Assessment of potential miRNA biomarkers of VERO-cell tumorigenicity in a new line (AGMK1-9T7) of African green monkey kidney cells.

Patterns of microRNA expression appear to delineate the process of spontaneous neoplastic development-transformation (SPNDT) occurring in the African green monkey kidney (AGMK) VERO cell line (Teferedegne et al., 2010). Analysis of microarray data identified 6 microRNAs whose high-level of expression peaked when the World Health Organization 10-87 VERO cells became tumorigenic at passage (p) 190. Six miRNAs were identified as potential biomarkers for the expression of the VERO-cell tumorigenic phenotype (Teferedegne et al., 2014). However, the question remained whether these miRNA biomarkers are specific for VERO cells or can be generalizable to other cells originating from African green monkey kidneys. To examine miRNA expression patterns in AGMK cells at lower passage levels and to re-examine the identified miRNAs as biomarkers associated with tumorigenic phenotype of VERO cells in another independently-derived line, we established a new line of African green monkey kidney cells (AGMK1-9T7) by serially passaging kidney cells from another AGM. The AGMK1-9T7 cells became tumorigenic in nude mice at p40. Evaluation of miRNA expression at intervals from p1 to p40 revealed similarities between the evolution of miRNA expression during SPNDT in the AGMK1-9T7 cells and the 10-87 VERO cells. Four of the 6 potential biomarker miRNAs (miR-376a, miR-654-3p, miR-543, miR-134) in our earlier reports were detected by microarray in the AGMK1-9T7 cells; RT-qPCR analysis detected all 6 miRNAs. All 6 of these miRNAs have been associated with human tumors. Detection of the same miRNAs associated with the tumorigenic p40 AGMK1-9T7 cells and tumorigenic 10-87 VERO cells confirmed our proposal that these miRNA represent biomarkers for the tumor-forming ability of AGMK/VERO cells. The similarities of expression of miRNAs in different AGMK cell lines that were established 50years apart suggest that the process of SPNDT in these non-human primate cells in tissue culture is based upon similar genetic and epigenetic mechanisms.

Culex mosquitoes are experimentally unable to transmit Zika virus.

We report that two laboratory colonies of Culex quinquefasciatus and Culex pipiens mosquitoes were experimentally unable to transmit ZIKV either up to 21 days post an infectious blood meal or up to 14 days post intrathoracic inoculation. Infectious viral particles were detected in bodies, heads or saliva by a plaque forming unit assay on Vero cells. We therefore consider it unlikely that Culex mosquitoes are involved in the rapid spread of ZIKV.

Potential toxic effects of iron oxide nanoparticles in in vivo and in vitro experiments.

The aim of this study was to determine the potential toxic effects of iron(II,III)oxide nanoparticles (IONPs). In in vivo experiments, the toxic effects of IONPs were monitored in adult male Wistar rats by morphological methods after a single intratracheal instillation. For the control group 1 ml of physiological saline per animal was given, and the treatment group received the same volume of a suspension containing 1 and 5 mg kg⁻¹ body weight IONPs. Lungs and internal organs underwent histopathological examination after 1, 3, 7, 14 and 30 days. The mutagenic effect of these nanoparticles was evaluated by the bacterial reverse mutation assay on Salmonella typhimurium TA98, TA100, TA1535 and TA1537 strains, and on Escherichia coli WP2uvrA strain, in the presence and absence of the mammalian metabolic activation system S9. The in vitro cytotoxic effect of IONPs was also examined in Vero cells after short-term (4 h) and long-term (24 h) exposure. There were no pathological changes in examined internal organs, except a very weak pulmonary fibrosis developing by the end of the first month in the treated rats. While in vitro MTT assay showed a moderate cytotoxic effect, IONPs proved to be devoid of mutagenic effect in the bacterial systems tested. The results may be a useful extension of our knowledge on the safety of magnetite nanoparticles in view of their possible medical applications, such as in hyperthermia and magnetic resonance imaging.

The open reading frame 3a protein of severe acute respiratory syndrome-associated coronavirus promotes membrane rearrangement and cell death.

The genome of the severe acute respiratory syndrome-associated coronavirus (SARS-CoV) contains eight open reading frames (ORFs) that encode novel proteins. These accessory proteins are dispensable for in vitro and in vivo replication and thus may be important for other aspects of virus-host interactions. We investigated the functions of the largest of the accessory proteins, the ORF 3a protein, using a 3a-deficient strain of SARS-CoV. Cell death of Vero cells after infection with SARS-CoV was reduced upon deletion of ORF 3a. Electron microscopy of infected cells revealed a role for ORF 3a in SARS-CoV induced vesicle formation, a prominent feature of cells from SARS patients. In addition, we report that ORF 3a is both necessary and sufficient for SARS-CoV-induced Golgi fragmentation and that the 3a protein accumulates and localizes to vesicles containing markers for late endosomes. Finally, overexpression of ADP-ribosylation factor 1 (Arf1), a small GTPase essential for the maintenance of the Golgi apparatus, restored Golgi morphology during infection. These results establish an important role for ORF 3a in SARS-CoV-induced cell death, Golgi fragmentation, and the accumulation of intracellular vesicles.

[The apoptosis of vero cells induced by Shiga toxin 2e].

OBJECTIVE: Shiga-like toxin-producing Escherichia coli (STEC) causes edema disease in piglets and hemolytic uremic syndrome in human. Shiga-like toxins (Stxs) produced by STEC induce mammalian cells death via either necrosis or apoptosis. However, the ability of stx2e, separated from edema disease (Stx2e), to trigger apoptosis and the sequence of intracellular signaling events have not yet been completely defined. In this study we investigated the apoptotic effects of Stx2e on Vero cells. METHODS: Vero cells were treated with different concentrations of Stx2e for different time and the apoptotic cells were characterized by acridine orange and ethidium bromide fluorescent dye staining. The fragmentation of chromatin from Vero cells treated with Stx2e were detected by agarose gel electrophoresis. The expression patterns of apoptosis-associated factors were assayed by Western blotting. RESULTS: Stx2e-treated cells showed characteristic features of apoptosis, including membrane blebbing, DNA fragmentation, chromatin condensation, and the formation of apoptotic bodies, whereas ricin did not induce apoptosis of Vero cells even at a high dose. Fluorescent dye staining showed that Stx2e induced apoptosis of Vero cells in dose- and time-dependent manners. Caspase-3 was activated whereas expression levels of bcl2 associated X protein (Bax) and caspase-9 had no change compared with the negative control. CONCLUSION: Stx2e induced intensively apoptosis of Vero cells, which was mediated through the mitochondrion-independent pathway and might be throught a receptor-dependent pathway.

High-level expression of glycoprotein D by a dominant-negative HSV-1 virus augments its efficacy as a vaccine against HSV-1 infection.

Using the T-REx (Invitrogen, Carlsbad, CA) gene switch technology, we previously generated a dominant-negative herpes simplex virus (HSV)-1 recombinant, CJ83193, capable of inhibiting its own replication as well as that of wild-type HSV-1 and HSV-2. It has been further demonstrated that CJ83193 is an effective vaccine against HSV-1 infection in a mouse ocular model. To ensure its safety and augment its efficacy, we generated an improved CJ83193-like HSV-1 recombinant, CJ9-gD, which contains a deletion in an HSV-1 essential gene and encodes an extra copy of gene-encoding glycoprotein D (gD) driven by the tetO-bearing human cytomegalovirus major immediate-early promoter. Unlike CJ83193, which exhibits limited plaque-forming capability in Vero cells and expresses little gD in infected cells, CJ9-gD is completely replication defective, yields high-level expression of gD following infection, and cannot establish detectable infection in mouse trigeminal ganglia following intranasal and ocular inoculation. Mice immunized with CJ9-gD produced 3.5-fold higher HSV-1 neutralizing antibody titer than CJ83193-immunized mice, and were completely protected from herpetic ocular disease following corneal challenge with wild-type HSV-1. Moreover, immunization of mice with CJ9-gD elicited a strong HSV-1-specific T-cell response and led to an 80% reduction in latent infection by challenge wild-type HSV-1 compared with the mock-immunized control.

In vitro assessment of the cytotoxic and mutagenic potential of perfluorooctanoic acid.

Perfluorooctanoic acid (PFOA) is a perfluorinated compound ubiquitously detected in the environment, including wildlife and humans. Despite the available information, research on the cytotoxicity of PFOA in non-tumoral mammalian cells is relatively limited. In this work, two in vitro toxicity systems were employed to provide further insight into the cytotoxic and mutagenic potential of PFOA. The cytotoxicity of the chemical towards Vero cells was assessed using biochemical and morphological parameters, while mutagenicity was evaluated according to Ames test. High doses of PFOA cause oxidative stress in Vero cells, that was closely linked to cell cycle arrest at the G1 phase and induction of apoptosis. Our results corroborate previous findings in human tumoral cells and suggest that the mode of action of this perfluorinated compound is not a peculiarity among mammalian cell types. On the other hand, the compound was not mutagenic in the Ames test, using four strains of Salmonella typhimurium in the presence or absence of rat S9 metabolic activation system.

Split mCherry as a new red bimolecular fluorescence complementation system for visualizing protein-protein interactions in living cells.

Bimolecular fluorescence complementation (BiFC) is a recently developed technique for detection of protein-protein interactions in living cells. In this study, a new red BiFC system was developed by splitting mCherry, a mutant monomeric red fluorescent protein, into two fragments between amino acids 159-160 and was verified using a pair of interacting proteins, SV40 large T antigen (LTag), and human p53 protein. By combined use of the mCherry-based red BiFC system with a Venus-based yellow BiFC system, the interaction between LTag and p53 as well as the interaction between sp100 and promyelocytic leukemia protein (PML), were detected simultaneously in Vero cells. The brilliant redness, short maturation time, and the long excitation and emission wavelengths (587/610 nm) of mCherry make the new BiFC system an excellent candidate for analyzing protein-protein interactions in living cells and for studying multiple protein-protein interactions when coupled with other BiFC systems.

Enhancement of cytotoxicity against Vero E6 cells persistently infected with SARS-CoV by Mycoplasma fermentans.

We previously reported that cells with persistent severe acute respiratory syndrome coronavirus (SARS-CoV) infection were established after apoptotic events. In the present study, we investigated the cytopathic effects of dual infection with SARS-CoV and Mycoplasma fermentans on Vero E6 cells. Dual infection completely killed cells and prevented the establishment of persistent SARS-CoV infection. M. fermentans induced inhibition of cell proliferation, but the cells remained alive. Apoptosis was induced easily in M. fermentans-infected cells, indicating that they were primed for apoptosis. These results indicated that M. fermentans enhances apoptosis in surviving cells that have escaped from SARS-CoV-induced apoptosis.

Cytoprotective and antioxidant role of diallyl tetrasulfide on cadmium induced renal injury: an in vivo and in vitro study.

Cadmium (Cd) is an environmental and industrial pollutant that affects various organs in humans and animals. A body of evidence has accumulated implicating the free radical generation with subsequent oxidative stress in the biochemical and molecular mechanisms of Cd toxicity. Since kidney is the critical target of Cd toxicity, we carried out this study to investigate the effects of diallyl tetrasulfide (DTS), an organosulfur compound derived from garlic on Cd induced toxicity in the kidney of rats and also in the kidney cell line (vero cells). In experimental rats, subcutaneous administration of Cd (3 mg/kg bw/day) for 3 weeks induced renal damage, which was evident from significantly increased levels of serum urea and creatinine with significant decrease in creatinine clearance. A markedly increased levels of lipid peroxidation markers (thiobarbituric acid reactive substances and lipid hydroperoxides) and protein carbonyl contents with significant decrease in nonenzymic antioxidants (total sulphydryl groups, reduced glutathione, vitamin C and vitamin E) and enzymic antioxidants (superoxide dismutase, catalase, glutathione peroxidase and glutathione-S-transferase) as well as glutathione metabolizing enzymes (glutathione reductase, and glucose-6-phosphate dehydrogenase) were also observed in Cd intoxicated rats. Coadministration of DTS (40 mg/kg bw/day) and Cd resulted in the reversal of the kidney function accompanied by a significant decrease in lipid peroxidation and increase in the antioxidant defense system. In vitro studies with vero cells showed that incubation of DTS (5-50 microg/ml) with Cd (10 microM) significantly reduced the cell death induced by Cd. DTS at 40 microg/ml effectively blocked the cell death and lipid peroxidation induced by Cd (10 microM) indicating its cytoprotective property. Further, the flow cytometric assessment on the level of intracellular reactive oxygen species using a fluorescent probe 2', 7'-dichlorofluorescein diacetate (DCF-DA) confirmed the Cd induced intracellular oxidative stress in vero cells, which was significantly suppressed by DTS (40 microg/ml). The histopathological studies in the kidney of rats also showed that DTS (40 mg/kg bw/day) markedly reduced the toxicity of Cd and preserved the architecture of renal tissue. The present study suggests that the cytoprotective potential of DTS in Cd toxicity might be due to its antioxidant and metal chelating properties, which could be useful for achieving optimum effects in Cd induced renal damage.

Oxidative stress in vero cells infected with vesicular stomatitis virus.

Viral-induced apoptosis might be mediated by oxidative stress. It has already been described that cell death in vesicular stomatitis virus (VSV)-infected cells occurs by apoptosis. In this study, oxidative stress parameters present in VSV-infected Vero cells were analyzed. Lipid peroxides (LP) were evaluated in cellular extracts and expressed as thiobarbituric acid-reactive substances. LP levels exhibited a rise at different times post infection, according to the multiplicity of infection (MOI), while the presence of cycloheximide determined a reduction on LP. Also, an increase in protein degradation products and a decrease in polyunsaturated fatty acids content was observed, indicating that cellular proteins and lipids began to be susceptible to degradation during VSV infection. In addition, we analyzed cell viability of VSV-infected Vero cells, which were incubated in the presence of butylated hydroxyanisole. This antioxidant was able to protect Vero cells, at least at MOIs assayed in this study, and to reduce viral yield only when VSV infection was done at MOI 0.05. Further, superoxide dismutases, which occupy the first step within the antioxidant enzyme cascade, also exhibit a rise in VSV-infected Vero cells, at different MOI. These results suggest that both an oxidative stress and an antioxidative cell response precede the induction of apoptosis by VSV.

Cytotoxic effects in mammalian Vero cells exposed to pentachlorophenol.

The effects of pentachlorophenol have been studied on diverse biological systems both in vivo and in vitro, however the cellular basis of the pronounced cytotoxicity of this organochlorine compound is poorly understood. In this work, morphological and biochemical analyses were carried out to identify the primary targets of pentachlorophenol toxicity in mammalian cells. Our results show that pentachlorophenol is a very potent cytotoxic drug that displays an unusual and interesting mode of action in Vero cells. Although this compound is a powerful uncoupler of oxidative phosphorylation, we present the novel finding that lysosome destabilization is an early cytotoxic response that precedes the mitochondrial dysfunction. In addition, soon after exposure to moderate doses of pentachlorophenol, a significant number of cells initiate an apoptotic death process identified by the condensed and fragmented state of their nuclei. These results demonstrate that there are multiple potential targets of PCP-induced toxicity in mammalian cells, and the need to develop further experimental studies for the risk assessment of this environmental pollutant.

RC-IAL cell line: sensitivity of rubella virus grow.

OBJECTIVE: The rapid growth of the rubella virus in RC-IAL2 with development of cytopathic effect, in response to rubella virus infection, is described. For purposes of comparison, the rubella virus RA-27/3 strain was titered simultaneously in the RC-IAL, Vero, SIRC and RK13 cell lines. METHODS: Rubella virus RA-27/3 strain are inoculated in the RC-IAL cell line (rabbit Kidney, Institute Adolfo Lutz). Plates containing 1.5x10(5) cells/ml of RC-IAL line were inoculated with 0.1ml s RA-27/3 strain virus containing 1x 10(4)TCID50/0.1ml. A 25% cytopathic effect was observed after 48 hours and 100% after 96 hours. The results obtained were compared to those observed with the SIRC, Vero and RK13 cell lines. Rubella virus was detected by immunohistochemistry. RESULTS: With the results, it was possible to conclude that the RC-IAL cell line is a very good substrate for culturing rubella virus. The cells inoculated with rubella virus were examined by phase contrast microscopy and showed the characteristic rounded, bipolar and multipolar cells. The CPE in RC-IAL was observed in the first 48 hours and the curve of the increased infectivity was practically the same as observed in other cell lines. CONCLUSIONS: These findings are important since this is one the few cell lines described in the literature with a cytopathic effect. So it can be used for antigen preparation and serological testing for the diagnosis of specific rubella antibodies.

Outbreak of fatal childhood viral infection in Sarawak, Malaysia in 1997: inocula of patients' clinical specimens induce apoptosis in vitro.

Identification of the aetiologic agent(s) associated with an outbreak of fatal childhood viral infection in Sarawak, Malaysia, in mid 1997 remains elusive. It is reported here that African green monkey kidney (Vero) and human monocytic (U937) cells treated with inocula derived from clinical specimens of some of these fatal cases showed the presence of cellular genomic DNA degradation when the extracted DNA was separated by pulsed field gel electrophoresis (PFGE), oligonucleosomal DNA ladders characteristic of apoptotic cells when the infected cells' DNA was separated by agarose gel electrophoresis, and apoptotic cellular DNA fragmentation when cells were stained using terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL). These results suggest that inocula derived from the patients' clinical specimens contain factors which stimulate apoptotic cellular responses in vitro.

Detection of cytotoxic activity on Vero cells in clinical isolates of Serratia marcescens

Cytotoxin production was studied in 60 Serratia marcescens strains isolated from hospitalized patients. Association of cytotoxic activity with serotype, source of isolation and presence of plasmids was also evaluated. Thirteen of the 60 S. marcescens strains produced a cytotoxic effect of Vero cells. These strains were isolated from distinct clinical sources and classified into seven different serotypes (O1:H7; O4:NM; O10:NT; O19:NM; O6,14:H4; O6,14:NM and O6,14:H1). No relationship was observed between cytotoxic activity and clinical source or serotypes of the strains. Plasmids from five cytotoxin-producing S. marcescens strains were transferred to E. Coli K12/711. The transconjugants did not exhibit cytotoxicity, indicating that the cytotoxic effect is not plasmid-mediated among these strains. Although a cytotoxic activity was demonstrated in filtrates of some S. marcescens strains, further studies should be performed to assess the role of this toxin in pathogenesis.

Scanning and transmission electron microscopy of transformed Vero cells, with altered in vitro growth characteristics.

The Vero lineage, established from kidney cells of the green. African monkey, presented fibroblasts-like cells and growth in monolayers. Maintained in culture, the Vero cells presented behavioural and morphologic alterations, associated with cellular transformation. The morphological alterations were investigated using scanning and transmission electron microscopy. The study of proliferation and determination of the cellular doubling time was obtained from the growth curve. The initial population presented growth in a monolayer, while the altered cells grew in multilayers forming cellular aggregates, with flattened cells on the surface and globular cells in the inner region of the aggregate, together with extracellular matrix material. The cell surface of the altered population presented innumerable structures similar to little vesicles, microvilli and cytoplasmic prolongations. The cellular proliferation of both populations was very similar. Our results indicate that morphological and growth changes probably resulted from cellular transformation of the initial Vero cells. These transformed cells presented several characteristics associated with neoplastic growth, and can be used as a model for tumor cells studies in vitro.

Improved plaque assays for Rickettsia prowazekii in Vero 76 cells.

Typhus group rickettsiae, including Rickettsia prowazekii and R. typhi, produce visible plaques on primary chick embryo fibroblasts and low-passage mouse embryo fibroblasts but do not form reproducible plaques on continuous cell culture lines. We tested medium overlay modifications for plaque formation of typhus group rickettsiae on the continuous fibroblast cell line Vero76. A procedure involving primary overlay with medium at pH 6.8, which was followed 2 to 3 days later with secondary overlay at neutral pH containing 1 microgram of emetine per ml and 20 micrograms of NaF per ml, resulted in visible plaques at 7 to 10 days postinfection. A single-step procedure involving overlay with medium containing 50 ng of dextran sulfate per ml also resulted in plaque formation within 8 days postinfection. These assays represent reproducible and inexpensive methods for evaluating the infectious titers of typhus group rickettsiae, cloning single plaque isolates, and testing the susceptibilities of rickettsiae to antibiotics.

A modified invasion-3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide assay for quantitating tumor cell invasion.

Reconstituted basement membrane matrix (Matrigel) has been utilized for in vitro assay of tumor cell invasion in recent years. In the conventional chamber for the invasion assay, however, a large number of cells passed easily through the center of the Matrigel-coated filter because the Matrigel layer could not be completely uniform by the meniscus formation. To prevent the meniscus phenomenon of the Matrigel layer, we devised a water-repellent treatment of the inside wall of the assay chamber with paraffin. Consequently, very few erythrocytes passed through the Matrigel-coated filter of this modified chamber with the erythrocyte assay, which was used to demonstrate the evenness and uniformity of the Matrigel layer on the filter. For quantitating a small number of cells which invaded through the Matrigel-coated filter by the invasion assay, a tetrazolium-based colorimetric 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide) assay was used. The invasive abilities of the eight different cells were determined by this invasion-3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium+ ++ bromide assay using the modified chamber with a filter coated with 70 microliters of the 0.2-mg/ml Matrigel. After 72 h of incubation, the malignant cell lines significantly exceeded the normal cell lines in the percentage of invasion (P < 0.01). Therefore, the modified invasion-3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium+ ++ bromide assay provides a simple, easily reproducible in vitro assay for quantitating tumor cell invasion.