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Track-etch membranes (e.g., Nuclepore polycarbonate membranes) are marketed and used as sieve filters. The track-etch process produces pores with approximately cylindrical cross sections and relatively uniform diameters. These thin membranes filter primarily by stereohindrance; i.e., large particles cannot pass through smaller holes. They have been used to determine approximate sizes of some viruses (5, 10). During an investigation of virus sizes in which track-etch filters with different pore sizes were used, it was found that some viruses (e.g., herpes simplex virus type I) in phosphate-buffered saline did not readily pass through pores whose diameters were significantly larger than the virus diameters, as determined by electron microscopy (10). The reason for this apparent discrepancy was suggested by evidence that viruses passed through tortuous-path filters.

During passage through tortuous-path filters (solution-cast membranes, including nitrocellulose membranes), virus particles may adsorb. Wallis et al. (13) concluded that poliovirus adsorption to nitrocellulose filters required the presence of one of several different mono-, di-, or trivalent salts, including NaCl, in the carrier medium. This indicated that there is some ionic aspect to the adsorption process and implied that ions might be directly involved in the adsorption phenomenon. Additional, nonionic (e.g., hydrophobic) aspects are also apparent in adsorption of virus particles to nitrocellulose. Several bacteriophages (including X174 and PRD1) adsorb via nonionic interactions with nitrocellulose membranes (9, 11). This adsorption can be prevented or reversed by the presence of a nonionic surfactant (4, 9).

One way of explaining the ionic factor is to acknowledge that the filter membrane has a negative charge (7). Since most viruses are negatively charged at neutral pH (2), a negative charge on the membrane might prevent nonionic adsorption because of electrostatic repulsion of the virus particles. The electrostatic forces between charged virus particles and a like-charged membrane can be modified by changing the ionic concentration in the intervening fluid; i.e., adding ions shields the charges (Debye-Hückel screening), thereby decreasing the effective electrostatic force (6). If the ionic concentration is high enough, the electrostatic repulsion could be reduced to the point that nonionic adsorption may occur.

In order to assess how this balance between ionic repulsion and nonionic adsorption might affect the transmission of viruses through track-etch membranes, virus passage was determined as a function of salinity and in the presence and absence of a nonionic surfactant. This was done by passing two bacteriophages, X174 and PRD1, through track-etch membranes with different nonionic adsorption properties and negative surface charges.

The two bacteriophages were chosen because of their different adsorption properties. X174 is an approximately spherical virus with a diameter of about 27 nm (3); its bacterial host is Escherichia coli C. With a pI (pH at which the net charge is zero) of 6.6, X174 has a slight negative charge at neutral pH (1). PRD1 also is an approximately spherical virus and has a diameter of about 63 nm (3), and it is more negatively charged and more strongly adsorptive through nonionic interactions than X174 is (9); its bacterial host is Salmonella typhimurium LT2. Both viruses were assayed biologically by plaque formation by using the double-agar layer technique (8).

The viruses were suspended in different concentrations of Dulbecco's phosphate-buffered saline without Ca²⁺ or Mg²⁺ (DPBS), which has a sodium ion (Na⁺) concentration of 160 mM when it is not diluted. A neutral pH was maintained after dilution, as shown in Table 1. The viruses were diluted to a concentration of about 1,500 PFU/ml with each final salt concentration just before filtration. Because of the possible importance of nonionic adsorption, in some experiments we added 0.1% Tween 80 (Sigma) to the DPBS to minimize such adsorption (9). Since the virus titers were about the same (±15%) in the presence and absence of surfactant, we believe that virus aggregation was not a significant factor in the filtration results.

View this table: [in this window] [in a new window]   TABLE 1.   pH values of DPBS after dilution with double-distilled water

The following two standard commercially available 0.2-µm-pore-size track-etch membrane filters were used: Nuclepore polycarbonate and Nuclepore polyester membrane filters (Corning Incorporated, Acton, Mass.). The polycarbonate membrane (which was coated by the manufacturer with polyvinylpyrrolidone to create minimal hydrophilic membrane adsorption) had a low negative surface charge (7). The chemistry of the polyester membrane resulted in a higher negative charge. In addition, 0.2-µm-pore-size track-etch Nuclepore polyvinylpyrrolidone-free polycarbonate filters were treated so that they had sodium dodecyl sulfate (SDS)-coated surfaces (12). This treatment was accomplished by dipping the filters into a solution containing 15% isopropyl alcohol and 0.3% SDS in distilled water. The hydrophobic end of the SDS molecule bound to the polycarbonate surface; this left the negatively charged sulfate end free, which resulted in a coated membrane surface, including coated pores (7, 12). The coated filters were thoroughly rinsed by dipping them into distilled water to remove any excess SDS that had not bound to the filters, and then they were air dried. The SDS coating provided hydrophilic filters with a negatively charged surfaces.

To determine the extent of virus passage through a membrane, 3 ml of a virus suspension was passed through the filter by using a 10-ml plastic disposable syringe (Becton Dickinson and Company, Franklin, N.J.) and a syringe pump (model 230; KD Scientific Inc., Boston, Mass.) to provide a constant flow rate of 2 ml/min, and the sample was collected in a glass test tube. To determine the percentage of virus transmission, the virus concentrations in the filtrate and the original suspension were compared.

Effect of salinity on passage through track-etch membranes. Transmission curves were determined for both viruses as a function of salinity (Na⁺ concentration). The data for X174 (Fig. 1A) revealed that essentially complete passage through the two commercial membranes (polycarbonate and polyester) occurred at the Na⁺ concentrations investigated. This indicates that there was no significant adsorption of X174 to either commercial membrane and confirmed that there was no evidence of virus aggregation at the Na⁺ concentrations used. However, X174 transmission through the SDS-coated membrane was substantially reduced at the higher Na⁺ concentrations.

View larger version (18K): [in this window] [in a new window]   FIG. 1.   Transmission of X174 (A) and PRD1 (B) through track-etch membranes with 0.2-µm-diameter pores in the presence of different Na+ concentrations, prepared by diluting Dulbecco's phosphate-buffered saline. Symbols: , Nuclepore polycarbonate (PC) membrane; , Nuclepore polyester (PE) membrane; , 0.3% SDS-coated polycarbonate membrane. The flow rate was 2 ml/min. The data are means of values from three to five experiments. The error bars indicate standard errors of the means.

Effect of a nonionic surfactant on virus passage. Increased virus passage in the presence of a nonionic surfactant (e.g., Tween 80) indicates that nonionic adsorption has occurred (9). When virus passage was reduced in the presence of physiological saline (160 mM Na⁺), 0.1% Tween 80 was added to DPBS to determine the extent of nonionic adsorption. The results are shown in Table 2. In each case, the presence of the nonionic surfactant increased passage of the virus so that passage was nearly complete, which indicated that nearly all of the adsorption of either virus to either membrane was primarily nonionic in nature.