Document Type

Report

Publication Date

January 1979

Abstract

A new procedure for the detection of viral antigens in fecal material was developed. The test is performed by first diluting a fecal sample with phosphate buffered saline to give a liquid consistency. The pH is then adjusted to 8.5-9.0 and the solids are allowed to settle for five minutes. Supernatant fluid from above the fecal sediment is placed on the upper surface of a well of an inverted Immulon microtiter plate and incubated for one hour at 37 degrees C to allow virus to adsorb to the plastic. The Immulon plate is then washed three times with a Tween 20 solution and dried. Adsorbed virus is stained with fluorescein labled antiviral antibody containing Evan's Blue dye. The stained preparations are examined by epi-fluorescence microsopy for the presence of viral aggregates and virus-containing cellular membranes. The test is applied in a continuous water monitoring procedure that can be used to upplement methods in which infectious viruses are isolated from water. In another study a protamine sulfate procedure for concentrating and an immunofluorescent cell procedure for assaying infectious virus (IV, reovirus that is infectious without proteolytic enzyme treatment), and potentially infectious virus (PIV, enzyme enhanceable reovirus) from polluted waters have been developed. The presence of PIV inthe environment had not previously been investigated. In following these procedures, protamine sulfate concentratiosn of 0.005 percent for the first precipitation of the sample, and 0.0025 percent for the second were used. With these protamine concentrations and 0.25 percent fetal bovine serum, IV and PIV are concentrations over 500-fold from river water inoculated with virus. Virus recoveries are between 80 and 100 percent. The IV and PIV fractions are assayed respectively before and after treatment with 200 ug fo chymotrypsin per millileter. When PIV is precipitated from river water, and the precipitate is dissolved and stored at 20 degrees C as a protamine-virus concentrate, only 5 percent of the viral infectivity is lost after 14 days. Therefore, reovirus can be precipitated from water at the sampling site, and only the protamine concentrate needs to be taken to the laboratory to be examined for virus content. When reoviruses are treated with chlorine, PIV is more resistant to inactivation thatn IV, and PIV appears to be at least as resistant to chlorination as poliovirus and coxsackievirus A-2. Granular media filtration systems (i.e., sand, anthracite coal and sand; anthracite coal; sand and garnet) are ineffectual in the removal of the acteriophage MS 2 from water when used as in-line direct filters. Batch assays have indicated a 93 percent reduction of MS 2 can occur when polyelectrolytes are added to the water. In addition, alum concentrations of 20, 30, 40, and 50 mg/1 remove 80 to 98 percent of the virus by precipitation. No reduction of MS 2 was observed at alum concentrations from 1 to 10 mg/1.