Viral aggregation: mixed suspensions of poliovirus and reovirus.

The aggregation of mixtures of two dissimilar viruses, poliovirus I (Mahoney) and reovirus III (Dearing), was followed by electron microscopy under conditions known to induce either aggregation or dispersion of each virus separately. Neither virus aggregated at pH 7 in an appropriate buffer, and no mixed aggregates were formed. Under conditions of lowered ionic strength (by dilution into distilled water) poliovirus became aggregated, whereas reovirus did not, and again no mixed aggregates were formed. At pH 6, however, poliovirus again aggregated and, although reovirus did not, it attached to poliovirus aggregates. Thus, some inducement toward aggregation was necessary to cause formation of mixed aggregates. This inducement probably took the form of a reduction of the ionic double layer surrounding the particles, which is known to occur at low pH. At pH 5 and below both viruses aggregated severely, and large mixed aggregates were formed. These mixed aggregates could be broken up by neutralization of the suspension, although small aggregates of poliovirus remained. Reovirus showed a marked tendency to attach to large clumps of poliovirus, but the reverse tendency was not observed. The results indicate that mixed aggregates may be of significance in the isolation of viruses from water or wastewater.     

Aggregation of poliovirus and reovirus by dilution in water.

Poliovirus and reovirus were found to aggregate into clumps of up to several hundred particles when diluted 10-fold into distilled water from a stock preparation of minimal aggregation in 0.05 M phosphate buffer, pH 7.2, plus 22 to 30% sucrose. Reovirus was also found to aggregate when diluted into phosphate-buffered saline. The aggregation was concentration dependent and did not occur when either virus was diluted into water 100-fold or greater. The aggregation of poliovirus was reversible by further addition of saline and produced a dispersed preparation of virus. Reovirus aggregation was not reversible. Both viruses aggregated when diluted into buffers at pH 5 and 3, and poliovirus aggregated at pH 6, and this aggregation of both viruses was reversible when returned to pH 7. Aggregation did not occur at alkaline pH values. Aggregation at low pH could be caused aggregation of either virus at pH 7. Calcium ions, however, were found to aggregate both viruses at a concentration of 0.01 M.     

Aggregation of poliovirus and reovirus by dilution in water.

Poliovirus and reovirus were found to aggregate into clumps of up to several hundred particles when diluted 10-fold into distilled water from a stock preparation of minimal aggregation in 0.05 M phosphate buffer, pH 7.2, plus 22 to 30% sucrose. Reovirus was also found to aggregate when diluted into phosphate-buffered saline. The aggregation was concentration dependent and did not occur when either virus was diluted into water 100-fold or greater. The aggregation of poliovirus was reversible by further addition of saline and produced a dispersed preparation of virus. Reovirus aggregation was not reversible. Both viruses aggregated when diluted into buffers at pH 5 and 3, and poliovirus aggregated at pH 6, and this aggregation of both viruses was reversible when returned to pH 7. Aggregation did not occur at alkaline pH values. Aggregation at low pH could be caused aggregation of either virus at pH 7. Calcium ions, however, were found to aggregate both viruses at a concentration of 0.01 M.     

Viral aggregation: buffer effects in the aggregation of poliovirus and reovirus at low and high pH.

The effects of the buffer employed in maintaining a given pH value were tested on the aggregation of two viruses, poliovirus and reovirus. Poliovirus was found to aggregate at pH values of 6 and below, but not at pH 7 or above, except in borate buffer. Reovirus aggregated at pH 4 and below, but was found to aggregate only in acetate or tris(hydroxymethyl)aminomethane-citrate buffers at pH 5. Other buffers tested for aggregation of reovirus at pH 5 (succinate, citrate, and phosphate-citrate) induced little aggregation. No significant aggregation was found for reovirus at pH 6 and above. For both viruses, the most effective aggregation was induced by buffers having a substantial monovalently charged anionic component, such as acetate at pH 5 and 6 or citrate at pH 3. Cationic buffers at low pH, such as glycine, were generally weaker in aggregating ability than anionic buffers at the same pH. These results, when correlated with the isoelectric point of the viruses (poliovirus at pH 8.2; reovirus at pH 3.9) indicated that both viruses aggregated strongly when their overall charge was positive, but only under certain circumstances when their overall charge was negative. Although reovirus aggregated massively at its isoelectric point, poliovirus remained dispersed at its isoelectric point. The conclusion can be drawn that those pH and buffer conditions which induced aggregation of one virus do not necessarily induce it in another.     

Viral aggregation: buffer effects in the aggregation of poliovirus and reovirus at low and high pH.

The effects of the buffer employed in maintaining a given pH value were tested on the aggregation of two viruses, poliovirus and reovirus. Poliovirus was found to aggregate at pH values of 6 and below, but not at pH 7 or above, except in borate buffer. Reovirus aggregated at pH 4 and below, but was found to aggregate only in acetate or tris(hydroxymethyl)aminomethane-citrate buffers at pH 5. Other buffers tested for aggregation of reovirus at pH 5 (succinate, citrate, and phosphate-citrate) induced little aggregation. No significant aggregation was found for reovirus at pH 6 and above. For both viruses, the most effective aggregation was induced by buffers having a substantial monovalently charged anionic component, such as acetate at pH 5 and 6 or citrate at pH 3. Cationic buffers at low pH, such as glycine, were generally weaker in aggregating ability than anionic buffers at the same pH. These results, when correlated with the isoelectric point of the viruses (poliovirus at pH 8.2; reovirus at pH 3.9) indicated that both viruses aggregated strongly when their overall charge was positive, but only under certain circumstances when their overall charge was negative. Although reovirus aggregated massively at its isoelectric point, poliovirus remained dispersed at its isoelectric point. The conclusion can be drawn that those pH and buffer conditions which induced aggregation of one virus do not necessarily induce it in another.     

States of aggregation of the dahlemense strain of tobacco mosaic virus protein and their relation to crystal formation.

The aggregation of the protein of the dahlemense strain of tobacco mosaic virus has been studied by electron microscopy and ultracentrifugation. The aggregates formed are similar to those formed by the vulgare strain, although the particular conditions for their formation are often rather different. Helix formation by dialysis of A protein at pH 8 to acid pH is much more efficient if an intermediate step at pH 7 is introduced. The 20 S particle or two-layer disk is stable over a wide range of pH and ionic strength values. There is no tendency to form short stacks of disks at high ionic strength; instead, 30 S particles are formed that correspond to a pair of interlocked disks giving a “figure-of-eight” appearance in electron micrographs. These particles appear to be the “building blocks” of the protein crystal.     

Influence of water quality on enteric virus concentration by microporous filter methods

Four enteric viruses, poliovirus type 1, echovirus type 1, reovirus type 3, and simian adenovirus SV-11, were concentrated from seeded 1.3-liter volumes of raw, finished, and granular activated carbon-treated waters by adsorption to 47-mm-diameter (17 cm2), electropositive ( Virosorb 1MDS ) filters at pH 7.5 or electronegative ( Filterite ) filters at pH 3.5 with and without 5 mM added MgCl2, followed by elution with 0.3% beef extract in 50 mM glycine at pH 9.5. Removal of particulates from raw and finished waters by 0.2-micron prefiltration before virus addition and pH adjustment had little effect on virus concentration efficiencies. Soluble organic compounds reduced virus adsorption efficiencies from both raw and finished waters compared with granular activated carbon-treated water, but the extent of interference varied with virus type and adsorption conditions. For electropositive 1MDS filters, organic interference was similar with all virus types. For Filterite filters, organic interference was evident with poliovirus and echovirus, but could be overcome by adding MgCl2. Reovirus and SV-11 were not adversely affected by organics during adsorption to Filterite filters. Elution of reovirus and adenovirus was inefficient compared with that of poliovirus and echovirus. None of the three adsorption schemes ( 1MDS at pH 7.5 and Filterite with and without 5 mM MgCl2 at pH 3.5) could be judged superior for all viruses and water types tested.     

Influence of water quality on enteric virus concentration by microporous filter methods.

Four enteric viruses, poliovirus type 1, echovirus type 1, reovirus type 3, and simian adenovirus SV-11, were concentrated from seeded 1.3-liter volumes of raw, finished, and granular activated carbon-treated waters by adsorption to 47-mm-diameter (17 cm2), electropositive ( Virosorb 1MDS ) filters at pH 7.5 or electronegative ( Filterite ) filters at pH 3.5 with and without 5 mM added MgCl2, followed by elution with 0.3% beef extract in 50 mM glycine at pH 9.5. Removal of particulates from raw and finished waters by 0.2-micron prefiltration before virus addition and pH adjustment had little effect on virus concentration efficiencies. Soluble organic compounds reduced virus adsorption efficiencies from both raw and finished waters compared with granular activated carbon-treated water, but the extent of interference varied with virus type and adsorption conditions. For electropositive 1MDS filters, organic interference was similar with all virus types. For Filterite filters, organic interference was evident with poliovirus and echovirus, but could be overcome by adding MgCl2. Reovirus and SV-11 were not adversely affected by organics during adsorption to Filterite filters. Elution of reovirus and adenovirus was inefficient compared with that of poliovirus and echovirus. None of the three adsorption schemes ( 1MDS at pH 7.5 and Filterite with and without 5 mM MgCl2 at pH 3.5) could be judged superior for all viruses and water types tested.     

Endoribonuclease activity associated with animal RNA viruses.

A specific endoribonucleolytic activity was detected when detergent-lysed vesicular stomatitis of Sendai virus was incubated with the precursor to Escherichia coli tRNA Tyr. The cleavage products produced and the characteristics of the reaction were similar to those previously reported for human KB cell RNase NU. Like RNase NU, the virus-associated reaction generates 5'-hydroxyl and 3'-phosphate groups at the cleavage sites. At protein concentrations similar to those used to test vesicular stomatitis and Sendai viruses, virions of Sindbis virus and poliovirus also exhibited endoribonucleolytic activity, but reovirus, simian virus 40, and minute virus of mice did not. This endoribonuclease may be of physiological relevance to some of the viruses we tested.     

Ultraviolet Devitalization of Eight Selected Enteric Viruses in Estuarine Water

The effect of ultraviolet (UV) radiation on the devitalization of eight selected enteric viruses suspended in estuarine water was determined. The surviving fractions of each virus were calculated and then plotted against the UV exposure time for purposes of comparison. Analytical assessment of the survival data for each virus consisted of least squares regression analysis for determination of intercepts and slope functions. All data were examined for statistical significance. When the slope function of each virus was compared against the slope function of poliovirus type 1, the analytical findings indicated that poliovirus types 2 and 3, echovirus types 1 and 11, and coxsackievirus A-9 exhibited similar devitalization characteristics in that no statistically significant difference was found (P > 0.05). Conversely, the devitalization characteristics of coxsackievirus B-1 and reovirus type 1 were dissimilar from those of poliovirus type 1 in that a statistically significant difference was found between the slope functions (P < 0.05). This observed difference in devitalization of coxsackievirus B-1 and reovirus type 1 was attributed primarily to the frequency distribution of single and aggregate virions, the geometric configuration, the size of the aggregates, and the severity of aggregation. The devitalization curve of coxsackievirus B-1 was characteristic of a retardant die-away curve. The devitalization curve of reovirus type 1 was characteristic of a multihittype curve. The calculated devitalization half-life values for poliovirus types 1, 2, and 3; echovirus types 1 and 11; coxsackievirus types A-9 and B-1; and reovirus type 1 were 2.8, 3.1, 2.7, 2.8, 3.2, 3.1, 4.0, 4.0 sec, respectively. These basic data should facilitate an operative extrapolation of the findings to the applied situation. It was concluded that UV can be highly effective and provide a reliable safety factor in treating estuarine water.     

Poliovirus aggregates and their survival in water.

Inactivation of aggregated poliovirus by bromine is characterized by a continuously decreasing reaction rate. Poliovirus released from infected cells in these experiments by alternate freezing and thawing in water without electrolytes has always been aggregated. The aggregates persist even on 7,000-fold dilution in ion-free water. Virus similarly released into phosphate-buffered saline solution may be well dispersed, but it aggregates when sedimented into a salt-free sucrose gradient or when it is diluted as little as 10-fold in water. Large one-step dilutions of dispersed virus in water remain dispersed. Aggregated virus was not dispersed by one-step dilution (7,000-fold) in distilled or untreated lake water but was dispersed if phosphate-buffered saline or clarified secondary sewage plant effluent was used as diluent. Dispersed virus aggregates at all dilutions in alum-treated, finished water from the city filter plant. This may be the result of complex formation with insoluble material rather than virion-virion aggregation. A simple procedure is described for rendering a very dilute suspension of mixed virion aggregates into a three-part spectrum of sizes.     

Poliovirus aggregates and their survival in water.

Inactivation of aggregated poliovirus by bromine is characterized by a continuously decreasing reaction rate. Poliovirus released from infected cells in these experiments by alternate freezing and thawing in water without electrolytes has always been aggregated. The aggregates persist even on 7,000-fold dilution in ion-free water. Virus similarly released into phosphate-buffered saline solution may be well dispersed, but it aggregates when sedimented into a salt-free sucrose gradient or when it is diluted as little as 10-fold in water. Large one-step dilutions of dispersed virus in water remain dispersed. Aggregated virus was not dispersed by one-step dilution (7,000-fold) in distilled or untreated lake water but was dispersed if phosphate-buffered saline or clarified secondary sewage plant effluent was used as diluent. Dispersed virus aggregates at all dilutions in alum-treated, finished water from the city filter plant. This may be the result of complex formation with insoluble material rather than virion-virion aggregation. A simple procedure is described for rendering a very dilute suspension of mixed virion aggregates into a three-part spectrum of sizes.     

Inactivation of coxsackieviruses B3 and B5 in water by chlorine

The inactivation rates of coxsackievirus B3 (CB3) and B5 (CB5) by chlorine in dilute buffer at pH 6 were very nearly the same and about half that of poliovirus (Mahoney) under similar conditions. Purified CB3, like the poliovirus, aggregated in the acid range but not at pH 7 and above. Purified CB5 aggregated rapidly at all pH values; still, the graph of log surviving infectivity versus time was a straight line. No chlorine inactivation data were obtained with dispersed CB5, for it could be dispersed only by addition of diethylaminoethyl dextran, which would react with the chlorine. Addition of 0.1 M NaCl to the buffer at pH 6 did not influence the aggregation of CB5 or the rate of chlorine action on either of the coxsackie-viruses, but at pH 10 it increased the disinfection activity of OCl- for both viruses roughly 20-fold. Cesium chloride had a similar but smaller effect. KCl was the most active of the three in this respect, making the inactivating effect of OCl- at pH 10 about equal to that of HOCl at pH 6.     

Inactivation of coxsackieviruses B3 and B5 in water by chlorine.

The inactivation rates of coxsackievirus B3 (CB3) and B5 (CB5) by chlorine in dilute buffer at pH 6 were very nearly the same and about half that of poliovirus (Mahoney) under similar conditions. Purified CB3, like the poliovirus, aggregated in the acid range but not at pH 7 and above. Purified CB5 aggregated rapidly at all pH values; still, the graph of log surviving infectivity versus time was a straight line. No chlorine inactivation data were obtained with dispersed CB5, for it could be dispersed only by addition of diethylaminoethyl dextran, which would react with the chlorine. Addition of 0.1 M NaCl to the buffer at pH 6 did not influence the aggregation of CB5 or the rate of chlorine action on either of the coxsackie-viruses, but at pH 10 it increased the disinfection activity of OCl- for both viruses roughly 20-fold. Cesium chloride had a similar but smaller effect. KCl was the most active of the three in this respect, making the inactivating effect of OCl- at pH 10 about equal to that of HOCl at pH 6.     

Self-assembly of brome mosaic virus protein into capsids: Initial and final states of aggregation

The pH and ionic strength dependence of the states of aggregation of brome mosaic virus protein has been investigated by small angle neutron scattering, quasielastic light-scattering, analytical centrifugation and electron microscopy. At pH above neutrality, protein oligomers are found in dynamical equilibrium, comprising monomers, dimers and aggregates of higher molecular weight. By lowering the pH. capsids assemble spontaneously with dimensions in solution which depend on ionic strength. If formed by dialysis, they contain 180 monomers, but are 30 Å larger in diameter than the native virus. If formed by pH-jump, they contain less monomers; the deficiency decreases with decreasing the final pH and the initial protein concentration. Upon dehydration for electron microscopy, capsids contract by 10%.     

Mechanism of Resistance to Enteroviruses of Some Primate Cells in Tissue Culture

Two cell lines, M10-45-2 and L-41, were studied, each of which possessed specific resistance either to poliovirus or to coxsackievirus. Infection of M10-45-2 cells with poliovirus ribonucleic acid (RNA) and L-41 cells with infectious coxsackievirus RNA was accompanied by production of complete viruses in each of the resistant cell lines. During incubation of the cells with the virus to which they were resistant, the amount of infectious virus did not decrease. Treatment with glycine-HCl buffer solution (pH 2.5) of resistant M10-45-2 cells after incubation with poliovirus at 0 C did not result in recovery of infectious virus, although such release did take place after treatment of sensitive M10 cells. Infection of resistant cells with virus containing poliovirus RNA and coxsackievirus proteins resulted in production of poliovirus in M10-45-2 cells but not in L-41 cells. The resistant cells are apparently unable to adsorb the virus to which they are resistant.     

Equilibrium and kinetic studies of the aggregation of porphyrins in aqueous solution.

An investigation of the behavior of protoporphyrin IX, deuteroporphyrin IX, haematoporphyrin IX and coproporphyrin III in aqueous solution revealed extensive and complex aggregation processes. Protoporphyrin appears to be highly aggregated under all conditions studied. At concentrations below 4 muM, aggregation of deutero-, haemato- and coproporphyrin is probably restricted to dimerization. At approx. 4muM each of these three porphyrins exhibits sharp changes in spectra consistent with a "micellization" process to form large aggregates of unknown size. This critical concentration increases with increasing temperature and pH, but is not very sensitive to variation in ionic strength. Temperature-jump kinetic studies on deuteroporphyrin also imply an initial dimerization process, the rate constants for which are comparable with those for various synthetic porphyrins, followed by a further extensive aggragation. The ability of a particular porphyrin to dimerize appears to parallel that of the corresponding iron(III) complexes (ferrihaems), although it is thought that ferrihaems do not exhibit further aggregation under these conditions.     

Viral aggregation: effects of salts on the aggregation of poliovirus and reovirus at low pH.

As a first step toward the understanding of virus particle interactions in water, we have used the modified single particle analysis test to follow the aggregation of poliovirus and reovirus as induced by low pH in suspensions containing varying amounts of dissolved salts. Salts composed of mono-, di-, and trivalent cations and mono- and divalent anions were tested for their ability to reduce or increase the aggregation of these viruses in relation to that obtained by low pH alone. Mono- and divalent cations in concentrations covering those in natural waters were generally found to cause a decrease in aggregation, with the divalent cations having a much greater effectiveness than the monovalent cations. Trivalent ions (Al3+), in micromolar concentrations, were found to cause aggregation over that at low pH alone. Anions, whether monovalent or divalent, had little ability to produce inhibition of viral aggregation, and thus the overall effects were due almost exclusively to the cation. This was true regardless of whether the overall charge on the virus particle was positive or negative, as determined by the relation between the isoelectric point and the pH at which the tests were carried out. Thus, whereas virus particles conform to classical colloid theory in many respects, there are specific exceptions which must be taken into account in the design of any experiment in which viral aggregation is a factor.     

pH modification of the effects of detergents on the stability of enteric viruses.

The effect of detergents on the stability of enteric viruses was found to be highly dependent on pH. This was demonstrated primarily with two ionic detergents, sodium dodecyl sulfate (an anionic detergent) and dodecyltrimethylammonium chloride (a cationic detergent). Both detergents were shown to be potent virucidal agents for reovirus, but the effects of sodium dodecyl sulfate were minimal near neutrality and much more pronounced at low than at high pH values. Dodecyltrimethylammonium chloride was extremely virucidal at high pH's but had little observable effect on reovirus stability at low pH values. In contrast, both detergents protected enteroviruses against heat at neutral and alkaline pH's. However, as was found with reovirus, sodium dodecyl sulfate was extremely virucidal at pH values below 5, even when the virus samples were incubated in ice. At different pH's the effects of detergents on the stabilities of coliphages T4, f1, and Q beta were qualitatively similar to those found with reovirus. Differences in viral stability in these experiments appeared to be due to the effects of pH on the ionic states of the viral capsid proteins.     

pH modification of the effects of detergents on the stability of enteric viruses.

The effect of detergents on the stability of enteric viruses was found to be highly dependent on pH. This was demonstrated primarily with two ionic detergents, sodium dodecyl sulfate (an anionic detergent) and dodecyltrimethylammonium chloride (a cationic detergent). Both detergents were shown to be potent virucidal agents for reovirus, but the effects of sodium dodecyl sulfate were minimal near neutrality and much more pronounced at low than at high pH values. Dodecyltrimethylammonium chloride was extremely virucidal at high pH's but had little observable effect on reovirus stability at low pH values. In contrast, both detergents protected enteroviruses against heat at neutral and alkaline pH's. However, as was found with reovirus, sodium dodecyl sulfate was extremely virucidal at pH values below 5, even when the virus samples were incubated in ice. At different pH's the effects of detergents on the stabilities of coliphages T4, f1, and Q beta were qualitatively similar to those found with reovirus. Differences in viral stability in these experiments appeared to be due to the effects of pH on the ionic states of the viral capsid proteins.