Cytopathic effect viruses

A sulphur containing pyrimidine (XLIX) is reported to be active against poliovirus (RNA virus) in vitro, reducing virus cytopathic effect by 70 per cent at 10 pg/ml and virus yield 3-4 logic units at SO pg/ml. The maximum... 

Bablanian, R., 1970, Studies on the mechanisms of vaccinia virus cytopathic effects. Effect of inhibitors of RNA and protein synthesis on early virus-induced cell damage, J. Gen. Virol. 6 221. 

Bablanian, R., 1972, Mechanisms of virus cytopathic effects, Symp. Soc. Gen. Microbiol. 22 359. 

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.)... 

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. 

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]. 

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. 

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. 

Incubate at least four series, cells with three or more different concentrations of the preparation to be examined and the reference preparation in a microtitre plate and include in each series appropriate controls of untreated cells. Choose the concentrations of the preparations such that the lowest concentration produces some protection and the largest concentration produces less than maximal protection against the viral cytopathic effect. At a suitable time add the cytopathic virus to the wells with the exception of a sulScient number of wells in ah series, which are left with uninfected control cells. Determine the cytopathic effect of virus quantitatively with a suitable method. Calculate the potency of the preparation to be examined by the usual statistical methods for a parallel line assay. 

There are two general approaches to cDNA expression, transient expression and stable expression. Transient expression systems are typically based on a viral vector the host cells are infected with cDNA-bearing virus and the cDNA-derived protein is produced. At some point a maximum level of cDNA-derived protein expression is obtained and the protein is harvested for use in incubations. Viral vectors often have cytopathic effects on the host cells which usually precludes analysis of xenobiotic-induced toxicity to the host cell. Stable expression systems can be based on integrating or episomal vectors. With both stable expression approaches, homogeneous, clonally derived populations of cells stably expressing the cDNA are identified and characterized. The properties, advantages and disadvantages of the two approaches are discussed below. 

Certain scrapie-like diseases of sheep in Iceland and some chronic encephalopathies in man (including kuru, the laughing disease of New Guinea) are the result of slow virus infections with long incubation periods. 1-Methylisatin 3-thiosemicar-bazone has been found to reduce the reverse transcriptase activity and cytopathic effect of the RNA slow viruses producing the sheep diseases and hence may provide a chemotherapeutic method for their control118. ... 

EC50 Inhibitor concentration that reduces the cytopathic effect of the virus by 50%... 

The RMK cell line, which exhibits cytopathic effect (CPE) in the presence of Influenza A(H1N1) or Avian Influenza A (H3N2) virus, was used as the indicator cell line in the infectivity assays. Cells in multiwell culture dishes were inoculated in quadruplicate with 0.1 ml of the dilutions prepared from test and control groups. Uninfected indicator cell cultures (cell controls) were inoculated with test medium alone. The cultures were incubated at 36-38° C. in a humidified atmosphere of 5-7% C02 in sterile disposable cell culture labware. The cultures were scored periodically for approximately seven days for the absence or presence of CPE, cytotoxicity and for viability. 

Insect cell lines present different susceptibilities to virus replication. A cell line is called permissive when it can support full virus replication and all stages of viral cycle are completed, leading to polyhedra production and lysis of the host cell. Semi-permissive cell lines allow partial virus replication, possibly due to a restriction at a particular viral cycle stage (Bilimoria et al., 1992). Abortive cell lines are those in which the resulting virus may have a cytopathic effect, but there are no infective particles produced (Carpenter and Bilimoria, 1983 Liu and Bilimoria, 1990). 

Since the possibility was recognised that bovine serum may be contaminated with several viruses (e.g. bovine herpesvirus and parainfluenza 3-virus) sera are now routinely tested by the manufacturer. The effectiveness of this screening has, however, been questioned (Kniazeff, 1973), and the use of serum-free media ( 5.8) is recommended as the only secure method of eliminating serum-borne viral contamination cytopathic effect. The problem is more serious, however, if no cytopathic effect is seen but the virus continues to replicate at a slow rate or in a few cells only. Thus primary cells from children with Burkitt s lymphoma show no signs of Epstein-Barr virus (EBV) until several passages when a few cells show positive immunofluorescence (Henle and Henle, 1966). 

Mitsuya H, Broder S. Inhibition of the in vitro infectivity and cytopathic effect of human T-lymphotrophic virus type... 

The virus reduction studies of the three process steps discussed here were performed with HFV-l, Bovine viral diarrhea virus (BVDV), Pseudorabies virus (PRV), Reovirus type 3 (Reo), Hepatitis A virus (HAV), and Porcine parvovirus (PPV). HIV-1 was included as a relevant enveloped virus, while BVDV and PRV were tested as specific model viruses for HCV and HBV, respectively (Table 1). Reo was chosen as a non-specific model non-enveloped virus, HAV was included as a relevant virus and PPV was used as a surrogate for human parvovirus B19. All viruses were propagated using standard cell culture conditions. " The appropriate cell lines were infected, at a low multiplicity of infection, and incubated until 4-1- cytopathic effects were observed. The infected cells were frozen and thawed three times to release virus, centrifuged at low speed to remove cell debris and the clarified supernatants were removed for use as virus spikes. 

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