Viruses suspensions

Plaques may be obtained for animal viruses by using animal cell-culture systems as hosts. A monolayer of cultured animals cells is prepared on a plate or flat bottle and the virus suspension overlayed. Plaques are revealed by zones of destruction of the animal cells. 

Antibody, murine IgG-2b/K, against tobacco mosaic virus Suspension Nicotiana tabacum (tobacco) A. tumefaciens transformation of leaf explant CaMV 35 S Murine 15 4g g 1 wet weight (i) 45 4g g 1 wet weight (i) with amino acids 62... 

Fig. 1.36.1. Electrical resistance (ER) as function of temperature during cooling and rewarming of a virus suspension. The suspension subcools from -10 °C to approx. -46 °C and freezes at -60 °C to -65 °C. During rewarming the resistance drops clearly at approx. -33 °C. This product should be freeze dried at rice = -40 °C or a little higher (Fig. 7 from [1.27]).

Fig. 1.36.2. Photographs of the virus suspension (Fig. 1.36.1.) by a cryomicroscope during slowly increased temperature left -40 °C, right -30 °C. In the right photo, bright inclusions in the dark lower zone can be seen. These represent partially molten product between the remaining ice. Water is also diffused into the already dried product, partially dissolving it (Fig. 8 from f 1.27]).

During the freeze drying tests the virus suspensions are either basic salt medium (BSM) or BSM plus calcium lactobionate (CL) plus serum albumin (SA) frozen at -76 °C and dried either at 0 °C or at -40 °C. The activity has been evaluated after 30 days storage at -4 °C or -65 °C. Rehydratation was done with distilled water at 0 °C. The results with the freeze dried viruses indicate ... 

Often medicinal plants known from folk-lore are picked up and their extracts tested against known plant viruses by mixing them with the inoculum and doing half-leaf experiments. Each half of the leaf is rubbed with virus suspension. 

A1 DANN virus suspensions in BSM changed only slightly. 

Viruses are much smaller, being are about 20-300 nm. Aggregation phenomena are important in virus suspensions because irreversible aggregation of virus particles results in precipitation and loss of biological activity. Conversely, a key challenge in the development and commercial production of viruses for human gene therapy... 

A 4.5 ml aliquot of each concentration of the test substance was dispensed into separate tubes and each was mixed with a 0.5 ml aliquot of the stock virus suspension. The mixtures were vortex mixed for a minimum of 10 seconds and held for the remainder of the specified exposure times at the appropriate temperature. Immediately following each exposure period, a 0.1 m aliquot was removed from each tube and the mixtures were titered by 10-fold serial dilutions (0.1 ml+0.9 ml test medium) and assayed for the presence of virus. Note to decrease the product cytotoxicity, the first dilution may be made in fetal bovine serum or other appropriate neutralizer with the remaining dilutions in test medium. 

A 0.5 ml aliquot of the stock virus suspension was exposed to a 4.5 ml aliquot of test medium instead of test substance and treated as previously described under Treatment of Virus Suspension. A virus control was performed for each exposure time tested. All controls employed the same neutralizer utilized in the test. The virus control titer was used as a baseline to compare the percent and log reduction of each test parameter following exposure to the test substance. 

Sterilize and clarify (to remove cellular debris) the virus suspension by consecutive filtrations through 1.2-, 0.8-, and 0.45-pm filters. 

Titration ef viruses. HSV-1 was assayed by using monolayer cultures of BSC-1 cells grown in 6-well multidishes. The cells were plated 3x10 cells/well in MEM(E) with 10% FBS and 1.1 g/1 sodium bicarbonate. After 24 hours, the cell sheet was about 75% confluent and was inoculated with 0.2 ml of the virus suspension to be assayed and incubated for 1 hour to permit viral adsorption. The cell sheets were then overlaid with 3 ml medium containing 0.5% methocel and incubated for another two days. 

Monolayers of susceptible cells are inoculated with small aliquots of serial dilutions of the virus suspension to be titrated. Whenever viral particles infect cells, progeny virus particles are produced, released and immediately infect adjoining cells. This process is repeated until after 2-12 incubation days or more. Areas of infected cells develop plaques that can be seen with a naked eye. Agar is frequently incorporated in the medium to ensure that the liberated progeny virus particles in the medium do not diffuse away and initiate separate or secondary plaques. The infected cells must differ in some recognizable manner from non infected ones, i.e., they must... 

Some viruses destroy cells they infect but do not produce the necessary CPE for visible plaque formation. These viruses are titrated by serial dilution end point method. Serial dilutions of virus suspensions are inoculated into cell monolayers which are then incubated until the cell sheets show clear signs of cell s destruction. The end point is the dilution that gives a positive (cell-destroying) reaction and originally contains at least one infectious unit. 

Sandwich ELISA for mAh profiling. All wells of the plates are coated with polyclonal anti-FMDV-type specific serum. A mixture of rabbit antibodies against A5, A22, and A24 serotypes is used. After incubation and washing, a single dilution of different virus suspensions is added (in blocking buffer) as shown, in duplicate rows for test samples (B, C D, E and F, G). 

Rod-shaped vimses can form hquid crystalline phases, which can be controlled by factors like virus suspension concentration, ionic strength of solution, external fields, etc. For example, Ml 3 phages are found to randomly orientated in an isotropic concentration range but transform to nematic, cholesteric, and smectic phases with... 

Collect medium covering the cells. Spin tnbe at 1,000 for 10 min. Transfer the supernatant to a new tube and discard the pellet. The supernatant contains viruses that are referred as the PO stock virus. Keep the virus suspension at 4°C covering the tube with aluminum foil. 

A standard fluorimetric assay is used to measure influenza virus NA activity. The substrate MUNANA (2 -(methylumbelliferyl)-a-D-acetylneuraniinic acid) is cleaved by NA to yield a fluorescent product, which can be quantified. The reaction mixture containing the test compounds/extracts and NA enzyme or a virus suspension in 32.5 mM MES buffer with 4 mM CaCl (pH 6.5) is incubated for 40 nun at 37°C. After incubation, the reaction is terminated by the addition of 34 mM NaOH. The fluorescence is quantified at an excitation wavelength of 360 ran and enussion wavelength of 450 ran. The 50% inhibitory concentration (IC ) is defined as the concentration of NA inhibitor necessary to reduce the NA activity by 50% relative to that in a reaction mixture containing virus but no inhibitor (Liu et al. 2008a). 

Standardization of virus activity by bioassay against larvae of a susceptible host is a more successful method of standardizing virus formulations. However, they are time consuming, expensive and data are often less precise than desired (22, 23), The most commonly used bioassay is diet surface treatment (24). A recently developed bioassay is a virus suspension feeding method (25). For some situations this method may be preferred to the diet method, but although larvae older than neonates need to be starved prior to exposure for consistent results. The most successful standardization protocols have employed both polyhedral counts and bioassay. ... 

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