Effects of ionic strength on bacteriophage MS2 behavior and their implications for the assessment of virus retention by ultrafiltration membranes

Abstract

Bacteriophage MS2 is widely used as a surrogate to estimate pathogenic virus elimination by membrane filtration processes used in water treatment. Given that this water technology may be conducted with different types of waters, we focused on investigating the effects of ionic strength on MS2 behavior. For this, MS2 was analyzed while suspended in solutions of various ionic strengths, first in a batch experiment and second during membrane ultrafiltration, and quantified using (i) quantitative reverse transcriptase PCR (qRT-PCR), which detects the total number of viral genomes, (ii) qRT-PCR without the RNA extraction step, which reflects only particles with a broken capsid (free RNA), and (iii) the PFU method, which detects only infectious viruses. At the beginning of the batch experiments using solutions containing small amounts of salts, losses of MS2 infectivity (90%) and broken particles (20%) were observed; these proportions did not change during filtration. In contrast, in high-ionic-strength solutions, bacteriophage kept its biological activity under static conditions, but it quickly lost its infectivity during the filtration process. Increasing the ionic strength decreased both the inactivation and the capsid breakup in the feed suspension and increased the loss of infectivity in the filtration retentate, while the numbers of MS2 genomes were identical in both experiments. In conclusion, the effects of ionic strength on MS2 behavior may significantly distort the results of membrane filtration processes, and therefore, the combination of classical and molecular methods used here is useful for an effective validation of the retention efficiency of ultrafiltration membranes.

FIG. 1.

Schematic representation of the laboratory cross-flow mode filtration apparatus.

FIG. 2.

Standard (A) and melting (B) curves of bacteriophage MS2 obtained using quantitative RT-PCR. (A) Results are expressed as means ± standard deviations of results from triplicate experiments. (B) The peak at 84.5°C represents MS2-specific amplification, while the peak at 77°C represents nonspecific amplicons that are observed in negative controls and in samples analyzed by qRT-PCR without the RNA extraction step. The quantification and detection limits of the qRT-PCR correspond to 10² and 10¹ equivalents of PFU/ml, respectively. Experiments were performed in triplicate. dT, deoxyribosylthymine.

FIG. 3.

Batch experiments of bacteriophage MS2 suspended in ultrapure water (A), tap water (B), and PBS (C). C/C₀ is the ratio of the quantity of total viral genomes (circles), broken particles (triangles), or infectious phage (squares) at time x over the initial quantity of total viral genomes in experimental suspension (1 × 10⁸ PFU/ml) after homogenization (time zero). Results are expressed as means ± standard deviations of results from triplicate experiments.

FIG. 4.

Retentates of membrane filtration experiments of bacteriophage MS2 suspended in tap water (A) and PBS (B). C/C₀ is the ratio of the quantity of total viral genomes (circles), broken particles (triangles), or infectious phage (squares) at time x over the initial quantity of total viral genomes in feed suspension (1 × 10⁸ PFU/ml) after homogenization (time zero). Results are expressed as means ± standard deviations of results from duplicate experiments. The detection limit of the PFU method corresponds to −8 on the y axis.

FIG. 5.

Batch experiments with bacteriophage MS2 suspended in tap water in the presence of increasing concentrations of NaCl added (0 to 6 g/liter). The quantity of total viral genomes (circles), broken particles (triangles), or infectious phage (squares) in each solution was quantified at the beginning of the experiment, after homogenization (time zero). Results are expressed as means ± standard deviations of results from triplicate experiments. The quantification limit of the qRT-PCR is 10² eq PFU/ml.

FIG. 6.

Batch experiments (A) and retentates of membrane filtration experiments (B) with bacteriophage MS2 suspended in tap water containing 5 g/liter of NaCl. C/C₀ is the ratio of the quantity of total viral genomes (circles), broken particles (triangles), or infectious phage (squares) at time x over the initial quantity of total viral genomes in the experimental suspension (1 × 10⁸ PFU/ml) after homogenization (time zero). Results are expressed as means ± standard deviations of results from triplicate experiments. The detection limit of the PFU method corresponds to −8 on the y axis.