In vitro effects of selenium deficiency on West Nile virus replication and cytopathogenicity.

BACKGROUND: Selenium (Se) deficiency plays an important role in viral pathogenesis. To understand the effects of Se deficiency on West Nile virus (WNV) infection, we analyzed cytopathogenicity, apoptosis and viral replication kinetics, using a newly developed Se-deficient cell culture system. RESULTS: Both Vero and SK-N-SH cells grown in Se-deficient media exhibited a gradual loss of glutathione peroxidase (GPx1) activity without any significant effect on cell growth and viability. In SK-N-SH cells, Se deficiency had no effect on the expression of key antioxidant enzymes, including manganese- and copper-zinc superoxide dismutase (MnSOD and CuZnSOD), catalase and inducible nitric oxide synthase, whereas Vero cells demonstrated a significant increase in the expression of MnSOD and an overall increase in oxidative stress (OS) at day 7 post-induction of Se deficiency. At 2 days after infection with WNV, CPE and cell death were significantly higher in WNV-infected Se-deficient Vero cells, compared to WNV-infected control cells. Furthermore, WNV-induced apoptosis was significantly heightened in Se-deficient cells and was contributed by loss of mitochondrial membrane potential and increased caspase activity. However, no significant difference was found in WNV copy numbers between control, Se-adequate and Se-deficient cell cultures. CONCLUSION: Overall results demonstrate that the in vitro Se-deficient model can be used to study responses of WNV to this essential nutrient. Although Se deficiency has no in vitro effect on WNV replication kinetics, adequate Se is presumably critical to protect WNV-infected cells against virus-induced cell death.

Expression and functional characterisation of the putative SARS coronavirus non-structural proteins X1-X5.

1. We produced mammalian expression vectors encoding the SARS coronavirus (SARS-CoV) accessory proteins with or without the fluorescence protein tag and cell lines with stable expression of these proteins. 2. The cellular localisation and function of the SARS-CoV accessory proteins was determined. 3. SARS 6 and SARS 8b proteins are localised to the endoplasmic reticulum and nucleus/cytoplasm, respectively, and both proteins stimulate host cell DNA synthesis.

Molecular and genetic characterisation of the SARS coronavirus auxiliary protein X1 in Drosophila.

1. We have generated monoclonal antibodies against the SARS coronavirus (SARS-CoV) X1/3a protein (3a), which are suitable for western blotting, immunocytochemistry, and immunohistochemistry. 2. We have established and characterised an in-vivo 3a transgenic Drosophila model, and demonstrated its usefulness in studying SARS-CoV 3a gene function. 3. We validated our in-vivo findings on 3a gene function in mammalian Vero E6 cells. 4. Our findings raise the possibility of using ion channel blockers as a novel approach to suppress SARS-CoV-induced cell death.

Transcriptional and posttranscriptional regulation of exogenous human beta interferon gene in simian cells defective in interferon synthesis.

We determined that the defect in beta interferon induction in Vero cells is due to the absence of the simian beta interferon (IFN-beta) gene. Nevertheless, the human IFN-beta gene or a hybrid gene, in which the human IFN-beta promoter-regulatory region directs expression of the chloramphenicol acetyltransferase gene (pIFN-CAT), could be induced in transfected Vero cells, and these cells also regulated IFN-beta mRNA (but not pIFN-CAT mRNA) posttranscriptionally. These results indicate that the instability in the human IFN-beta gene is coded for by the coding or 3'-end region of IFN-beta mRNA and that the human IFN-beta gene is regulated in Vero and human cells in an identical manner.

[Microtubules are necessary for lamellae retraction of Vero cells].

Behavior of Vero cells under the 2,3-butaneodione monoxime (BDM) treatment was examined using video-microscopy with contrast enhancement. After addition of BDM to the culture medium the area of cell contact with substratum gradually reduced--within 5 min of treatment cell lamellae became thicker, after 60 min the cell area decreased approximately 70 %, and the cells became nearly rounded. At the same time actin bundles (stress fibers) depolymerized, and microtubule network became denser. Partial depolymerization of microfilaments by treatment with latrunculin B at a concentration of 5 nM resulted in complete loss of stress fibers, yet cells slightly change their form, and microtubule system remained the same as in the control cells. However, after addition of BDM in the presence of latrunculin B cells retracted their lamellae more quickly then under BDM sole treatment. To evaluate the role of microtubules in the process of cell retraction we depolymerized them with nocodazole taken at the concentration of 5 ng/ml. Under nocodazole treatment the cell area decreased approximately 20 %, and stress fibers became more thick and abandon. The cells did not change their form, and stress fibers depolymerized very slowly under BDM treatment in the absence of microtubules. After 1 h of BDM treatment in the presence ofnocodazole stress fibers were still more numerous than in the control cells. Complete depolymerization of stress fibers happened in 90 % of cells only in 24 h after addition of BDM. When nocodazole had been washed out of the culture medium in the presence of BDM, lamellae started shrinking in 6 min. This time corresponds to the time required for the partial restoration of microtubule system. On the bases of the results obtained we conclude that retraction of the lamellae in Vero cells is guided rather mainly by microtubules, than stress-fibers.

Dual effects of microwaves on single Ca(2+)-activated K+ channels in cultured kidney cells Vero.

Using the patch voltage-clamp method, possible effects of millimetre microwaves (42.25 GHz) on single Ca(2+)-activated K+ channels in cultured kidney cells (Vero) were investigated. It was found that exposure to the field of non-thermal power (about 100 microW/cm2) for 20-30 min greatly modifies both the Hill coefficient and an apparent affinity of the channels for Ca2+(i). The data suggest that the field alters both cooperativity and binding characteristics of the channel activation by internal Ca2+. The effects depend on initial sensitivity of the channels to Ca2+ and the Ca2+ concentration applied.

Vero cells stimulate human sperm motility in vitro.

OBJECTIVE: To study human sperm motility in a coculture system for in vitro fertilization (IVF). DESIGN: We studied the viability and motility (percentage and curvilinear velocity) of human spermatozoa after incubation in: (1) medium 199 supplemented with fetal calf serum (M199/FCS) together with Vero cells; (2) Vero cell conditioned M199/FCS; (3) M199/FCS supplemented with Vero cell extract; and (4) some control media. In a second experiment, FCS was substituted by sera from different IVF patients. SETTING: Semen samples were obtained from the fertility laboratory of the St. Radbound Hospital, Nijmegen, The Netherlands. PATIENTS: Twelve men of couples with fertility problems. INTERVENTIONS: None. MAIN OUTCOME MEASURE: The motility parameters were determined with a computerized motility-analyzing system, after 24 hours' incubation at 37 degrees C and 5% CO2. Viability was determined after eosin Y staining. RESULTS: Sperm viability was the same in all media. In the coincubation system, the Vero cell-conditioned medium, and the experiment with human sera, the sperm motility parameters were higher (P less than 0.005) than in the control media. Vero cell extract did not have this positive effect. CONCLUSIONS: Coincubation has a positive effect on sperm motility and may be beneficial to IVF, intrauterine insemination, and artificial insemination.

Comparison of results after in vitro fertilized human embryos are cultured in routine medium and in coculture on Vero cells: a randomized study.

OBJECTIVE: To investigate whether coculture of human embryos on Vero cells improves embryo viability compared with a routine culture method. SETTING: In vitro fertilization Clinic of the Hôpital Cantonal Universitaire de Genève, Geneva, Switzerland. PATIENT SELECTION: Couples who had given informed consent, had undergone < 3 IVF cycles with ET and where the male had normal semen parameters were selected. Patients who had undergone > or = 3 IVF cycles with ET were allocated directly to coculture. DESIGN: Patients were randomly allocated to have their embryos cultured in a routine embryo culture medium or in coculture with Vero cells. RESULTS: There was no difference in pregnancy rates between the two culture groups. Coculture gave a high (> 50%) rate of blastocyst formation. In 16 cycles where patients had previously undergone > or = 3 IVF cycles 4 patients became pregnant. CONCLUSIONS: Coculture provides no benefit for patients that are performing their initial treatment cycles in IVF.

Microcalorimetric measurements on tissue cells attached to microcarriers in stirred suspension.

Vero cells growing on microcarriers in stirred suspension were observed calorimetrically using a vessel designed for use with the LKB 'BioActivity Monitor'. Rates of formation of carbon dioxide and lactate were followed in parallel. The results showed that the power and rate of lactate formation could be correlated to both cell number and amount of protein, while the rate of carbon dioxide formation was slightly better correlated to cell number. The power per cell was 27.4 +/- 2.1 pW. Only 33% of this power could be accounted for by the formation of lactate and carbon dioxide.

[Influence] of enzymatic treatment on Junin virus--Vero cells interaction]. / Influencia del tratamiento enzimático sobre la interacción virus Junín-células Vero.

The chemical nature of cellular structures involved in the attachment of Junin virus (wild type XJC13 and host range mutant Cl 67) to Vero cells was investigated. Enzyme treatment of cells before virus infection indicated that whereas lipids are not directly involved in virus attachment, cellular proteins play a significant role in early interaction with JV. Moreover aromatic residues, leucine and basic amino acid seem to actively participate in this interaction with different affinity for the assayed strains.

Detection of apoptotic cells in Vero cell cultures inoculated with samples derived from fatal cases of Sarawak acute childhood viral infection.

Infectious agent(s) causing the fatal Sarawak acute childhood viral infection (SACVI) has not been identified. In the present study, results indicating that inocula prepared from the fatal cases of SACVI induced apoptosis in Vero cell cultures are presented. These findings suggest the possible involvement of apoptotic cellular responses in SACVI.

Dynamic scaling analysis of two-dimensional cell colony fronts in a gel medium: a biological system approaching a quenched Kardar-Parisi-Zhang universality.

The interfacial two-dimensional spreading dynamics of quasilinear Vero cell colony fronts in methylcellulose (MC)-containing culture medium, under a constant average front displacement velocity regime, was investigated. Under comparable experimental conditions, the average colony front displacement velocity becomes lower than that reported for a standard culture medium. Initially, the presence of MC in the medium hinders both the colony spreading, due to a gradual change in the average size and shape of cells and their distribution in the colony, and the cell motility in the gelled medium. Furthermore, at longer culture times enlarged cells appear at random in the border region of the colony. These cells behave as obstacles (pinning sites) for the displacement of smaller cells towards the colony front. The dynamic scaling analysis of rough fronts yields the set of exponents α=0.63±0.04,ß=0.75±0.05, and z=0.84±0.05, which is close to that expected for a quenched Kardar-Parisi-Zhang model.

The effects of confluency on cell mechanical properties.

Mechanical properties of cells depend on various external and internal factors, like substrate stiffness and surface modifications, cell ageing and disease state. Some other currently unknown factors may exist. In this study we used force spectroscopy by AFM, confocal microscopy and flow cytometry to investigate the difference between single non-confluent and confluent (in monolayer) Vero cells. In all cases the stiffness values were fitted by log-normal rather than normal distribution. Log-normal distribution was also found for an amount of cortical actin in cells by flow cytometry. Cells in the monolayer were characterized by a significantly lower (1.4-1.7 times) Young's modulus and amount of cortical actin than in either of the single non-confluent cells or cells migrating in the experimental wound. Young's modulus as a function of indentation speed followed a weak power law for all the studied cell states, while the value of the exponent was higher for cells growing in monolayer. These results show that intercellular contacts and cell motile state significantly influence the cell mechanical properties.

Development of an in situ detachment protocol of Vero cells grown on Cytodex1 microcarriers under animal component-free conditions in stirred bioreactor.

Subcultivation of Vero cells grown in a proprietary animal component-free medium named IPT-AFM, on microcarriers, was studied. TrypLE Select, a non-animal-derived protease, was used as an alternative to trypsin for cell passaging. We first studied the effect of increasing concentrations of TrypLE Select toward cell growth and then studied the inactivation of the protease using either soybean trypsin inhibitor (STI) or the soy hydrolysate Hypep 1510, in six-well plates. Data showed that cell growth was impaired by residual level of TrypLE Select; STI was identified as an efficient agent to neutralize this effect. To restore cell growth and inactivate TrypLE Select, STI should be added to the medium at least at 0.2 g L(-1). Cells were also grown in spinner flask on 2 g L(-1) Cytodex1 in IPT-AFM. In these conditions, the cell detachment yield was equal to 78 ± 8 %. Furthermore, cells exhibited a typical growth profile when using the dislodged cells to seed a new culture. A cell detachment yield of 70 ± 19 % was also achieved when the cells were grown in a 2-L stirred bioreactor in IPT-AFM, on 3 g L(-1) Cytodex1. This protocol can be of great interest to scale-up the process of Vero cells cultivation in IPT-AFM on Cytodex1 from one stirred bioreactor culture to another.

[In vitro inhibitory mechanism of respiratory syncytial virus with solution prescription of zhidanhuayu].

OBJECTIVE: To study the effective part of solution prescription of Zhidanhuayu (ZDHY) against respiratory syncytial virus (RSV) in vitro. METHODS: Observe the pathology of RSV to Hep-2 under the condition of different concentrations and each effective part of ZDHY. RESULTS: The concentration limit causing celluar toxicity of ZDHY is 5.5 mg/ml. The ZDHY failed to block the absorption of RSV to Hep-2 within this concentration, and consequently the cell fell into the full pathological changes. During the concentration of 2.75-5.50 mg/ml, the ZDHY directly destroyed virus array,meanwhile, the infected cells that treated by the medicine kept healthy also. CONCLUSION: ZDHY could not defend the infection of RSV, but is able to destroy the RSV directly and inhibit the RSV inhabiting in the cell.

African green monkey kidney Vero cells require de novo protein synthesis for efficient herpes simplex virus 1-dependent apoptosis.

During HSV-1 infection, IE gene expression triggers apoptosis, but subsequent synthesis of infected cell proteins blocks apoptotic death from ensuing. This "HSV-1-dependent" apoptosis was identified in HEp-2/HeLa cells infected with wild-type HSV-1 in the presence of an inhibitor of protein synthesis or a virus lacking ICP27 {HSV-1(vBSDelta27)}. Unlike HEp-2/HeLa cells, vBSDelta27-infected Vero cells fail to exhibit dramatic apoptotic morphologies at times prior to 24 hpi. Here, we examined the basis of these different apoptotic responses to HSV-1. We found that infected Vero cells take substantially longer than HEp-2/HeLa cells to display membrane blebbing, chromatin condensation, DNA laddering, and PARP cleavage. Vero, but not HEp-2/HeLa, cells required de novo protein synthesis to exhibit efficient HSV-1-dependent apoptosis, which included changes in mitochondrial membrane potential, and these factors were produced prior to 3 hpi. Vero cells infected with recombinant viruses devoid of the ICP27 and ICP4 proteins alone or both the ICP27 and ICP22 proteins were apoptotic. These results indicate a requirement for cellular or other viral protein synthesis in Vero cells and provide insight into cell type differences in HSV-1-dependent apoptosis.

Anchorage-dependent mammalian cell culture using polyurethane foam as a new substratum for cell attachment.

Anchorage-dependent mammalian cells were cultivated at high cell density in a novel culture system using polyurethane foam (PUF) as a substratum for cell attachment. PUF has a macroporous structure giving a high surface area to volume ratio. Monkey kidney cells (Vero) and Chinese hamster ovary cells (CHO-K1) attached to the internal surface of PUF and grew to a high cell density (1.04 X 10(8) cells/cm3 PUF and 3.5 X 10(7) cells/cm3 PUF, respectively) in PUF stationary cultures. In addition, we have designed a PUF-particle packed-bed culture system for high density mass cell culture. A maximum cell density of 2.4 X 10(7) cells/cm3 culture vessel volume was obtained in a packed-bed culture of Vero cells.

On the cyto-toxicity caused by quantum dots.

Quantum dots (QDs) such as CdSe QDs have been introduced as new fluorophores. The QDs conjugated with antibody are starting to be widely used for immunostaining. However there is still not sufficient analysis of the toxicity of QDs in the literature. Therefore we evaluated the cell damage caused by the quantum dots for biological applications. We performed cell viability assay to determine the difference in cell damage depending on the sizes and colors of mercapto-undecanoic acid (MUA) QDs and the cell types. The results showed that the cell viability decreased with increasing concentration of MUA-QDs. But in the case of Vero cell (African green monkey's kidney cell) with red fluorescence QD (QD640), the cell damage was less than for the others. Furthermore through the flow cytometry assay we found that this cell damage caused by MUA-QD turned out to be cell death after 4-6-hr incubation. From the two assays described above, we found that there is a range of concentration of MUA-QDs where the cell viability decreased without cell death occurring and thus we conclude that attention should be given when MUAQDs are applied to living organisms even in low concentrations.