Craig Wallis

The role of serum and fetuin in the growth of monkey kidney cells in culture

Abstract The function of serum in the growth medium for primary monkey kidney cells has been shown to be inhibition of proteolytic enzymes. Serum is required to inactivate the residual trypsin remaining from enzymatic digestion of the kidneys and the proteolytic enzymes subsequently synthesized by the cells. Freshly trypsinized cells could be grown to monolayers in the absence of serum provided that they were repeatedly washed to remove residual trypsin. In the absence of serum, cell growth ceased on the 4–5th day after initiation of the culture, at which time the culture fluids became active proteolytically. When the 5th day fluids were replaced with fresh serum-free medium, cell growth was accelerated and a monolayer was attained by the 7th day. If cells were grown in the absence of whole serum but in the presence of medium containing alpha globulins or fetuin which inhibit both trypsin and cell proteases, such cultures grew as well as cultures containing serum. Human and animal sera were found to contain stable factors against trypsin and labile factors against vervet- and rhesus-synthesized proteases. The growth-promoting quality of serum was shown to be related to its titer against these enzymes. The references in the literature to “toxic lots of serum” may in fact be a measure of the low concentration or absence of anti-enzyme components. “Inactivation” of serum may result in the loss of these enzyme-inactivators and decrease in growth-promoting quality. A direct relationship between the presence of anti-protease factors and growth of cells was shown with a wide variety of sera from different animals and man. In this regard, human sera obtained from alpha 1 -deficient donors failed to support initial growth of conventionally trypsinized cells which require serum. Normal human serum supported initiation of cell growth, but proved to be inefficient for continued growth of primary cells. The cells synthesized an activator for plasminogen, and were subsequently proteolysed by plasmin. Plasminogen-free serum was a substantially more effective factor for growth of primary monkey kidney cells.

The hazards of incorporating charcoal filters into domestic water systems

Abstract Charcoal filters used in domestic and commercial applications for the removal of objectionable taste and odor support the growth of bacteria to alarmingly high counts. The charcoal beds concentrate both bacteria and organic nutrients that are present in water at low concentration. This results in marked growth of bacteria during the overnight period when the water is stagnant and in their subsequent release into the morning flow.

Characteristics of humic acid and organic compounds concentrated from tapwater using the aquella virus concentrator

Abstract Humic acid and other organic compounds present in eluates from filters used to process large volumes of treated tapwater using the Aquella virus concentrator were found to share certain properties. Both humic acid and the other organic compounds in the filter eluates adsorbed to membrane filters at low pH, were eluted from membrane filters at high pH, interfered with virus adsorption to membrane filters, formed flocs at low pH, and were removed by anion-exchange resins.

Detection of viruses in large volumes of natural waters by concentration on insoluble polyelectrolytes

Abstract A method is described for recovering small amounts of virus from very large volumes of water. When poliovirus was added to large volumes of tap water, it could be recovered by adsorbing the virus onto thin layers of an insoluble polyelectrolyte (a crosslinked copolymer of isobutylene maleic anhydride), with subsequent concentration into small volumes of eluent for assay purposes. Virus contained in 25, 50, 75 and 100 gal of water was recovered with efficiencies ranging from 60 to 80 per cent. Poliovirus added to a 17,000-gal swimming pool was recovered with an efficiency of about 40 per cent by passing 300 gal of the pool water through a thin 3 mm polyelectrolyte layer 293 mm in diameter, with subsequent elution of the virus from the layer, and reconcentration into a smaller volume for assay.

A portable virus concentrator for testing water in the field

Abstract A system is described for concentrating viruses from large volumes of water. The system consists of a water pump, an electric generator, a series of clarifiers, a virus adsorbent, a virus reconcentrator, a 5- and a 1-gal pressure vessel with a small tank of nitrogen as a source of positive pressure, and ancillary equipment, all mounted on 2-wheel carts for easy portability. Standardization of the system was achieved by use of minute amounts of poliovirus. The virus was added to dechlorinated city tap water so that it could not be detected unless the virus was first concentrated. In the system, raw tap water containing virus is serially passed through clarifying filters of porosities of 1–5 μm to remove particulate matter, and then through a 1-μm cotton textile filter to electrostatically remove submicron ferric and other heavy metallic complexes. These filters do not detectably remove virus. Salts are then added to the running tap water to enhance the adsorption of virus to a fibreglass or cellulose acetate filter. Raw water could be processed at the rate of 300 gallons per hour, with total virus removal from the water and with 80 per cent elution of the virus from the adsorbent.

APPLICATION OF PHOTODYNAMIC OXIDATION TO THE DISINFECTION OF TAPWATER, SEA WATER, AND SEWAGE CONTAMINATED WITH POLIOVIRUS

Abstract. Poliovirus when added to tapwater, sewage or seawater was readily photoinactivated by methylene blue and visible light. Typically, almost 2.5 logs of virus could be inactivated upon a 5‐min exposure to 670 nm light (20 W/m2) in solutions containing 13 μM methylene blue at pH 10.0. A biphasic inactivation curve was produced for poliovirus, regardless of dye concentration, pH, temperature, sensitization time, nature of suspending solution or sequence of light exposure. These results indicated that a multi‐hit inactivation event was occurring. Preincubation of the dye‐virus mixture at 24°C increased the rate of virus photoinactivation. Dye concentrations above 26 μM have little advantage in increasing the amount of virus photoinactivated. Significant inactivation of the virus in the dark occurred at high dye concentrations (52–130μM).

Methods for concentrating viruses from large volumes of estuarine water on pleated membranes

Abstract The efficiency of cartridge filters with pleated membranes was determined for the recovery of poliovirus from large volumes of seawater. Flow rates of about 6 gal min−1 were obtained when a combination of a 3- and a 0.45-μm pleated filter were incorporated into the Wallis—Melnick virus concentrator. The filters adsorbed poliovirus when water was acidified at pH 3.5 and aluminum chloride added at a final concentration of 0.0015 m . The filters retained their capacity to adsorb virus particles, even after 600 gal (22681.) of very turbid estuarine water had been filtered. Viruses were eluted from the pleated filters with pH 11.5 glycine buffer and reconcentrated by precipitation with ferric chloride or aluminum chloride. Virus from 100-gal samples was concentrated into a final eluate volume of 20–100 ml, with virus recovery of about 50% being achieved in the material that had been concentrated 20,000–100,000 times.

Virus concentration from sewage

Abstract Optimal conditions for concentrating poliovirus from large volumes of raw sewage were established. Solids 1 μm or larger, present in the raw sewage, were removed by textile clarifying filters without significant retention of virus. By acidification of the clarified sewage and addition of salts to enhance virus attachment to the adsorbent, virus in the sewage was concentrated on a fibre glass depth filter, with subsequent elution of virus into small volumes suitable for assay. An 80–95 per cent efficiency of virus concentration was effected.

Photodynamic Inactivation of Herpesvirus

Photodynamic inactivation has been known since Raab, at the beginning of the 20th century, observed that acridine was harmless to paramecia in the dark but was lethal when the organisms were exposed to visible light (1). Three decades later viruses were shown to be photosensitive (2, 3). However, assay methods in the 1930s were crude, and no quantitative results were reported. In 1958, Yamamoto (4) reported the first quantitative studies on photodynamic inactivation of bacterial virus; in 1960, Hiatt et al. (5) extended this work to a number of DNA-containing animal viruses, but found that RNA-containing enteroviruses were resistant to photosensitization. We have since learned that naturally photoresistant viruses can be made photosensitive if the virus is grown in cells maintained with medium containing proflavine, neutral red, or acridine orange. During replication of the virus, the photoreactive dye becomes incorporated within the virus structure (6–10). If one looks through the literature, one can see that in most laboratories virus titers could be reduced markedly by “dye-light” treatment, but usually some virus persisted. The point is that, as usually practiced, photoinactivation may not be complete. Transformation by such preparations in which some infectious virus is still present (11) cannot be said to be caused by photoinactivated virus. However, if the procedures described in the papers from our laboratory are carefully followed, photoinactivation can be made total (12–17).

Herpesvirus neutralization: Induction of the persistent fraction by insufficient antibody☆

Abstract Previous work has shown that naturally aggregated herpesvirus particles are responsible for the nonneutralizable (persistent) virus fraction. A second cause of the persistent fraction has now been shown to result from “insufficient” antibody. When undiluted monodispersed herpesvirus was treated with antibody in amounts insufficient to completely neutralize the total virus population, infectious virus-antibody aggregates were produced. Subsequent treatment with excess antiserum did not abolish the persistent fraction formed by these immunoaggregates. However, if the incompletely neutralized mixture was first differentially filtered to remove the infectious aggregates but to permit monodispersed virus to pass into the filtrate, then the virus in the filtrate could be completely neutralized by additional antiserum. Essentially the same results were obtained with vaccinia virus.