLQP/CH/EG/14 and NDV/F460/RLQP/CH/EG/13. Regarding evaluation of the prepared experimental vaccines, it was found that the NDV infected tissue culture fluid, were completely inactivated with 3% BEI at 30 °C for 18 hrs post treatments showing no pathological changes in the ECE and no CPE in the BHK cell culture. Humoral immune response of chickens to the prepared vaccines showed that vaccinated chicks with NDV-Montanide ISA 70 adjuvanted vaccine exhibited antibody titer of 8.3 log2 three weeks post vaccination recording the higher titer of (10.6 log2) at the 6th week post vaccination with and 3rd week post challenge test. The high antibodies values were persist till the 12th weeks post NDV-Montanide ISA 70 vaccination followed by gradual decrease till the end of the experiment (24th weeks). On the other hand, vaccination of chicks with inactivated NDV isolate without adjuvant failed to induce protective HI antibodies (3 log2) all over the experiment. Challenge test showed that chickens vaccinated with ISA-70 adjuvant vaccine showed no sign of disease and no mortalities while the antigen without adjuvant could not provide any protective efficiency to SPF chickens.

Comparison of leucine-rich repeat-containing G protein-coupled receptor 5 expression in different cancer and normal cell lines.

Evaluating the expression of leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5) may be useful for predicting the best models and achieving more accurate results in cancer research. Therefore, the aim of the present study was to analyze the LGR5 expression levels in different cell lines. Eight commonly used cell lines were assessed (COS-7, NIH3T3, HEK293, VERO, HeLa, BHK, HepG2 and AGS). All the cell lines were cultured in RPMI-1640 medium contain 10% fetal calf serum at 37°C in humidified conditions with 5% CO2. According to the western blotting results, LGR5 was expressed in all cell lines. Densitometry results of LGR5 expression in the different cell lines showed that high LGR5 expression levels were apparent in BHK, AGS, VERO and NIH3T3 cell lines compared with the other cell lines. The results indicate that for the normal and cancer cell lines, BNK and AGS may be a better choice, respectively, for in vitro cancer studies.

RT-PCR based detection and adaptation of foot and mouth disease virus serotype “A” in BHK-21 cell line

The economic consequences of an outbreak of Foot and Mouth Disease (FMD) in Bangladesh is devastating. A sensitive, reliable and rapid diagnosis is crucial for the effective control of FMD. The present research was conducted for quick molecular detection and adaptation of FMD virus (FMDV) in BHK-21 (Baby Hamster Kidney) cell line. A two-step RT-PCR method was applied for the detection of the FMDV. Without prior adaption into BHK-21 cell culture, it is difficult to detect FMDV directly from the field samples by RT-PCR method. Samples were collected from the tongue epithelium (N=19) and inter digital tissues (N=4) of the suspected animals in Sujanagar, Bera, Santhia of Pabna district and Kotowali, Trishal of Mymensingh districts of Bangladesh during the year 2014. BHK-21 cell subculture was done from a previously cultured bottle containing BHK-21 cells. Prepared inocula were inoculated into BHK-21 cell culture and incubated at 37ºC for 24 h. After 36 h, cytopathic effects (CPE) were observed in BHK-21 cell line characterized by rounding and flattening of the cells, multinucleated giant cells formation, breaking down of the intracellular bridges and finally cell death indicated the presence of FMDV. Clear infectious BHK-21 cell culture fluid was collected and preserved at -20ºC temperature for virus detection by RT-PCR with serotype specific primers. Viral RNA was extracted from the clear infectious cell culture fluid for cDNA synthesis and used for PCR. Out of 23 samples tested, 3(13.04%) were positive for FMDV serotype A. The findings of this study can be helpful for the selection of vaccine having specific FMDV type, and it may help in controlling FMD in Bangladesh.Progressive Agriculture 27 (1): 64-69, 2016

ABCA1-Mediated Cholesterol Efflux Capacity to Cerebrospinal Fluid Is Reduced in Patients With Mild Cognitive Impairment and Alzheimer's Disease.

BackgroundAnimal and human studies indicate that ABCA1‐mediated cholesterol transport is important in Alzheimer's disease (AD). We hypothesized that the efficiency of cerebrospinal fluid (CSF) to facilitate ABCA1‐mediated cholesterol efflux would be reduced in participants with mild cognitive impairment (MCI) or AD compared with cognitively healthy participants.Methods and ResultsCSF was collected from a cross‐sectional study of cognitively healthy participants (n=47) and participants with MCI (n=35) or probable AD (n=26).The capacity of CSF to mediate cholesterol transport was assessed using a BHK cell line that can be induced to express the ABCA1 transporter. ABCA1‐mediated cholesterol efflux capacity was 30% less in participants with MCI or AD compared with cognitively healthy participants (P<0.001 for both). Cholesterol efflux capacity correlated with CSF cholesterol content (r=0.37, P<0.001). CSF phosphatidylcholine decreased in participants with MCI and AD compared with cognitively healthy participants (9% less in MCI and 27% less in AD compared with cognitively healthy participants, P=0.01) and correlated with CSF efflux capacity (r=0.3, P=0.001). CSF sphingomyelin also correlated with the efflux capacity (r=0.24, P=0.02). Concentrations of CSF apoA‐I and apoE did not significantly correlate with measures of efflux capacity.ConclusionsIn people with MCI and AD, the capacity of CSF to facilitate ABCA1‐mediated cholesterol efflux is impaired. This lesser cholesterol efflux in MCI supports a pathophysiological role for ABCA1‐mediated cholesterol transport in early neurodegeneration.

Safety evaluation of Acalypha wilkesiana in albino rats and BHK-21 cell line

Acalypha wilkesiana is a member of the spurge family, genus Acalypha, that is widely used in folklore medicine. The aim of this study was to screen aqueous leaf extract of A. wilkesiana for toxic effects in vitro and in vivo. We examined the phytochemical profile, cytotoxic effects on baby hamster kidney cell line (BHK-21), and oral subacute toxicity of A. wilkesiana leaf decoction in rats. Rats were given 0, 300, 600, and 1200 mg/kg body weight of A. wilkesiana leaf extract, daily, orally for 14 days. The phytochemical profile showed the presence of flavonoids, saponin, cardiac glycosides, and tannins. It caused apoptosis in BHK-21 cell line at concentrations of 0, 25, 50, 100, and 200 μg/ml. There was significant increase in the levels of serum AST, ALT, creatinine, urea, Na+, K+, and Cl− levels in all the test groups compared to the control. Histology of the liver revealed centrilobular degeneration and necrosis with sinusoidal dilatation as well as polymorphonuclear and mononuclear infiltration. The kidney showed severe glomerular and tubular degeneration and necrosis with hemorrhage at all doses administered. We conclude that the plant was toxic at the doses tested in vitro and in vivo, and care should be exercised in its use in herbal medicine.

Preparation of Inactivated Trivalent FMD Vaccine and Determination of Antibody Titre in Vaccinated Cattle

Aims: This research work was conducted for isolation and molecular detection of Foot and Mouth Disease (FMD) virus from the field samples, development of inactivated trivalent FMD vaccine, and determination of antibody titer in vaccinated cattle. Methodology: A total of 10 samples (tongue epithelium) were collected from FMD affected cattle from Gazipur, Mymensingh, and Pabna districts of Bangladesh during May 2014. Inoculum was prepared from the sample and the associated FMDV was propagated in BHK-21 cell lines. Besides, viral RNA was extracted for molecular detection (RT-PCR) of the serotypes involved. The isolated serotypes were used as seed virus in preparation of binary ethyleneimine (BEI) inactivated trivalent FMD vaccine using saponin and oil adjuvants. For the determination of antibody production in Original Research Article Chowdhury et al.; IJTDH, 16(2): 1-8, 2016; Article no.IJTDH.25870 2 response to our trivalent FMD vaccine, a total of 25 cattle aging 11-22 months were used. Sera of vaccinated cattle were collected on day 0, 21, 35, 49, 63. The sera were subjected for the determination of antibody level using enzyme-linked immunosorbent assay (ELISA). The titer values were statistically analyzed using one-way ANOVA to see immune response against the serotypes. Results: Three serotypes of FMDV were detected; these were FMD serotype-O, A and Asia-1, of which serotype-O was mostly prevalent, followed by serotype-A and Asia-1. The highest mean antibody titer was found on day 63 in all serotypes. Sixteen cattle (80%) out of 20 vaccinated cattle obtained protective antibody titer. The titer values of the vaccinated cattle were statistically significant against O, A and Asia-1 serotypes. Conclusion: FMD serotype-O, A, and Aisa-1 are prevailing in Bangladesh. A trivalent inactivated FMD vaccine has been prepared successfully using circulating virus of Bangladesh. The vaccine can be used to combat FMD in Bangladesh.

Discovery of berberine, abamectin and ivermectin as antivirals against chikungunya and other alphaviruses

Chikungunya virus (CHIKV) is an arthritogenic arbovirus of the Alphavirus genus, which has infected millions of people after its re-emergence in the last decade. In this study, a BHK cell line containing a stable CHIKV replicon with a luciferase reporter was used in a high-throughput platform to screen approximately 3000 compounds. Following initial validation, 25 compounds were chosen as primary hits for secondary validation with wild type and reporter CHIKV infection, which identified three promising compounds. Abamectin (EC50 = 1.5 μM) and ivermectin (EC50 = 0.6 μM) are fermentation products generated by a soil dwelling actinomycete, Streptomyces avermitilis, whereas berberine (EC50 = 1.8 μM) is a plant-derived isoquinoline alkaloid. They inhibited CHIKV replication in a dose-dependent manner and had broad antiviral activity against other alphaviruses - Semliki Forest virus and Sindbis virus. Abamectin and ivermectin were also active against yellow fever virus, a flavivirus. These compounds caused reduced synthesis of CHIKV genomic and antigenomic viral RNA as well as downregulation of viral protein expression. Time of addition experiments also suggested that they act on the replication phase of the viral infectious cycle.

In vitro evaluation of antiviral activity of leaf extracts of Azadirachta indica, Moringa oleifera, and Morus alba against the foot and mouth disease virus on BHK-21 cell line

In vitro evaluation of antiviral activity of leaf extracts of Azadirachta indica, Moringa oleifera, and Morus alba against the foot and mouth disease virus on BHK-21 cell line

SA14-14-2Rep: A Replicon Vector Derived from the Live-Attenuated Japanese Encephalitis Vaccine SA14-14-2 Virus for Human and Veterinary Medicine

Japanese encephalitis (JE), an acute inflammatory disease of the brain, is caused by infection with Japanese encephalitis virus (JEV). Children are most susceptible to JEV, which is commonly found in Asia. JEV is an enveloped RNA virus that is genetically similar to West Nile, dengue, and yellow fever viruses. No specific antiviral drugs are currently available to treat JE disease, but four types of vaccine are licensed locally to prevent JEV infection. However, the disease continues to be an important global health priority. Of the four vaccines, the most widely used in JE-endemic countries is the live-attenuated vaccine SA14-14-2. In our previous work, we cloned a genomelength cDNA of SA14-14-2 and rescued recombinant viruses entirely from the cloned cDNA (pBac/SA14-14-2). In the present study, we engineered this functional SA14-14-2 cDNA to construct a self-replicating subgenomic replicon (pBac/SA14-14-2Rep) by deleting the coding region for its two viral envelope glycoproteins, prM and E. After transfection of BHK-21 cells with the replicon RNA transcribed in vitro from pBac/SA14-14-2Rep, a combination of confocal microscopic imaging and immunoblotting showed the transient replication of the replicon RNA in the cytoplasm of RNA-transfected cells. Moreover, transfection of the SA14-14-2 replicon RNA into a BHK-21 cell line stably expressing all three JEV structural proteins (C, prM and E) led to the production of singleround infectious particles that packaged the replicon RNA, with a titter of ~105 infectious units per ml. Our SA14-14-2-based replicon represents a valuable tool for studying JEV RNA replication in low-level bio containment facilities and provides a useful platform for developing new vaccine vectors against an array of human and animal pathogens.

Nano-Micelle of Moringa Oleifera Seed Oil Triggers Mitochondrial Cancer Cell Apoptosis

Cancer, a worldwide epidemic disease with diverse origins, involves abnormal cell growth with the potential to invade other parts of the body. Globally, it is the main cause of mortality and morbidity. To overcome the drawbacks of the commercially available chemotherapies, natural products-loaded nano-composites are recommended to improve cancer targetability and decrease the harmful impact on normal cells. This study aimed at exploring the anti-cancer impacts of Moringa oleifera seed oil in its free- (MO) and nano-formulations (MOn) through studying whether it mechanistically promotes mitochondrial apoptosis-mediating cell death. Mitochondrial-based cytotoxicity and flow cytometric-based apoptosis analyses were performed on cancer HepG2, MCF7, HCT 116, and Caco-2 cell lines against normal kidney BHK-21 cell line. The present study resulted that MOn triggered colorectal cancer Caco-2 and HCT 116 cytotoxicity via mitochondrial dysfunction more powerful than its free counterpart (MO). On the other side, MOn and MO remarkably induces HCT 116 mitochondrial apoptosis, while sparing normal BHK-21 cells with minimal cytotoxic effect. The present results concluded that nano-micelle of Moringa oleifera seed oil (MOn) can provide a novel therapeutic approach for colorectal and breast cancers via mitochondrial-mediated apoptosis, while sparing normal and even liver cancer cells a bit healthy or with minimal harmful effect. Intriguingly, MOn induced breast cancer not hepatocellular carcinoma cell death.

Intrinsic, extrinsic and endoplasmic reticulum stress-induced apoptosis in RK13 cells infected with equine arteritis virus

The modulation of the expression of caspases by viruses influences the cell survival of different cell types. Equine arteritis virus (EAV) induces apoptosis of BHK21 and Vero cell lines, but it is not known whether EAV induces apoptosis in RK13 cells, a common cell line routinely used in EAV diagnosis and research. In this study, we determined that caspase-3 expression was triggered after infection of RK13 cells with EAV in a time- and dose-dependent manner. We also detected caspase-8 and caspase-9 activation, indicating the stimulation of both extrinsic and intrinsic apoptosis pathways. Finally, we found caspase-12 activation, an indicator of endoplasmic reticulum stress-induced apoptosis. The variability observed in the apoptotic response in the different cell lines demonstrates that apoptosis depends on the distinctive sensitivity of each cell line used for investigation.

Molecular and immunogenic characterization of BHK-21 cell line adapted CVS-11 strain of rabies virus and future prospect in vaccination strategy.

Development of a cost effective quality vaccine is a key issue in rabies control programme in developing countries. With this perspective, in the present study, challenge virus standard (CVS)-11 strain of rabies virus was adapted to grow in BHK-21 cells, characterized, compared with other viruses including global vaccine strains and field isolates from Indian subcontinent and China at molecular level. This cell adapted virus was evaluated for the production of cost effective veterinary vaccine. The maximum virus titre achieved was 10(7)fluorescent focus unit (FFU)/mL at 10th passage level. There was no nucleotide difference in the nucleoprotein (N) and glycoprotein (G) genes after adaptation in cell line. Phylogenetic analysis showed that adapted virus was grouped with global vaccine strains, closest being with other CVS strains but distinct from the Indian field isolates. Global vaccine strains including cell adapted CVS-11 virus have 83-87% identity at nucleotide level of G gene with Indian field viruses. Growth kinetics of cell culture adapted virus showed that the optimum virus titer (around 10(7)FFU/mL) could be obtained at around 48h post infection by co-cultivation method using 0.1 multiplicity of infection inoculums at 37°C. These findings can be used for up scaling of vaccine production. The protective efficacy of test vaccine produced using 10(6.95)FFU/mL cell culture harvest showed 1.17IU/mL relative potency by NIH test. Further, adapted virus was found to be suitable for use in rapid fluorescent focus inhibition test.

Validation of γ-radiation and ultraviolet as a new inactivators for foot and mouth disease virus in comparison with the traditional methods.

Aim:The present work deals with different methods for foot and mouth disease virus (FMDV) inactivation for serotypes O/pan Asia, A/Iran05, and SAT-2/2012 by heat, gamma radiation, and ultraviolet (UV) in comparison with the traditional methods and their effects on the antigenicity of viruses for production of inactivated vaccines.Materials and Methods:FMDV types O/pan Asia, A/Iran05, and SAT-2/2012 were propagated in baby hamster kidney 21 (BHK21) and titrated then divided into five parts; the first part inactivated with heat, the second part inactivated with gamma radiation, the third part inactivated with UV light, the fourth part inactivated with binary ethylamine, and the last part inactivated with combination of binary ethylamine and formaldehyde (BEI+FA). Evaluate the method of inactivation via inoculation in BHK21, inoculation in suckling baby mice and complement fixation test then formulate vaccine using different methods of inactivation then applying the quality control tests to evaluate each formulated vaccine.Results:The effect of heat, gamma radiation, and UV on the ability of replication of FMDV “O/pan Asia, A/Iran05, and SAT-2/2012” was determined through BHK cell line passage. Each of the 9 virus aliquots titer 108 TCID50 (3 for each strain) were exposed to 37, 57, and 77°C for 15, 30, and 45 min. Similarly, another 15 aliquots (5 for each strain) contain 1 mm depth of the exposed samples in petri-dish was exposed to UV light (252.7 nm wavelength: One foot distance) for 15, 30, 45, 60, and 65 min. Different doses of gamma radiation (10, 20, 25, 30, 35, 40, 45, 50, 55, and 60 KGy) were applied in a dose rate 0.551 Gy/s for each strain and repeated 6 times for each dose. FMDV (O/pan Asia, A/Iran05, and SAT-2/2012) were inactivated when exposed to heat ≥57°C for 15 min. The UV inactivation of FMDV (O/pan Asia and SAT-2) was obtained within 60 min and 65 min for type A/Iran05. The ideal dose for inactivation of FMDV (O/pan Asia, A/Iran05, and SAT-2/2012) with gamma radiation were 55-60 and 45 kGy, respectively. Inactivation of FMDV with binary was 20, 24 and 16 hr for O/pan Asia, A/Iran05, and SAT-2/2012, respectively while inactivation by (BEI+FA) was determined after 18, 19 and 11 hr for O/pan-Asia, A/Iran 05, and SAT-2/2012, respectively. The antigenicity of control virus before inactivation was 1/32, it was not changed after inactivation in case of gamma radiation and (BEI+FA) and slightly decrease to 1/16 in case of binary and declined to 1/2, 1/4 in case of heat and UV inactivation, respectively. The immune response induced by inactivated FMD vaccines by gamma radiation and (BEI+FA) lasted to 9 months post-vaccination, while the binary only still up to 8 months post-vaccination but heat and UV-inactivated vaccines were not effective.Conclusion:Gamma radiation could be considered a good new inactivator inducing the same results of inactivated vaccine by binary with formaldehyde (BEI+FA).

Studies of the physicochemical and structural properties of self-assembling cationic pyridine derivatives as gene delivery agents

New amphiphilic pyridine derivatives containing dodecyloxycarbonyl substituents at positions 3 and 5 and cationic moieties at positions 2 and 6 have been designed and synthesised. Compounds of this type can be considered as synthetic lipids. The corresponding 1,4-dihydropyridine (1,4-DHP) derivatives have earlier been proposed as a promising tool for plasmid DNA (pDNA) delivery in vitro. In this work studies of the self-assembling properties of amphiphilic pyridine derivatives leading to the formation of liposomes, determination of particle size, zeta-potential and critical micelle concentration (CMC) with dynamic light scattering (DLS) measurements are described. Furthermore, thermal analysis of pyridine derivatives was performed using thermogravimetry analysis (TGA) and differential thermal analysis (DTA) as well as the ability to deliver the pEGFP-C1 plasmid DNA (that encodes GFP reporter) into the Baby hamster kidney-derived (BHK-21) cell line was used for evaluation of gene delivery properties. We have revealed that the new pyridine derivatives possessed self-assembling properties which were proved by formation of nanoparticles with the average size from 115 to 743nm, the zeta-potentials in the range of 48–79mV and CMC values in the range of 2–67μM. DTA data showed that all processes were endothermic for all compounds. Additionally, we established that among the tested pyridines the representatives with N-methylpyrrolidinium or pyridinium moieties as cationic head-group at the positions 2 and 6 possessed higher pEGFP-C1 transfection activity into the BHK-21 cell line. Nevertheless, the obtained results indicated that correlation of the physicochemical, structural properties and gene delivery activities of the tested compounds were not completely elucidated yet. On the other hand, the synthesised pyridines as possible metabolites of promising delivery systems on the 1,4-DHP core possessed lower pDNA transfection activity than the corresponding 1,4-DHP amphiphiles.

Isolation of buffalo poxvirus from clinical case and variations in the genetics of the B5R gene over fifty passages.

Outbreaks of buffalopox affect udder and teats, which may ultimately lead to mastitis in dairy buffalo and can significantly compromise the production. In this study, we report isolation of buffalo poxvirus and sequence analysis of the B5R gene collected from the buffalo clinically suspected to be poxvirus infected. The virus was isolated on BHK-21 cell line and was passaged for 50 times, B5R gene was amplified and sequenced using gene-specific primers, and analyzed at both nucleotide and amino acid levels. Phylogenetically, the isolate can be classified close to the previously reported Pakistani and Indian isolates with certain level of differential clustering patterns. Three significant putative mutations (I2K, N64D, and K111E) were observed in the B5R protein. The K111E was common with previous human isolate from Karachi, Pakistan in 2005. These mutations differed from poxviruses reported from the neighboring countries. Some deletion mutations were observed which were recovered in upcoming passages. The K111E mutation suggests potential to cause zoonotic infection in human all over the country.

Preparation and characterization of a stable BHK-21 cell line constitutively expressing the Schmallenberg virus nucleocapsid protein

Schmallenberg virus (SBV) is a newly emerged orthobunyavirus that predominantly infects livestock such as cattle, sheep, and goats. Its nucleocapsid (N) protein is an ideal target antigen for SBV diagnosis. In this study, a stable BHK-21 cell line, BHK-21-EGFP-SBV-N, constitutively expressing the SBV N protein was obtained using a lentivector-mediated gene transfer system combined with puromycin selection. To facilitate the purification of recombinant SBV N protein, the coding sequence for a hexa-histidine tag was introduced into the C-terminus of the SBV N gene during construction of the recombinant lentivirus vector pLV-EGFP-SBV-N. The BHK-21-EGFP-SBV-N cell line was demonstrated to spontaneously emit strong enhanced green fluorescent protein (EGFP) signals that exhibited a discrete punctate distribution throughout the cytoplasm. SBV N mRNA and protein expression in this cell line were detected by real-time RT-PCR and western blot, respectively. The expressed recombinant SBV N protein carried an N-terminal EGFP tag, and was successfully purified using Ni–NTA agarose by means of its C-terminal His tag. The purified SBV N protein could be recognized by SBV antisera and an anti-SBV monoclonal antibody (mAb) 2C8 in an indirect enzyme-linked immunosorbent assay and western blot analyses. Indirect immunofluorescence assays further demonstrated that the stable cell line reacts with SBV antisera and mAb 2C8. These results suggest that the generated cell line has the potential to be used in the serological diagnosis of SBV.