Concept: Bacteriophage MS2
To use a MS2 bacteriophage model to compare three hand-drying methods, paper towels (PT), a warm air dryer (WAD) and a jet air dryer (JAD), for their potential to disperse viruses and contaminate the immediate environment during use.
We show that viruslike particles (VLPs) reassembled in vitro with the RNA bacteriophage MS2 coat protein and an RNA conjugate encompassing a siRNA and a known capsid assembly signal can be targeted to HeLa cells by covalent attachment of human transferrin. The siRNA VLPs protect their cargoes from nuclease, have a double-stranded conformation in the capsid and carry multiple drug and targeting ligands. The relative efficiency of VLP reassembly has been assessed, and conditions have been determined for larger scale production. Targeted VLPs have been purified away from unmodified VLPs for the first time allowing improved analysis of the effects of this synthetic virion system. The particles enter cells via receptor-mediated endocytosis and produce siRNA effects at low nanomolar concentrations. Although less effective than a commercial cationic lipid vector at siRNA delivery, the smaller amounts of internalized RNA with VLP delivery had an effect as good as if not better than the lipid transfection route. This implies that the siRNAs delivered by this route are more accessible to the siRNA pathway than identical RNAs delivered in complex lipid aggregates. The data suggest that the MS2 system continues to show many of the features that will be required to create an effective targeted drug delivery system. The fluorescence assays of siRNA effects described here will facilitate the combinatorial analysis of both future formulations and dosing regimes.
Ferrate [Fe(VI); FeO(4)(2-)] is an emerging oxidizing agent capable of controlling chemical and microbial water contaminants. Here, inactivation of MS2 coliphage by Fe(VI) was examined. The inactivation kinetics observed in individual batch experiments was well described by a Chick-Watson model with first-order dependences on disinfectant and infective phage concentrations. The inactivation rate constant k(i) at a Fe(VI) dose of 1.23 mgFe/L (pH 7.0, 25 °C) was 2.27(±0.05) L/(mgFe × min), corresponding to 99.99% inactivation at a Ct of ∼4 (mgFe × min)/L. Measured k(i) values were found to increase with increasing applied Fe(VI) dose (0.56-2.24 mgFe/L), increasing temperature (5-30 °C), and decreasing pH conditions (pH 6-11). The Fe(VI) dose effect suggested that an unidentified Fe byproduct also contributed to inactivation. Temperature dependence was characterized by an activation energy of 39(±6) kJ mol(-1), and k(i) increased >50-fold when pH decreased from 11 to 6. The pH effect was quantitatively described by parallel reactions with HFeO(4)(-) and FeO(4)(2-). Mass spectrometry and qRT-PCR analyses demonstrated that both capsid protein and genome damage increased with the extent of inactivation, suggesting that both may contribute to phage inactivation. Capsid protein damage, localized in the two regions containing oxidant-sensitive cysteine residues, and protein cleavage in one of the two regions may facilitate genome damage by increasing Fe(VI) access to the interior of the virion.
Reverse-transcription loop-mediated isothermal amplification (RT-LAMP) has frequently been proposed as an enabling technology for simplified diagnostic tests for RNA viruses. However, common detection techniques used for LAMP and RT-LAMP have drawbacks, including: poor discrimination capability, inability to multiplex targets, high rates of false positives, and (in some cases) the requirement of opening reaction tubes post-amplification. Here, we present a simple technique that allows closed-tube, target-specific detection, based on inclusion of a dye-labeled primer that is incorporated into a target-specific amplicon if the target is present. A short, complementary quencher hybridizes to unincorporated primer upon cooling down at the end of the reaction, thereby quenching fluorescence of any unincorporated primer. Our technique, which we term QUASR (for Quenching of Unincorporated Amplification Signal Reporters, read “quasar”), does not significantly reduce the amplification efficiency or sensitivity of RT-LAMP. Equipped with a simple LED excitation source and a colored plastic gel filter, the naked eye or a camera can easily discriminate between positive and negative QUASR reactions, which produce a difference in signal of approximately 10:1 without background subtraction. We demonstrate that QUASR detection is compatible with complex sample matrices such as human blood, using a novel LAMP primer set for bacteriophage MS2 (a model RNA virus particle). Furthermore, we demonstrate single-tube duplex detection of West Nile virus (WNV) and chikungunya virus (CHIKV) RNA.
The objective of this study was to characterize human norovirus (hNoV) GI and GII reductions during disinfection by peracetic acid (PAA) and monochloramine in secondary wastewater (WW) and phosphate buffer (PB) as assessed by reverse transcription-qPCR (RT-qPCR). Infectivity and RT-qPCR reductions are also presented for surrogate viruses murine norovirus (MNV) and bacteriophage MS2 under identical experimental conditions to aid in interpretation of hNoV molecular data. In WW, RT-qPCR reductions were less than 0.5 log10 for all viruses at concentration-time (CT) values up to 450 mg-min/L except for hNoV GI, where 1 log10 reduction was observed at CT values of less than 50 mg-min/L for monochloramine and 200 mg-min/L for PAA. In PB, hNoV GI and MNV exhibited comparable resistance to PAA and monochloramine with CT values for 2 log10 RT-qPCR reduction between 300 to 360 mg-min/L. Less than 1 log10 reduction was observed for MS2 and hNoV GII in PB at CT values for both disinfectants up to 450 mg-min/L. Our results indicate that hNoVs exhibit genogroup dependent resistance and that disinfection practices targeting hNoV GII will result in equivalent or greater reductions for hNoV GI. These data provide valuable comparisons between hNoV and surrogate molecular signals that can begin the process of informing regulators and engineers on WW treatment plant design and operational practices necessary to inactivate hNoVs.
Households that lack piped water supply are often forced to meet water needs by storing in the home, leaving water vulnerable to contamination by viruses. Storage in copper containers can potentially prevent this type of contamination, but the inactivation kinetics of viruses by copper need to be described to make appropriate storage recommendations. This work characterized inactivation kinetics of bacteriophage MS2 as a surrogate for enteric viruses by dissolved ionic copper in water. Reduction of MS2 increased with increasing doses of copper. At 0.3 mg/L, there was a 1.8-log10 reduction of MS2 within 6 h. At 1 and 3 mg/L, 2-2.5 log10 inactivation could be achieved between 6 and 24 h. Parameters for the Chick-Watson, Hom, and One Hit-Two Population models of inactivation were calculated and evaluated, all of which demonstrated strong goodness-of-fit and predictability at various contact times. Copper inactivates MS2 under controlled conditions at doses between 0.3 and 3 mg/L. Although requiring longer contact times than conventional disinfectants, it is a candidate for improving the safety of stored drinking water.
The aim of this study was to accurately quantify the impact of hydrodynamic cavitation on the infectivity of bacteriophage MS2, a norovirus surrogate, and to develop a small scale reactor for testing the effect of hydrodynamic cavitation on human enteric viruses, which cannot be easily prepared in large quantities. For this purpose, 3 mL scale and 1 L scale reactors were constructed and tested. Both devices were efficient in generating hydrodynamic cavitation and in reducing the infectivity of MS2 virus. Furthermore, they reached more than 4 logs reductions of viral infectivity, thus confirming the scalability of hydrodynamic cavitation for this particular application. As for the mechanism of page inactivation, we suspect that cavitation generated OH(-) radicals formed an advanced oxidation process, which could have damaged the host’s recognition receptors located on the surface of the bacteriophage. Additional damage could arise from the high shear forces inside the cavity. Moreover, the effectiveness of the cavitation was higher for suspensions containing low initial viral titers that are in similar concentration to the ones found in real water samples. According to this, cavitation generators could prove to be a useful tool for treating virus-contaminated wastewaters in the future.
Effect of membrane property and feed water organic matter quality on long-term performance of the gravity-driven membrane filtration process
- Environmental science and pollution research international
- Published 2 months ago
This study investigated the influence of membrane property and feed water organic matter quality on the permeate flux and water quality during gravity-driven membrane (GDM) filtration. GDM filtration was continuously carried out over 500 days at hydrostatic pressure of 65 mbar in dead-end mode without any back-flushing or membrane cleaning. Three ultrafiltration (UF) membranes (PES-100 kDa, PVDF-120 kDa, and PVDF-100 kDa) and one microfiltration (MF) membrane (PTFE-0.3 μm) were tested for treating lake water with varied organic matter qualities due to algal growth. The fluxes of the four membranes rapidly decreased to ~8 L/(m(2) × h) within a week of GDM filtration. The flux variations were quite similar for the four membranes during the entire GDM filtration, indicating that membrane property has a little effect on the flux. The flux strongly depends on the feed water organic matter quality. The average flux in treating low organics containing water (7-60 days) was ~5 L/(m(2) × h) and decreased to ~2 L/(m(2) × h) in treating high organics containing water (60-300 days). The accumulation of algal-derived biopolymers was mainly responsible for the flux decline by forming biofilms with high permeation resistance. The average flux in 300-500 days increased to ~3.5 L/(m(2) × h) when the feed water contained lower levels of biopolymers and higher levels of easily biodegradable organics, which created open and heterogeneous biofilms with lower permeation resistance. Removal efficiency for Escherichia coli was more than 5 log, while the removal efficiency for total bacterial cells was 1 log-2 log for the four membranes, indicating some bacterial regrowth after the filtration. Removal efficiency for the MS2 phage was 2.4 log and 1.5 log for the fouled PES-UF and PTFE-MF membranes.
Contact transmission of pathogens from personal protective equipment is a concern within the healthcare industry. During public health emergency outbreaks, resources become constrained and the reuse of personal protective equipment, such as N95 filtering facepiece respirators, may be needed. This study was designed to characterize the transfer of bacteriophage MS2 and fluorescein between filtering facepiece respirators and the wearer’s hands during three simulated use scenarios. Filtering facepiece respirators were contaminated with MS2 and fluorescein in droplets or droplet nuclei. Thirteen test subjects performed filtering facepiece respirator use scenarios including improper doffing, proper doffing and reuse, and improper doffing and reuse. Fluorescein and MS2 contamination transfer were quantified. The average MS2 transfer from filtering facepiece respirators to the subjects' hands ranged from 7.6-15.4% and 2.2-2.7% for droplet and droplet nuclei derived contamination, respectively. Handling filtering facepiece respirators contaminated with droplets resulted in higher levels of MS2 transfer compared to droplet nuclei for all use scenarios (p = 0.007). MS2 transfer from droplet contaminated filtering facepiece respirators during improper doffing and reuse was greater than transfer during improper doffing (p = 0.008) and proper doffing and reuse (p = 0.042). Droplet contamination resulted in higher levels of fluorescein transfer compared to droplet nuclei contaminated filtering facepiece respirators for all use scenarios (p = 0.009). Fluorescein transfer was greater for improper doffing and reuse (p = 0.007) from droplet contaminated masks compared to droplet nuclei contaminated filtering facepiece respirators and for improper doffing and reuse when compared improper doffing (p = 0.017) and proper doffing and reuse (p = 0.018) for droplet contaminated filtering facepiece respirators. For droplet nuclei contaminated filtering facepiece respirators, the difference in MS2 and fluorescein transfer did not reach statistical significance when comparing any of the use scenarios. The findings suggest that the results of fluorescein and MS2 transfer were consistent and highly correlated across the conditions of study. The data supports CDC recommendations for using proper doffing techniques and discarding filtering facepiece respirators that are directly contaminated with secretions from a cough or sneeze.
Tailed bacteriophages specific for Gram-negative bacteria encounter lipopolysaccharide (LPS) during the first infection steps. Yet, it is not well understood how biochemistry of these initial interactions relates to subsequent events orchestrating phage adsorption and tail rearrangements to initiate cell entry. For many phages, long O-antigen chains found on the LPS of smooth bacterial strains serve as essential receptor recognized by their tailspike proteins (TSP). Many TSP are depolymerases and O-antigen cleavage was described as necessary step for subsequent orientation towards a secondary receptor. However, O-antigen specific host attachment must not always come along with O-antigen degradation. In this issue of Molecular Microbiology Prokhorov et al. report that coliphage G7C carries a TSP that deacetylates O-antigen but does not degrade it, whereas rough strains or strains lacking O-antigen acetylation remain unaffected. Bacteriophage G7C specifically functionalizes its tail by attaching the deacetylase TSP directly to a second TSP that is nonfunctional on the host’s O-antigen. This challenges the view that bacteriophages use their TSP only to clear their way to a secondary receptor. Rather, O-antigen specific phages may employ enzymatically active TSP as a tool for irreversible LPS membrane binding to initiate subsequent infection steps. This article is protected by copyright. All rights reserved.