cell culture processes for viral vaccine production are mainly based on adherent cell culture systems using serum, which are associated with expensive and labor-intensive processes to produce large amounts of viral vaccine strains. In this study, we investigated whether Vero cells could be grown in serum-free and shaking suspension conditions. Furthermore, we assessed the ability of the Vero cell suspension culture system to produce adenovirus type 5 (Ad5), compared to that of the adhesive Vero cell culture system.MATERIALS AND METHODS: We tested the feasibility of commercial serum-free media for Vero cell culture. For the adaptation of Vero cells in suspension culture, adhesive Vero cells were added in the early phase of shaking suspension culture, and 50 days after shaking suspension culture, suspension-adapted Vero cells were subcultured continuously. To assess the virus production ability of Vero cells in suspension, the cells were infected with Ad5-green fluorescent protein and evaluated based on their fluorescence intensity.RESULTS: The Vero cells grown in OptiPRO serum-free medium showed no changes in morphology and growth rate, but MRC-5 and FRhk-4 cells showed morphological changes and decreased growth rate, respectively. The Vero cells were well adapted to the suspension culture system. The Vero cells in suspension showed a better Ad5 production ability than the adherent Vero cells.CONCLUSION: Vero cells can be grown in OptiPRO serum-free medium. Further, our suspension culture-adapted Vero cells may be suitable to produce viral vaccine strains due to their high ability to produce viruses such as Ad5.]]>
Adenoviridae ; Adhesives ; Cell Culture Techniques ; Culture Media, Serum-Free ; Fluorescence ; Vero Cells

Animals ; Antibodies, Monoclonal ; immunology ; Cell Line ; Cercopithecus aethiops ; Cricetinae ; Culicidae ; Dengue Virus ; immunology ; Encephalitis Virus, Japanese ; immunology ; Encephalitis, Japanese ; immunology ; Mice ; Mice, Inbred Strains ; Vero Cells ; Viral Vaccines ; immunology

To study on the molecular evolution of Coxsackie virus A16 (CVA16)isolated from clinical speci-mens of Hand, foot and mouth Disease( HFMD) patients in Inner Mongolia in 2010. A total of 921 clinical specimens including stools, throat swabs and vesicle fluids were collected from 888 HFMD patients in out-patient service in Inner Mongolia and viral isolation was then performed, the positive viral isolates were identified by using the real-time PCR method detecting CVA16. A total of 50 CVA16 isolates were selected from the patients presenting mild symptoms, severe symptoms and the death patients randomly, and the VP1 coding regions of representative CVA16 isolates were amplified and sequenced. Finally the phylogenetic tree was constructed among the VP1 coding regions of the different genotypes and subgenotypes of CVA16 strains. Eighty two viruses were isolated form 921 clinical specimens, the positive rate was 8. 90%, of which 3 viruses were isolated from severe cases and 1 viruses was from death cases. The nucleotide acid of 50 representative CVA16 strains in Inner Mongolia were closed to CVA16 strains isolated from mainland China since 1998, especially from Beijing in 2009 and from Henan in 2010, the identity were 96. 18% approximately 98. 88% and 94. 94a approximately 98. 76%, respectively. There was a little difference in the nucleotide acid between the CVA16 strains from Inner Mongolia in 2010 and in 2007, the identity were 91. 68% approximately 96. 52% The phylogenetic tree showed that all CVA16 strains clustered within Bla and B1b evolution branch of B1 genotype. There was slight difference in the nucleotide and the amino acid in VP1 region among the 50 Inner Mongolia CVA16 strains, the identity were 89. 99% approximately 100% and 98. 31% approximately 100%, respectively, indicating that these strains belonged to many different viral transmission chains. The CVA16 strains circulated in Inner Mongolia in 2010 were all belong to B1a and B1b evolution branch of B1 genotype, and the two evolutionary branchs of Coxsackie virus A16 were co-evolved and co-prevailed in Inner Mongolia Autonomous Region.
Adolescent ; Adult ; Animals ; Capsid Proteins ; genetics ; Cell Line, Tumor ; Cercopithecus aethiops ; Child ; Child, Preschool ; China ; epidemiology ; Coxsackievirus Infections ; epidemiology ; mortality ; virology ; Enterovirus ; classification ; genetics ; isolation & purification ; Evolution, Molecular ; Feces ; virology ; Female ; Genotype ; Hand, Foot and Mouth Disease ; epidemiology ; mortality ; virology ; Humans ; Infant ; Male ; Phylogeny ; RNA, Viral ; genetics ; Sequence Analysis, DNA ; Vero Cells ; Young Adult

Activities of 58 miRNAs for BHK21, HEK293, and Vero cell lines were screened using the high-throughput miRNA activity profiling method. miR-206 activity was found specifically high in BHK21. Considering miR-206 was recognized as a muscle-specific miRNA, we further detected the expression and activity level of miR-206 in BHK21 cells, with myoblast cells C2C12 as positive control and HEK293 cells as negative control. Then, we induced BHK21 by culturing with medium containing 2% horse serum (HS) and tested expression level of slow skeletal myosin heavy chain (MHC), activity and expression levels of miR-206, and expression level of Connexin43 (Cx43) which was reported negatively regulated by miR-206. Results demonstrated that activity and expression levels of miR-206 were both higher in BHK21 cells than in C2C12 cells. After induction of HS, MHC expression level was increased in BHK21 cells. The activity and expression levels of miR-206 were further enhanced. The Cx43 expression level was decreased. These results suggested that BHK21 had the characters of myoblast cells. In conclusion, we firstly discovered that miR-206 activity was specifically high in BHK21 cells, suggesting that BHK21 cells were derived from interstitial cells other than parenchyma cells of kidney from miRNA point of view. Our study also indicated that BHK21 cells were able to be used as models in vitro for research of miR-206 function.
Animals ; Cell Line ; Cercopithecus aethiops ; Connexin 43 ; metabolism ; Cricetinae ; Gene Expression Regulation ; HEK293 Cells ; Humans ; MicroRNAs ; genetics ; Myosin Heavy Chains ; metabolism ; Vero Cells

OBJECTIVETo develop a novel rapid protocol for the detection of occult disseminated tumor cells by a recombinant herpes simplex virus expressing GFP (HSV(GFP)).

METHODSTumor cells of seven cell lines were exposed to HSV(GFP) and then examined for GFP expression by fluorescence microscopy. Various numbers of tumor cells (10, 100, 1000, 10 000) were mixed into 2 ml human whole blood, separated with lymphocytes separation medium, exposed to HSV(GFP), incubated at 37°C for 6 - 24 h and then counted for the number of green cells under the fluorescence microscope. Some clinical samples including peripheral blood, pleural effusion, ascites, spinal fluid from tumor-bearing patients were screened using this protocol in parallel with routine cytological examination.

RESULTSHSV(GFP) was able to infect all 7 tumor cell lines indicating that the HSV(GFP) can be used to detect different types of tumor cells. The detection sensitivity was 10 cancer cells in 2 ml whole blood. In the clinical samples, there were 4/15 positive by routine cytological examination but 11/15 positive by HSV(GFP), indicating a higher sensitivity of this new protocol.

CONCLUSIONRecombinant herpes simplex virus-mediated green fluorescence is a simple and sensitive technique for the identification of occult disseminated cancer cells including circulating tumor cells (CTCs).

Animals ; Cell Line, Tumor ; Cercopithecus aethiops ; Green Fluorescent Proteins ; metabolism ; Humans ; Neoplastic Cells, Circulating ; metabolism ; pathology ; Recombinant Proteins ; metabolism ; Sensitivity and Specificity ; Simplexvirus ; metabolism ; Vero Cells

OBJECTIVETo investigate the cytotoxic effect of Elaeis guineensis methanol extract on MCF-7 and Vero cell.

METHODSIn vitro cytotoxicity was evaluated in by MTT assay. Cell morphological changes were observed by using light microscope.

RESULTSThe MTT assay indicated that methanol extract of the plant exhibited significant cytotoxic effects on MCF-7. Morphological alteration of the cell lines after exposure with Elaeis guineensis extract were observed under phase contrast microscope in the dose dependent manner.

CONCLUSIONSThe results suggest the probable use of the Elaeis guineensis methanol extract in preparing recipes for cancer-related ailments. Further studies on isolation of metabolites and their in vivo cytotoxicity are under investigation.

Animals ; Antineoplastic Agents, Phytogenic ; pharmacology ; Arecaceae ; chemistry ; Cell Proliferation ; drug effects ; Cercopithecus aethiops ; Dose-Response Relationship, Drug ; Humans ; MCF-7 Cells ; Methanol ; Plant Extracts ; pharmacology ; toxicity ; Plant Leaves ; chemistry ; Vero Cells

In order to find antiviral compounds with novel structures, geldanamycin and lamivudine with different antiviral mechanisms were conjunctively synthesized to acquire a new compound TC-GM, and the antiviral activity of TC-GM was measured. The antiviral activity against HIV-1 was examined by p24 antigen ELISA kit. The activity against HBV was examined by dotblot. The activity against HSV and CoxB virus was examined by CPE. TC-GM exhibited broad-spectrum antiviral activities similarly like geldanamycin. TC-GM inhibited the replication of different viruses, including HIV-1, HBV, HSV 1 and 2, CoxB6. TC-GM showed more potent inhibitory activity against HIV-1 and HBV than other detected virus.
Animals ; Anti-HIV Agents ; chemical synthesis ; chemistry ; pharmacology ; Antiviral Agents ; chemical synthesis ; chemistry ; pharmacology ; Benzoquinones ; chemical synthesis ; chemistry ; pharmacology ; Cell Line, Tumor ; Cercopithecus aethiops ; Enterovirus B, Human ; drug effects ; physiology ; HIV-1 ; drug effects ; physiology ; Hep G2 Cells ; Hepatitis B virus ; drug effects ; physiology ; Herpesvirus 1, Human ; drug effects ; physiology ; Herpesvirus 2, Human ; drug effects ; physiology ; Humans ; Lactams, Macrocyclic ; chemical synthesis ; chemistry ; pharmacology ; Lamivudine ; chemical synthesis ; chemistry ; pharmacology ; Madin Darby Canine Kidney Cells ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; pathology ; virology ; Vero Cells ; Virus Replication ; drug effects

Recently, BST-2 has been identified as an effective cellular factor that prevents the release of human immunodeficiency virus type 1 and other enveloped viruses by tethering virus particles to the cell surface. Here, we showed that the production of HIV-1 virus-like particles was markedly inhibited by BST-2. Both the transient and stable expressing of BST-2 had the same function and Vpu rescued the release of HIV-1 VLP in the presence of human BST-2. Consistent with a direct tethering mechanism, we confirmed that proteolysis releases restricted virions and further showed that this removed the ectodomain of BST-2 from the cell surface.
Animals ; Antigens, CD ; genetics ; metabolism ; Cell Line ; Cercopithecus aethiops ; Down-Regulation ; GPI-Linked Proteins ; genetics ; metabolism ; HIV Infections ; genetics ; metabolism ; virology ; HIV-1 ; genetics ; physiology ; Humans ; Vero Cells ; Virion ; genetics ; physiology ; Virus Replication

OBJECTIVETo research the cytotoxicity and in vitro antiproliferative effect of the six flavone compounds extracted from Laggera pterodonta.

METHODThe cytotoxicity on the normal cells and antiproliferative effect on tumor cells were tested by MTT assay, and then the preliminary structure-activity relationship was analysed. The phase distribution of the cell cycle and apoptosis rate were analyzed by flow cytometry.

RESULTThe results of MTT assay showed 5,7,3',4'-tetramethoxy-3-hydroxyflavone and chrysosplenetin B inhibited growth of A549 and Hela cells significantly with a dose dependent mode, while exhibited low cytotoxicity to the two normal cells, Vero and EVC304. Both compounds contain ortho-phenolic methoxyl moietys in their structures. Flow cytometry analysis revealed that Hela cells treated with increasing quantities of chrysosplenetin B increased the percentage of cells in the G2/M phase, and Hela and A549 cells treated with increasing quantites of the 5,7,3',4'-tetramethoxy-3-hydroxyflavone and chrysosplenetin B increased the apoptosis rates.

CONCLUSIONThe 5,7,3',4'-tetramethoxy-3-hydroxyflavone and chrysosplenetin B extracted from L. pterodonta showed high antiproliferative effect on cancer cells with low cytotoxicity on normal cells, and took the effects on A549 and Hela cells through the hold-up of the G2/M phase of the cell cycle and induction of the apoptosis.

Animals ; Asteraceae ; chemistry ; Cell Line ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cercopithecus aethiops ; Flavones ; chemistry ; pharmacology ; Flavonoids ; pharmacology ; Flow Cytometry ; HeLa Cells ; Humans ; Vero Cells

OBJECTIVETo find a sensitive cytotoxic response to reflect the bio-toxicity of trace organic pollutants, the sensitivity and reliability of morphological change and proliferation inhibition of Vero cells exposed to 2, 4, 6-trichlorophenol (TCP) and the leachate from products related to drinking water (PRDW) were compared, and the mechanism of the morphological change in Vero cells exposed to chemical pollutants was studied.

METHODSVero cells were treated by different concentration of TCP and the leachate from PRDW. Methylthiazol-2-yl-2, 5-diphenyl tetrazolium bromide (MTT) assay was carried out for proliferation inhibition. Bioluminescence method was carried out as another method to test the toxicity of TCP. Flow Cytometry assay was used to test cell Apoptosis and damage of cell-membrane.

RESULTS0.25 mg/L TCP had an effect on cell morphology, and the proportion of morphologically changed cells increased with increasing TCP concentration. At low TCP concentrations, inhibition of cell proliferation did not seem to correlate to TCP concentration, and was negative when TCP concentration was <1.0 mg/L. After exposure to leachate from PRDW extracted at different temperatures, the percentage of morphologically changed cells increased with extracting temperature, but the inhibition of cell proliferation failed to reflect the correlation between extracting temperature and proliferation inhibition of Vero cells. Although the Sensitivity of bioluminescence method seems to be similar to morphological change in Vero cells, the bacterial in this method is not homologous enough with human body cells to reflect the toxicity to human body. These imply cell morphological change is a more sensitive and reliable method to reflect bio-toxicity of organic pollutants than proliferation inhibition. Flow cytometry analysis and cell rejuvenation experiments indicated cell membrane damage, which results in cell morphological change, was an early and sensitive cytotoxic response comparing with necrosis.

CONCLUSIONThese results indicated that the cell membrane toxicity represented by morphological changes is a more sensitive and reliable method to indicate the composite bio-toxicity of trace chemicals than proliferation inhibition, inhibition on bioluminescence and necrosis. Nevertheless, the quantification of morphological change should be studied further.

Animals ; Cell Division ; drug effects ; Cell Survival ; drug effects ; Cercopithecus aethiops ; Vero Cells ; Water Pollutants, Chemical ; toxicity