Concentration and purification of enteroviruses by membrane chromatography

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Six laboratories actively involved in water virology research participated in a methods evaluation study, conducted under the auspices of the American Society for Testing and Materials Committee on Viruses in the Aquatic Environment, Task Force on Drinking Water. Each participant was asked to examine the Viradel (virus adsorption-elution) method with cartridge-type Filterite filters for virus adsorption and organic flocculation and aluminum hydroxide-hydroextraction for reconcentration. Virus was adsorbed to filter media at pH 3.5 and eluted with either glycine buffer (pH 10.5) or beef extract-glycine (pHG 9.0). Considerable variation was noted in the quantity of virus recovered from four 100-liter samples of dechlorinated tapwater seeded with low (350 to 860 PFU) and high (1,837 to 4,689 PFU) doses of poliovirus type 1. To have a more uniform standard of comparison, all the test samples were reassayed in one laboratory, where titers were also determined for the virus seed. Test results of the Viradel-organic flocculation method indicated that the average percentage of virus recovery for low-input experiments was 66%, with a range of 8 to 20% in two laboratories, 49 to 63% in three laboratories, and 198% in one laboratory. For the high-input experiments, two laboratories reported recoveries of 6 to 12%, and four laboratories reported recoveries of 26 to 46%. For the Viradel aluminum hydroxide-hydroextraction procedure, two laboratories recovered 9 to 11%, whereas four obtained 17 to 34% for low-input experiments. For the high-input tests, two laboratories reported a recovery of 3 to 5%, and four recovered 11 to 18% of the seeded virus.(ABSTRACT TRUNCATED AT 250 WORDS)

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

Round robin investigation of methods for the recovery of poliovirus from drinking water

Melnick¹,

Safferman²,

Rao³

et al. 1984

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“…The apparatus described in this report represents an adaptation of the techniques developed for water sampling to the problem of detecting airborne or aerosolized viruses. In this apparatus, air that contains viruses is drawn through a filter that has been moistened with glycine buffer, pH 3.5, since at this pH enteric viruses are adsorbed to the filter surface (6,9). Subsequently, adsorbed viruses are eluted from the filter and concentrated, and the concentrate is assayed for virus content.…”

Section: Discussionmentioning

confidence: 99%

“…Preparation of poliovirus stocks. Stocks of the attenuated type 1 poliovirus (strain LSc-1) were prepared by the adsorption-elution techniques previously described (9).…”

mentioning

confidence: 99%

Method for detecting viruses in aerosols

Wallis¹,

Melnick²,

Rao³

et al. 1985

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A simple method with poliovirus as the model was developed for recovering human enteric viruses from aerosols. Filterite filters (pore size, 0.45 ,um; Filterite Corp., Timonium, Md.) moistened with glycine buffer (pH 3.5) were used for adsorbing the aerosolized virus. No virus passed the filter, even with air flow rates of 100 liters/min. Virus recovery from the filter was achieved by rapid elution with 800 ml of glycine buffer, pH 10. The virus in the primary eluate was reconcentrated by adjusting the pH to 3.5, adding AIC13 to 0.0005 M, collecting the virus on a 0.25-,um-pore Filterite disk (diameter, 25 mm) and eluting with 6 ml of buffer, pH 10. With this method, virus could be detected regularly in aerosols produced by flushing when 3 x 108 PFU of poliovirus were present in the toilet bowl. Poliovirus-containing fecal material from two of four infants who had recently received oral polio vaccine also yielded virus in the aerosols when feces containing 2.4 x 107 to 4.5 x 107 PFU of virus had been added to the toilet bowl. Persons infected with a variety of natural enteric viruses are known to excrete this amount of virus in their daily stools. * Corresponding author. Virtually all of the aerosolized poliovirus in the stream of air is removed as it passes through the apparatus.

“…Negatively charged membrane filters have previously been used for the concentration of enteroviruses from CCH (7,12). Because the presence of membrane-coating components in the CCH interfered with virus adsorption to the filtration matrix, it was necessary to subject the sample to a series of time-consuming pretreatments.…”

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confidence: 99%

Rotavirus concentration from cell culture harvests: trypsin treatment followed by hydroextraction

Ramia¹,

Sattar²

1980

An 80-to 150-ml amount of calf or simian rotavirus-containing cell culture harvests of MA-104 cells were treated with 50 jig of trypsin per ml and hydroextracted overnight (4°C) with polyethylene glycol 6,000. The concentrate was resuspended in 8 to 10 ml of tryptose phosphate broth and plaque assayed. Between 85 and 97% of the input virus could be recovered with a concentration of up to 15-fold. Earlier work in our laboratory showed that polyethylene glycol hydroextraction is a useful method for the second-step concentration of entero-(9), reo-(9), and rotaviruses (11) from the water environment. Because of the relatively simple and efficient nature of this technique, we attempted to extend its use to the concentration of rotaviruses from cell culture harvests (CCH). This communication describes the findings of these experiments. Trypsin (1:250; GIBCO, Grand Island, N.Y.) and soybean trypsin inhibitor (Sigma Chemical Co., St. Louis, Mo.) were prepared separately as