Poliovirus inactivation by ammonia causes a slight reduction in the sedimentation coefficients of viral particles, but has no detectable effect on either the electrophoretic pattern of viral capsid proteins or the isoelectric points of inactivated particles. These virions still attach to cells, but are unable to repress host translation or stimulate the synthesis of detectable amounts of viral RNA. Although ammonia has no detectable effect on naked poliovirus RNA, it causes cleavage of this RNA

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... when still within viral particles. Therefore, the RNA genome appears to be the only component of poliovirus significantly affected by ammonia. During the course of an investigation on the newborn calf serum. Radioactively labeled virus prepeffects of wastewater sludge on poliovirus, it was arations made with either [3H]uridine or '4C-reconstifound that viral inactivation rates are consider-tuted protein hydrolysate (Schwarz mixture) were ably increased in anaerobically digested but not grown and purified as described in an earlier publicaraw sludge (16, 18). Subsequently, it was shown tion (16). that the sludge agent responsible for this effect Inactivation of poliovirus with ammonia. Purthat the sludge agent responsTi le for this effec ified preparations of poliovirus were diluted into 0.1 iS ammonia (15, 17). This compound IS not vl-M Tris at pH 9.5 either with or without (control) 0.5 rucidal as ammonium ion, and the pK for its M NH4Cl. After incubation at 40C for the times specconversion into virucidal ammonia is 9.2. Beified, loss of biological activity was determined by cause the pH of digested sludge is typically plaque assay (16) . about 2 pH units higher than that of raw sludge, Adsorption ofinactivated poliovirions to HeLa a far greater percentage of indigenous ammonia cells. After 24 h of incubation at 4°C in either the Tris is present in the virucidal state in digested or Tris plus NH4Cl described above, radioactively sludge. This explains why poliovirus inactivation labeled viral particles were diluted 10-fold with 0.1 M isaceertd. ndigested, but not raw, sludge. Tris (pH 7.0) and assayed for biological activity and S a e s * * ina r s o recoverable radioactivity. Samples of each preparation Situdies on the mactivation rates of other en-were then adsorbed at 370C on monolayer cultures of teric viruses with ammonia indicated that sen-HeLa cells and, after 30 min., unadsorbed virus was sitivity to this agent appears to be a general removed with a Pasteur pipette. The cells were washed property of viruses belonging to the enterovirus twice and suspended in phosphate-buffered saline group (17). Reovirus, an enteric virus of a differ-(PBS) before measuring cell-associated radioactivity. ent group, is insensitive to ammonia. The phys-The results were corrected for loss of radioactivity due ical or chemical diimilarity between enterovi-to quenching. ruses and reoviruses that causes this very differ-Analysis of poliovirions and subviral compoent response to ammonia is not immediately nents. Sedimentation analysis of viral particles, evident fresomnstructural ammomansideionA dter-Y phenol extraction of viral RNA, and sedimentation evident from structural considerations. A deter-studies and infectivity analysis of poliovirus RNA were mination of the particular property of enterovicarried out as previously described (16) . Isoelectric ruses that makes them sensitive to ammonia focusing of poliovirions was performed by the method should reveal why reovirus is insensitive, and of Korant and Lonberg-Holm (8). In short, 0.2 ml of may allow one to predict the susceptibilities of the virus preparation to be analyzed was mixed with other viruses to this compound as well. There-0.12 ml of 40% (wt/vol) sucrose containing 1% amphofore, the mechanism by which ammonia causes line (pH 3.5 to 10). These samples were added at the inactivation of poliovirus was determined. position of 15% sucrose during the generation of 10 to 40% sucrose gradients also containing 1% ampholine. MATERIALS AND METHODS AU sucrose gradients were made in glass tubes (13 by 0.6 cm) stoppered with a small piece of dialysis tubing. Virus and cells. Poliovirus type 1 strain CHAT For electrophoresis, the bottom reservoir contained obtained from the American Type Culture Collection 1% sulfuric acid (vol/vol) in 40% sucrose and the top was used throughout the course of this investigation. reservoir contained 2% 2-aminoethanol (vol/vol). This virus was both produced and assayed for biolog-After electrophoresis at a constant voltage of 500 V at ical activity in monolayer cultures of HeLa cells grown 40C for 6 h, fractions were collected from the bottom in Eagle minimal essential medium containing 5% of the tubes after perforation of the dialysis mem-299 on May 8, 2020 by guest