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.
Polychromatic ultraviolet (UV) irradiation is a common method of pathogen inactivation in the water treatment industry. To improve its disinfection efficacy, more information is necessary on the mechanisms of ultraviolet inactivation on microorganisms at wavelengths throughout the germicidal UV spectrum, particularly below 240 nm. This work examined UV inactivation of the bacteriophage MS2, a common surrogate for enteric pathogens, as a function of wavelength. The bacteriophage was exposed to monochromatic UV irradiation from a tunable laser at wavelengths between 210 nm and 290 nm. To evaluate the mechanisms of UV inactivation throughout this wavelength range, RT-qPCR (Reverse Transcription quantitative Polymerase Chain Reaction) was performed to measure genomic damage for comparison with genomic damage at 253.7 nm. The results indicate that the rates of RNA damage closely mirror the loss of viral infectivity across the germicidal UV spectrum. This demonstrates that genomic damage is the dominant cause of MS2 inactivation from exposure to germicidal UV irradiation. These findings contrast those of adenovirus, for which MS2 is used as a viral surrogate when validating polychromatic UV reactors.
Reverse transcription, quantitative PCR (RT-qPCR) is a sensitive method for quantification of specific RNA targets, but the first step of the assay, reverse transcription, is notoriously variable and sensitive to reaction conditions. In this study, we used purified Bacteriophage MS2 genomic RNA as a model virus target to test two different RT enzymes (SuperScript II and SuperScript III), two RT-priming strategies (gene-specific primers and random hexamers), and varying background RNA concentrations (0-50ngμl(-1)) to determine how these variables influence the efficiency of reverse transcription over a range of target concentrations (10(1)-10(7) copies μl(-1)). The efficiency of the RT reaction was greatly improved by increasing both background RNA and primer concentrations. At a given target concentration, similar RT efficiencies were achieved with gene-specific primers and random hexamers, but the latter required much higher concentrations. Using random hexamers, we observed a systematic variation in RT reaction efficiency as a function of target concentration. Using an RNA standard curve that was also subject to RT effectively normalized the RT variability, but the accuracy with which one can determine absolute target concentrations depends critically on the nature of the RNA standard.
MRI Compatible MS2 Nanoparticles Designed to Cross the Blood-Brain-Barrier: Providing a Path towards Tinnitus Treatment
- Nanomedicine : nanotechnology, biology, and medicine
- Published about 2 years ago
Fundamental challenges of targeting specific brain regions for treatment using pharmacotherapeutic nanoparticle (NP) carriers include circumventing the blood-brain-barrier (BBB) and tracking delivery. Angiopep-2 (AP2) has been shown to facilitate the transport of large macromolecules and synthetic nanoparticles across the BBB. Thus, conjugation of AP2 to an MS2 bacteriophage based NP should also permit transport across the BBB. We have fabricated and tested a novel MS2 capsid-based NP conjugated to the ligand AP2. The reaction efficiency was determined to be over 70%, with up to two angiopep-2 conjugated per MS2 capsid protein. When linked with a porphyrin ring, manganese (Mn2+) remained stable within MS2 and was MRI detectable. Nanoparticles were introduced intracerebroventricularly or systemically. Systemic delivery yielded dose dependent, non-toxic accumulation of NPs in the midbrain. Design of a multifunctional MRI compatible NP platform provides a significant step forward for the diagnosis and treatment of intractable brain conditions, such as tinnitus.
Laboratory testing to support the care of patients with highly infectious diseases may pose a risk for laboratory workers. However, data on the risk of virus transmission during routine laboratory testing conducted using standard personal protective equipment (PPE) are sparse. Our objective was to measure laboratory contamination during routine analysis of patient specimens. Remnant specimens were spiked with the nonpathogenic MS2 bacteriophage at 1.0 x 107PFU/mL and biomarker contamination was assessed using RT-PCR for MS2. Specimen containers were exteriorly coated with a fluorescent powder to enable visualization of gross contamination using UV light. Testing was performed by two experienced laboratory technologists using standard laboratory PPE and sample-to-answer instrumentation. Fluorescence was noted on the gloves, bare hands, and laboratory coat cuffs of the laboratory technologist in 36/36 (100%), 13/36 (16%) and 4/36 (11%) tests performed, respectively. Fluorescence was observed in the biosafety cabinet (BSC) in 8/36 (22%) tests, on 14/32 (44%) test cartridges/devices, and 29/32 (91%) testing accessory items. Fluorescence was not observed on or in laboratory instrumentation or adjacent surfaces. In contrast to fluorescence detection, MS2 detection was infrequent (3/286; 1%) and occurred during test setup for the FilmArray instrument and on FilmArray accessory equipment. The information from this study may provide opportunities to improve clinical laboratory safety practices to reduce the risk of pathogen transmission to laboratory workers.
Evaluating the reduction of virus load in water reclamation plants is important to ensuring the hygienic safety of the reclaimed water. A virus-spiking test is usually used to estimate virus reduction but is not practicable at large-scale plants. Thus, we evaluated virus reduction by ultrafiltration (UF) plus ultraviolet (UV) irradiation at a large-scale reclamation plant (1000 m3/d) by quantifying indigenous F-specific RNA bacteriophages (FRNAPHs). To detect the infectious FRNAPH, we used both plaque assay and integrated culture-reverse-transcription polymerase chain reaction combined with the most probable number assay, which can detect infectious FRNAPH genotypes. For comparison, we determined reductions of indigenous FRNAPHs and spiked MS2 at a small-scale pilot plant (10 m3/d) at the same time. Reductions by UF were not significantly different among the bacteriophages at pilot plants. This result suggests that indigenous bacteriophages could be used for evaluating virus reduction by UF at large-scale plants. Indigenous Genotype I (GI) FRNAPH showed the highest UV resistance, followed by GII, GIII, and GIV. The resistance of GI-FRNAPH was equivalent to that of spiked MS2. The reduction of the total infectious FRNAPHs determined by plaque assay was affected by the predominant FRNAPH genotype, presumably because of their different UV resistances. Our results reveal that indigenous GI-FRNAPH can be a good alternative indicator to spiked MS2 in view of virus reduction during water reclamation. The reclaimed water from our large-scale reclamation plant could be used for irrigation because the expected reduction (6.3 log10) of indigenous GI-FRNAPH achieved the Title 22 (>5 log10).
Norovirus outbreaks are associated with the consumption of contaminated shellfish, and so efficient methods to recover and detect infectious norovirus in shellfish are important. The Proteinase K digestion method used to recover norovirus from shellfish, as described in the ISO 15216, would be a good candidate but its impact on the virus capsid integrity and thus infectivity was never examined. The aim of this study was to assess the impact of the Proteinase K digestion method, and of the heat treatment component of the method alone, on norovirus (genogroups I and II) and MS2 bacteriophage capsid integrity. A slightly modified version of the ISO method was used. RT-qPCR was used for virus detection following digestion of accessible viral RNA using RNases. MS2 phage infectivity was measured using a plaque assay. The effect of shellfish digestive glands (DG) on recovery was evaluated. In the presence of shellfish DG, a reduction in MS2 phage infectivity of about 1 log10 was observed after the Proteinase K digestion method and after heat treatment component alone. For norovirus GII and MS2 phage, there was no significant loss of genome following the Proteinase K digestion method but there was a significant 0.24 log10 loss of norovirus GI. In the absence of shellfish DG, the reduction in MS2 phage infectivity was about 2 log10, with the addition of RNases resulting in a significant loss of genome for all tested viruses following complete Proteinase K digestion method and the heat treatment alone. While some protective effect from the shellfish DG on viruses was observed, the impact on capsid integrity and infectivity suggests that this method, while suitable for norovirus genome detection, may not completely preserve virus infectivity.