Characterization of Virus Adsorption by Using DEAE-Sepharose and Octyl-Sepharose†

Viruses were characterized by their adsorption to DEAE-Sepharose or by their elution from octyl-Sepharose by using buffered solutions of sodium chloride with different ionic strengths. Viruses whose adsorption to DEAE-Sepharose was reduced most rapidly by an increase in the sodium chloride concentration were considered to have the weakest electrostatic interactions with the solids; these viruses included MS2, E1, and X174. Viruses whose adsorption to DEAE-Sepharose was reduced least rapidly were considered to have the strongest electrostatic interactions with the column; these viruses included P1, T4, T2, and E5. All of the viruses studied adsorbed to octyl-Sepharose in the presence of 4 M NaCl. Viruses that were eluted most rapidly following a decrease in the concentration of NaCl were considered to have the weakest hydrophobic interactions with the column; these viruses included X174, CB4, and E1. Viruses that were eluted least rapidly from the columns after the NaCl concentration was decreased were considered to have the strongest hydrophobic interactions with the column; these viruses included f2, MS2, and E5.     

Influence of salts on virus adsorption to microporous filters

We investigated the direct and indirect effects of mono-, di-, and trivalent salts (NaCl, MgCl(2), and AlCl(3)) on the adsorption of several viruses (MS2, PRD-1, phiX174, and poliovirus 1) to microporous filters at different pH values. The filters studied included Millipore HA (nitrocellulose), Filterite (fiberglass), Whatman (cellulose), and 1MDS (charged-modified fiber) filters. Each of these filters except the Whatman cellulose filters has been used in virus removal and recovery procedures. The direct effects of added salts were considered to be the effects associated with the presence of the soluble salts. The indirect effects of the added salts were considered to be (i) changes in the pH values of solutions and (ii) the formation of insoluble precipitates that could adsorb viruses and be removed by filtration. When direct effects alone were considered, the salts used in this study promoted virus adsorption, interfered with virus adsorption, or had little or no effect on virus adsorption, depending on the filter, the virus, and the salt. Although we were able to confirm previous reports that the addition of aluminum chloride to water enhances virus adsorption to microporous filters, we found that the enhanced adsorption was associated with indirect effects rather than direct effects. The increase in viral adsorption observed when aluminum chloride was added to water was related to the decrease in the pH of the water. Similar results could be obtained by adding HCl. The increased adsorption of viruses in water at pH 7 following addition of aluminum chloride was probably due to flocculation of aluminum, since removal of flocs by filtration greatly reduced the enhancement observed. The only direct effect of aluminum chloride on virus adsorption that we observed was interference with adsorption to microporous filters. Under conditions under which hydrophobic interactions were minimal, aluminum chloride interfered with virus adsorption to Millipore, Filterite, and 1MDS filters. In most cases, less than 10% of the viruses adsorbed to filters in the presence of a multivalent salt and a compound that interfered with hydrophobic interactions (0.1% Tween 80 or 4 M urea).     

Minimized virus binding for tests of barrier materials.

Viruses are used to test the barrier properties of materials. Binding of virus particles during passage through holes in the material may yield misleading test results. The choices of challenge virus and suspending medium may be important for minimizing confounding effects that might arise from such binding. In this study, different surrogate viruses, as well as different support media, were evaluated to determine optimal test parameters. Two membranes with high-binding properties (nitrocellulose and cationic polysulfone) were used as filters to compare binding activities of different surrogate challenge viruses (MS2, phi X174, T7, PRD1, and phi 6) in different media. The media consisted of buffered saline with surfactants, serum, or culture broth as additives. In addition, elution rates of viruses that bound to the membranes were determined. The results suggest that viruses can bind by hydrophobic and electrostatic interactions, with phi X174 displaying the lowest level of binding by either process. The nonionic detergents Triton X-100 and Tween 80 (0.1%) equally minimized hydrophobic interactions. Neither anionic nor cationic surfactants were as effective at nontoxic levels. Serum was effective at reducing both hydrophobic and electrostatic binding, with 2% being sufficient for eliminating binding under our test conditions. Thus, phi X174 remains the best choice as a surrogate virus to test barrier materials, and Triton X-100 (0.1%) remains a good choice for reducing hydrophobic binding. In addition, binding of viruses by barrier materials is unlikely to prevent passage of blood-borne pathogens.     

Interaction of inorganic pyrophosphatase from yeast with alkylated derivatives of Sepharose

The adsorption of inorganic pyrophosphatase from baker's yeast by alkylated polysaccharides involves both electrostatic and hydrophobic interactions. The enzyme is eluted by NaCl solutions of different ionic strengths depending on the adsorbent hydrophobicity. The degree of purification on different adsorbents varies from 2- to 80-fold.     

Virus passage through track-etch membranes modified by salinity and a nonionic surfactant.

Why do viruses sometimes not pass through larger pores in track-etch filters? Increasing the salinity (0.8 to 160 mM Na+) decreased phiX174 and PRD1 passage through track-etch polycarbonate membranes (sodium dodecyl sulfate coated but not polyvinylpyrrolidone coated) and PRD1 passage through polyester membranes. Undiminished passage when 0.1% Tween 80 was added implied that nonionic virus adsorption occurred and indicated that high levels of salinity decreased virus passage by decreasing electrostatic repulsion that prevented adsorption.     

Calcium-dependent hydrophobic chromatography of calmodulin, S-100 protein and troponin-C

We have demonstrated calcium-dependent hydrophobic interactions among calmodulin, S-100 protein and troponin-C and a homologous series of omega-aminoalkyl-agaroses. The three Ca2+-binding proteins were retained on the column of agarose substituted with omega- aminooctyl or even longer with alkylamine, in the presence of Ca2+ and 0.15 M NaCl. As these proteins were not retained on the column with shorter alkylamine 'arms' (N = 2, 4), they are probably successively absorbed with a higher affinity to the hydrophobic agarose column. Calmodulin and S-100 protein were eluted from the aminoocytl -agarose column with 1 mM EGTA in the presence of 0.15 M NaCl and the elution of troponin-C was Ca2+-independently carried out with 0.3 M NaCl. On the other hand, S-100 and troponin-C were eluted Ca2+-dependently from aminodecyl -agarose in the presence of 1 M NaCl and half the amount of the calmodulin applied was eluted with 1 M NaCl. As there are obvious differences among the three Ca2+-binding proteins with regard to chromatographic behavior on omega-aminoalkyl-agarose columns, our results suggest that these three proteins expose different hydrophobic regions following Ca2+-induced conformational changes and, if so, such would explain the interaction with aminoalkyl-agaroses.     

Method for improving the detection of viruses in fecal samples.

A method is described that improved the detection of viruses in fecal samples by electron microscopy. The virus particles were concentrated, and much of the background debris was removed by adsorption of viruses on meat protein added to the fecal sample at a low pH and a low salt concentration. Viruses were eluted by raising the pH and the salt concentration. Further concentration was achieved by acid precipitation and vacuum dialysis.     

Method for improving the detection of viruses in fecal samples

A method is described that improved the detection of viruses in fecal samples by electron microscopy. The virus particles were concentrated, and much of the background debris was removed by adsorption of viruses on meat protein added to the fecal sample at a low pH and a low salt concentration. Viruses were eluted by raising the pH and the salt concentration. Further concentration was achieved by acid precipitation and vacuum dialysis.     

Influence of Salts on Virus Adsorption to Microporous Filters

We investigated the direct and indirect effects of mono-, di-, and trivalent salts (NaCl, MgCl₂, and AlCl₃) on the adsorption of several viruses (MS2, PRD-1, X174, and poliovirus 1) to microporous filters at different pH values. The filters studied included Millipore HA (nitrocellulose), Filterite (fiberglass), Whatman (cellulose), and 1MDS (charged-modified fiber) filters. Each of these filters except the Whatman cellulose filters has been used in virus removal and recovery procedures. The direct effects of added salts were considered to be the effects associated with the presence of the soluble salts. The indirect effects of the added salts were considered to be (i) changes in the pH values of solutions and (ii) the formation of insoluble precipitates that could adsorb viruses and be removed by filtration. When direct effects alone were considered, the salts used in this study promoted virus adsorption, interfered with virus adsorption, or had little or no effect on virus adsorption, depending on the filter, the virus, and the salt. Although we were able to confirm previous reports that the addition of aluminum chloride to water enhances virus adsorption to microporous filters, we found that the enhanced adsorption was associated with indirect effects rather than direct effects. The increase in viral adsorption observed when aluminum chloride was added to water was related to the decrease in the pH of the water. Similar results could be obtained by adding HCl. The increased adsorption of viruses in water at pH 7 following addition of aluminum chloride was probably due to flocculation of aluminum, since removal of flocs by filtration greatly reduced the enhancement observed. The only direct effect of aluminum chloride on virus adsorption that we observed was interference with adsorption to microporous filters. Under conditions under which hydrophobic interactions were minimal, aluminum chloride interfered with virus adsorption to Millipore, Filterite, and 1MDS filters. In most cases, less than 10% of the viruses adsorbed to filters in the presence of a multivalent salt and a compound that interfered with hydrophobic interactions (0.1% Tween 80 or 4 M urea).     

Affinity chromatography of DNA on nonporous copolymerized particles of styrene and glycidyl methacrylate with immobilized polynucleotide

Nonporous particles of microsize were prepared by the dispersion polymerization of styrene and glycidyl methacrylate and chemically modified to introduce amino groups on the surface by grafting with either hexamethylenediamine or N-methyl-1,3-propanediamine. Aminated particles were then coupled with phosphorylated single-stranded polynucleotides at the 5'-end through covalent linkages. The affinity columns packed with these prepared polynucleotide-immobilized particles effectively retained single-stranded DNA, which could base-pair with the immobilized sequence. Bound DNAs could be eluted to yield a sharp peak by using an aqueous solution of 0.4M NaOH. The nonspecific adsorption due to the electrostatic interaction between the polynucleotide and the residual amino groups on the particle surface via the amination with hexamethylenediamine was significant and could only be reduced by using a high salt (NaCl) concentration. A higher salt concentration in the elution solution could result in a portion of complementary polynucleotide eluted in the nonretained fraction. However, the nonspecific adsorption of polynucleotides was insignificant in the column packed with DNA-immobilized particles prepared via amination using N-methyl-1,3-propanediamine. The column was effective for microanalysis of sequence-specific DNA.     

DETECTION OF LISTERIA MONOCYTOGENES BY ADSORPTION AND ELUTION FROM HYDROPHOBIC MATRICES

The binding of L. monocytogenes Scott A strain to three hydrophobic matrices, octyl, phenyl and butyl Sepharose, was investigated. Optimal adsorption of L. monocytogenes to octyl Sepharose was obtained at pH 3.5 and 4 M NaCl. However, it was difficult to elute the bacteria from octyl Sepharose, even after changing the pH and lowering the salt concentration. Good adsorption of L. monocytogenes to phenyl Sepharose at pH 3.5 and 4 M NaCl was also observed. L. monocytogenes was found to adsorb weakly to butyl Sepharose, which is less hydrophobic than phenyl Sepharose. Bacteria were eluted under various conditions. The best elution was obtained with 10 mM sodium phosphate, followed by an increasing gradient of ethylene glycol. To test the potential application of hydrophobic chromatography for separating L. monocytogenes from food matrices, milk was inoculated with L. monocytogenes and then passed through a column of phenyl Sepharose at pH 3.5 and 4 M NaCl. Nearly all L. monocytogenes were bound to the hydrophobic gel and were eluted in a pure and viable form by changing the pH and lowering the salt concentration, and by using a polar reducing agent, ethylene glycol. This study shows that hydrophobic interaction chromatography can be used to separate L. monocytogenes from milk and may be applicable to other food suspensions. It is a gentle method that makes use of the hydrophobic surface properties of Listeria for attachment to hydrophobic gels, as well as using mild elution conditions to avoid inactivation of the organism.     

Use of modified diatomaceous earth for removal and recovery of viruses in water.

Diatomaceous earth was modified by in situ precipitation of metallic hydroxides. Modification decreased the negative charge on the diatomaceous earth and increased its ability to adsorb viruses in water. Electrostatic interactions were more important than hydrophobic interactions in virus adsorption to modified diatomaceous earth. Filters containing diatomaceous earth modified by in situ precipitation of a combination of ferric chloride and aluminum chloride adsorbed greater than 80% of enteroviruses (poliovirus 1, echovirus 5, and coxsackievirus B5) and coliphage MS2 present in tap water at ambient pH (7.8 to 8.3), even after filtration of 100 liters of tap water. Viruses adsorbed to the filters could be recovered by mixing the modified diatomaceous earth with 3% beef extract plus 1 M NaCl (pH 9).     

Use of modified diatomaceous earth for removal and recovery of viruses in water.

Diatomaceous earth was modified by in situ precipitation of metallic hydroxides. Modification decreased the negative charge on the diatomaceous earth and increased its ability to adsorb viruses in water. Electrostatic interactions were more important than hydrophobic interactions in virus adsorption to modified diatomaceous earth. Filters containing diatomaceous earth modified by in situ precipitation of a combination of ferric chloride and aluminum chloride adsorbed greater than 80% of enteroviruses (poliovirus 1, echovirus 5, and coxsackievirus B5) and coliphage MS2 present in tap water at ambient pH (7.8 to 8.3), even after filtration of 100 liters of tap water. Viruses adsorbed to the filters could be recovered by mixing the modified diatomaceous earth with 3% beef extract plus 1 M NaCl (pH 9).     

Concentration of Viruses from Sewage and Excreta on Insoluble Polyelectrolytes

The concentration of viruses from sewage by adsorption on and elution from an insoluble cross-linked copolymer of maleic anhydride is described. Viruses either added to sewage or naturally contained in sewage were preferentially adsorbed to this polyelectrolyte at a pH range of 5.0 to 6.0 and were eluted at pH 8.0 to 9.0. In a 2-month survey of viruses in sewage in the spring (April to May 1968), when viruses are at low levels, efficient and economical detection of these agents was accomplished with the polyelectrolyte-concentration method. This method lends itself to the detection of viruses present in minute amounts in fecal samples, urine, sewage, and other natural waters. Large volumes of these fluids can be treated with the polymer described, and virus can be concentrated sufficiently for detection.     

A New Microviridae Phage Isolated from a Failed Biotechnological Process Driven by Escherichia coli

Bacteriophages are present in every environment that supports bacterial growth, including manmade ecological niches. Virulent phages may even slow or, in more severe cases, interrupt bioprocesses driven by bacteria. Escherichia coli is one of the most widely used bacteria for large-scale bioprocesses; however, literature describing phage-host interactions in this industrial context is sparse. Here, we describe phage MED1 isolated from a failed industrial process. Phage MED1 (Microviridae family, with a single-stranded DNA [ssDNA] genome) is highly similar to the archetypal phage phiX174, sharing >95% identity between their genomic sequences. Whole-genome phylogenetic analysis of 52 microvirus genomes from public databases revealed three genotypes (alpha3, G4, and phiX174). Phage MED1 belongs to the phiX174 group. We analyzed the distribution of single nucleotide variants in MED1 and 18 other phiX174-like genomes and found that there are more missense mutations in genes G, B, and E than in the other genes of these genomes. Gene G encodes the spike protein, involved in host attachment. The evolution of this protein likely results from the selective pressure on phages to rapidly adapt to the molecular diversity found at the surface of their hosts.     

Rifampin inhibition of bacteriophage phiX174 parental replicative-form DNA synthesis in an Escherichia coli dnaC mutant.

The Escherichia coli dnaC protein is not absolutely required in vivo for bacteriophage phiX174 parental replicative-form synthesis (Kranias and Dumas, 1974). However, when rifampin is present at a concentration that inhibits DNA-dependent RNA polymerase, phiX174 parental replicative-form synthesis is dependent on the dnaC protein activity. We conclude that E. coli DNA-dependent RNA polymerase can substitute for the dnaC protein in phiX174 parental replicative-form DNA synthesis, presumably in its initiation. The implications of this result with respect to the in vitro synthesis of the complementary strand of phiX174 DNA are discussed.     

Penetration of different human pathogenic viruses into sand columns percolated with distilled water, groundwater, or wastewater.

The adsorption of several enteroviruses and rotavirus SA11 to sand from an aquifer in the Federal Republic of Germany was estimated in sand-filled columns loaded with ca. 10(7) PFU and run at a velocity of 2.5 m/day for 12 h. After either distilled water, groundwater, secondary effluent, or tertiary effluent was percolated, the sand core was slowly extruded out of the column and cut in 1-cm slices. The slices were eluted with nutrient broth, and the amount of viruses in the broth was estimated. The best adsorption was promoted by groundwater and tertiary effluent, followed by distilled water and secondary effluent. Similar experiments, carried out at different percolation rates, indicated that a 50-day underground stay of recharged water probably suffices to eliminate viruses in the groundwater-recharged tertiary effluent. However, when viruses and sand were incubated in the presence of the surfactants sodium dodecyl sulfate, nonyl phenol, dodigen 226, or alkylbenzylsulfonate, the adsorption of the viruses was substantially diminished. Experiments in the presence of nonyl phenol seem to indicate that hydrophobic interactions are involved in the adsorption of viruses to sand.     

Calmodulin interacts with cyclic nucleotide phosphodiesterase and calcineurin by binding to a metal ion-independent hydrophobic region on these proteins

Hydrophobic interaction chromatography is employed to determine if calmodulin might associate with its target enzymes such as cyclic nucleotide phosphodiesterase and calcineurin through its Ca2+-induced hydrophobic binding region. The majority of protein in a bovine brain extract that binds to a calmodulin-Sepharose affinity column also is observed to bind in a metal ion-independent manner to phenyl-Sepharose through hydrophobic interactions. Cyclic nucleotide phosphodiesterase activity that is bound to phenyl-Sepharose can be resolved into two activity peaks; one peak of activity is eluted with low ionic strength buffer, while the second peak eluted with an ethylene glycol gradient. Calcineurin bound tightly to the phenyl-Sepharose column and could only be eluted with 8 M urea. Increasing ethylene glycol concentrations in the reaction mixture selectively inhibited the ability of calmodulin to stimulate phosphodiesterase activity, suggesting that hydrophobic interaction is required for activation. Comparison of the proteins which are bound to and eluted from phenyl- and calmodulin-Sepharose affinity columns indicates that chromatography involving calmodulin-Sepharose resembles hydrophobic interaction chromatography with charged ligands. In this type of interaction, hydrophobic binding either is reinforced by electrostatic attractions or opposed by electrostatic repulsions to create a degree of specificity in the binding of calmodulin to certain proteins with accessible hydrophobic regions.     

Penetration of different human pathogenic viruses into sand columns percolated with distilled water, groundwater, or wastewater

The adsorption of several enteroviruses and rotavirus SA11 to sand from an aquifer in the Federal Republic of Germany was estimated in sand-filled columns loaded with ca. 10(7) PFU and run at a velocity of 2.5 m/day for 12 h. After either distilled water, groundwater, secondary effluent, or tertiary effluent was percolated, the sand core was slowly extruded out of the column and cut in 1-cm slices. The slices were eluted with nutrient broth, and the amount of viruses in the broth was estimated. The best adsorption was promoted by groundwater and tertiary effluent, followed by distilled water and secondary effluent. Similar experiments, carried out at different percolation rates, indicated that a 50-day underground stay of recharged water probably suffices to eliminate viruses in the groundwater-recharged tertiary effluent. However, when viruses and sand were incubated in the presence of the surfactants sodium dodecyl sulfate, nonyl phenol, dodigen 226, or alkylbenzylsulfonate, the adsorption of the viruses was substantially diminished. Experiments in the presence of nonyl phenol seem to indicate that hydrophobic interactions are involved in the adsorption of viruses to sand.     

Bacteriophage inactivation at the air-water-solid interface in dynamic batch systems

Bacteriophages have been widely used as surrogates for human enteric viruses in many studies on virus transport and fate. In this investigation, the fates of three bacteriophages, MS2, R17, and phiX174, were studied in a series of dynamic batch experiments. Both MS2 and R17 readily underwent inactivation in batch experiments where solutions of each phage were percolated through tubes packed with varying ratios of glass and Teflon beads. MS2 and R17 inactivation was the result of exposure to destructive forces at the dynamic air-water-solid interface. phiX174, however, did not undergo inactivation in similar studies, suggesting that this phage does not accumulate at air-water interfaces or is not affected by interfacial forces in the same manner. Other batch experiments showed that MS2 and R17 were increasingly inactivated during mixing in polypropylene tubes as the ionic strength of the solution was raised (phiX174 was not affected). By the addition of Tween 80 to suspensions of MS2 and R17, phage inactivation was prevented. Our data suggest that viral inactivation in simple dynamic batch experiments is dependent upon (i) the presence of a dynamic air-water-solid interface (where the solid is a hydrophobic surface), (ii) the ionic strength of the solution, (iii) the concentration of surface active compounds in the solution, and (iv) the type of virus used.