Cytopathicity

CarbocycHc 2/3 -didehydro-2/3 -dideoxyguanosine [118353-05-2] (carbovk, CBV, 66), C H 2N502, synthesized in 1988 (177), is a promising candidate for the chemotherapy of AIDS. CBV inhibits HIV repHcation and HIV-induced cytopathic effects in a variety of human T-lymphoblastoid ceU lines at concentrations approximately two hundred- to four hundredfold below its cytotoxic concentrations (177). CBV is as effective as AZT and DDC in reducing the expression of vkal antigen in HIV-infected CEM ceUs (177). The antivkal potency and selectivity of carbovk is comparable to the anti-HIV-1 potency and selectivity of 2/3 -dideoxyadenosine (178). The exact mode of antivkal action of carbovk has not yet been elucidated, but may be the modulating effect of intraceUular nucleotides on 5 -nucleotidase activity (179). 

Rhesus monkey kidney infected with Semliki Forest arborvirus gave interferon of tltre 1.5 log interferon units/2 ml. (The interferon unit, determined in a volume of 2 ml, is the dilution of interferon which produced a half-maximal score for degree of cytopathic effect In virus-infected tissue culture tubes at the time when the control without interferon first showed the maximal score.)... 

CaUebaut C, Jacotot E, Blanco J et al (1998) Increased rate of HIV-1 entry and its cytopathic effect in CD4+/CXCR4+ T cells expressing relatively high levels of CD26. Exp Cell Res 241 352-362 Campbell JJ, Qin S, Unutmaz D et al (2001) Unique subpopulations of CD56+ NK and NK-T peripheral blood lymphocytes identified by chemokine receptor expression repertoire. J Immunol 166 6477-6482... 

Inoculation of cell cultures with virus-containing material produces characteristic changes in the cells. The replication of many types of viruses produces the cytopathic effect (CPE) in which cells degenerate. This effect is seen as the shrinkage or sometimes ballooning of cells and the disruption of the monolayer by death and detachment of the cells (Fig. 3.6). The replicating virus can then be identified by inoculating a series of cell cultures with mixtures of the virus and different known viral antisera. If the virus is the same as one of the types used to prepare the various antisera, then its activity will be neutralized by that particular antiserum and CPE will not be apparent in that tube. Alternatively viral antisera labelled with a fluorescent dye can be used to identify the virus in the cell culture. 

Studies have demonstrated that one such method is to examine the effects of disinfectants on endogenous RNA-dependent DNA polymerase (i.e. reverse transcriptase) activity. In essence, HIV is an RNA virus after it enters a cell the RNA is converted to DNA under the influence of reverse transcriptase. The virus induces a cytopathic effect on T lymphocytes, and in the assay reverse transcriptase activity is determined after exposure to different concentrations of various disinfectants. However, it has been suggested that monitoring residual viral reverse transcriptase activity is not a satisfactory alternative to tests whereby infectious HIV can be detected in systems employing fresh human peripheral blood mononuclear cells. 

Viral vaccines present problems of safety testing far more complex than those experienced with bacterial vaccines. With killed viral vaccines the potential hazards are those due to incomplete virus inactivation and the consequent presence of residual live virus in the preparation. The tests used to detect such live virus consist of the inoculation of susceptible tissue cultures and of susceptible animals. The cultures are examined for cytopathic effects and the animals for symptoms of disease and histological evidence of infection at autopsy. This test is of particular importance in inactivated poliomyelitis vaccine, the vaccine being injected intraspinally into monkeys. At autopsy, sections of brain and spinal cord are examined microscopically for the histological lesions indicative of proliferating poliovirus. 

Cohen DI, Tani Y, Tian H, Boone E, Samelson LE, Lane HC. Participation of tyrosine phosphorylation in the cytopathic effect of human immunodeficiency virus-1. Science 1992 256(5056) 542-545. 

Fig. 8.5 Demonstration of IFNa2b functionality by the ability of IF-Na2b to protect HeLa cells against the cytopathic effect of encephalomyocarditis virus (EMC). Chloroplast derived IFNa2bwas as active as commercially produced Intron A.

One of the most popular bioassay for interferons is termed the cytopathic effect inhibition assay . This assay is based upon the ability of many interferons to render animal cells resistant to viral attack. It entails incubation of the interferon preparation with cells sensitive to destruction by a specific virus. That virus is then subsequently added, and the percentage of cells that survive thereafter is proportional to the levels of interferon present in the assay sample. Viable cells can assimilate certain dyes, such as neutral red. Addition of the dye followed by spectrophotometric quantitation of the amount of dye assimilated can thus be used to quantitate percentage cell survival. This type of assay can be scaled down to run in a single well of a microtitre plate. This facilitates automated assay of large numbers of samples with relative ease. 

Viral bioassays of various different formats have also been developed. One format entails incubation of the final product with cell lines sensitive to a range of viruses. The cells are subsequently monitored for cytopathic effects or other obvious signs of viral infection. 

We reported an extensive LIE study of 6 and 11 analogs that included consideration of the alternative amine epimers and protonation states.28 The MC simulations were initiated from the crystal structure for the complex of 8-C1-TIBO and HIV-1 RT.50 Partial charges came from RHF/6-31G CHELPG calculations for each inhibitor. The experimental data are IC50 values for the effective concentration required to achieve 50% protection of MT- 4 cells against the cytopathic effect of HIV-1.51... 

Researchers at SRMLSC recently developed a HTS that allowed the identification of potential inhibitors of the severe acute respiratory syndrome coronavirus (SARS CoV) from large compound libraries [34], The luminescent-based assay, which measured the inhibition of SARS CoV-induced cytopathic effects (CPE) in Vero E6 cells, was validated with two different diversity sets of compounds against the SARS CoV. The hit rate for both libraries was approximately 0.01%. 

Inhibition of HIV-1 Integrase 3 Processsing (3 -Proc), Strand Transfer (ST) and HIV-1RF Cytopathicity (HIV-1rf) by Some Guanine Quartets in Cell Cultures... 

A few nitrogen-substituted allenes themselves are known as biologically active compounds [154], For example, the 9-(4 -hydroxy-1, 2 -butadienyl)adenine (268a) was found to inhibit in vitro replication and cytopathic effects of human immunodeficiency viruses HIV-1 and HIV-2 [155], More recently, an increase in the anti-HIV activity in cell cultures using the adenallene phosphotriester derivative 268b was reported (Scheme 8.72) [156]. 

Gl. Gallo, R. C., Salahuddin, S. Z., Propovic, M., Shearer, G. M., Kaplan, N., and Palker, T. G., Frequent detection and isolation of cytopathic retrovirus (HTLV111) from patients with AIDS and at risk of AIDS. Science 224, 500-503 (1984). 

Given that oxidative injury plays an important role in central nervous system (CNS) degenerative diseases, novel drags that protect cells from cytopathic effects of ROS could conceivably be used to treat some of these devastating illnesses. To screen for possible neuroprotective drags, a variety of standardized test systems have been designed mostly based on the in situ generation of superoxide by xanthine/xanthine oxidase. Superoxide decomposition may be followed photometrically by the reduction of ferricytochrome c, as it was reported by McCord and Fridovich (McCord and Fridovich, 1969). 

Vandenbroeck et al.7 used an ELISA to determine the recovery of immu-noreactive porcine interferon-gamma (IFN-y) from E. coli inclusion bodies. The ELISA used a polyclonal coating antibody with detection by a MAb. The inclusion bodies were solubilized in diluted 6 M guanidine/HCl and IFN subsequently refolded by its removal. The antiviral activity of the interferon was measured with a bioassay using the cytopathic effect (CPE) of vesicular stomatitis virus (VSV) on bovine kidney cells. The results of this study showed that the immu-noreactivity measured by ELISA matched the biological activity measured by bioassay. 

The viral RNA polymerase first transcribes the incoming positive-stranded 42 S RNA into negative-stranded 42 S RNA, which in turn serves as a template for the synthesis of both new positive-stranded 42 S RNA molecules and 26 S RNA molecules. The transcription of the 26 S RNA is initiated internally on the negative-stranded 42 S RNA. RNA replication seems to take place on membranes in characteristic cytoplasmic vacuoles, called cytopathic vacuoles I (CPV I), which appear soon after infection and are not seen in uninfected cells (Grimley et al., 1968, 1972 Friedman et al., 1972). The detailed mechanism of the RNA replication will not be dealt with here (for reviews see Strauss and Strauss, 1977 Kaariainen and Soderlund, 1978 Kennedy, 1980). 

Classical bacterial exotoxins, such as diphtheria toxin, cholera toxin, clostridial neurotoxins, and the anthrax toxins are enzymes that modify their substrates within the cytosol of mammalian cells. To reach the cytosol, these toxins must first bind to different cell-surface receptors and become subsequently internalized by the cells. To this end, many bacterial exotoxins contain two functionally different domains. The binding (B-) domain binds to a cellular receptor and mediates uptake of the enzymatically active (A-) domain into the cytosol, where the A-domain modifies its specific substrate (see Figure 1). Thus, three important properties characterize the mode of action for any AB-type toxin selectivity, specificity, and potency. Because of their selectivity toward certain cell types and their specificity for cellular substrate molecules, most of the individual exotoxins are associated with a distinct disease. Because of their enzymatic nature, placement of very few A-domain molecules in the cytosol will normally cause a cytopathic effect. Therefore, bacterial AB-type exotoxins which include the potent neurotoxins from Clostridium tetani and C. botulinum are the most toxic substances known today. However, the individual AB-type toxins can greatly vary in terms of subunit composition and enzyme activity (see Table 2). 

Normanno, G., Celano, G., Dambrosio, A., Lassandro, L. and Buonavoglia, C., Enterotoxins of Staphylococcus aureus induce a cytopathic effect in cell lines. New Microbiol., 24, 341-346, 2001. 

Initial screening of lipophilic and hydrophilic extracts against influenza A/WY/03/2003 (H3N2) was selected from a library of diverse marine invertebrates, algae, and microorganisms. The primary influenza screen used in this study begins with a microscopic evaluation of the cytopathic effect of extracts on virus-infected mammalian cells and is quantified by an MTT stain. From 800 screened extracts, only one, well A4 in Fig. 1.1, which is the crude extract from G. 

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