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Human norovirus (hNoV) is an important etiology of gastrointestinal illness and can be transmitted via ingestion of contaminated water. Currently impractical to culture, hNoV detection is reliant on real-time polymerase chain reaction (RT-PCR)-based methods. This approach cannot distinguish between infective and inactivated viruses because intact regions of the RNA genome can amplify even if the damage is present in other regions of the genome or because intact genetic material is not contained within an infectious virion. Herein, we employ a multiple long-amplicon RT-qPCR extrapolation approach to assay genome-wide damage and an enzymatic pretreatment to study the impact of simulated sunlight on the infectivity of hNoV in clear, sensitizer-free water. Using MS2 coliphage as an internal control, the genome-wide damage extrapolation approach, previously successfully applied for UV-254 inactivation, vastly overestimated sunlight inactivation, suggesting key differences in photoinactivation under different spectral conditions. hNoV genomic RNA was more susceptible to simulated sunlight degradation per base compared to MS2 genomic RNA, while enzymatic pretreatment indicated that hNoV experienced more capsid damage than MS2. This work provides practical and mechanistic insight into the endogenous sunlight inactivation of single-stranded RNA bacteriophage MS2, a widely used surrogate, and hNoV GII.4 Sydney, an important health-relevant virus, in clear sensitizer-free water.

Virus adsorption to colloidal particles is an important issue in the water quality community. Namely, if viruses can quickly and strongly associate to colloids, this can potentially lead to significant implications for the management of biohazardous wastes at water reclamation facilities. This research evaluated the adsorption of bacteriophage MS2 to colloidal suspensions of kaolinite (KAO) and fiberglass (FG). Observed pseudo first-order MS2 removal rate constants were between 0.53 and 5.1 min−1 and between 2.4 and 3.5 min−1 for KAO and FG, respectively. These kinetics were at least an order of magnitude faster than previously reported values when compared to data retrieved at similar colloid concentrations. Fluorescent and bright field microscopic images showed clusters of MS2 on and around the edges of the colloids, and the majority of the bound MS2 was not readily removed during a vigorous wash step, suggesting comparatively strong, operationally relevant adsorption. MS2 aggregation was observed experimentally and predicted on the basis of interaction energies calculated with XDLVO models. When virus-containing biohazardous wastes are introduced into wastewater treatment plants, removing colloids is essential.

Despite the threat to human health posed by some single-stranded RNA viruses, little is understood about their assembly. The goal of this work is to introduce a new tool for watching an RNA genome direct its own packaging and encapsidation by proteins. Contrast variation small-angle X-ray scattering (CV-SAXS) is a powerful tool with the potential to monitor the changing structure of a viral RNA through this assembly process. The proteins, though present, do not contribute to the measured signal. As a first step in assessing the feasibility of viral genome studies, the structure of encapsidated MS2 RNA was exclusively detected with CV-SAXS and compared with a structure derived from asymmetric cryo-EM reconstructions. Additional comparisons with free RNA highlight the significant structural rearrangements induced by capsid proteins and invite the application of time-resolved CV-SAXS to reveal interactions that result in efficient viral assembly.

The secondary structures of the bacteriophage MS2 ssRNA genome, frozen in defined states, were determined with minimal perturbation using constraints from X-ray synchrotron footprinting (XRF). The footprints of the gRNA in the virion and as transcript are consistent with single, dominant but distinct conformations, and reveal the presence of multiple Packaging Signals potentially involved in assembly regulation that have not been detected by other techniques. XRF also reveals the dramatic effect of the unique Maturation Protein (MP) on both the capsid lattice, and the gRNA conformation inside the phage compared with a virus-like-particle composed only of coat protein subunits. Aspects of genome organisation in the phage, their impacts on the capsid shell, and the distortion of lattice geometry by MP, are hallmarks of molecular frustration. Phage assembly therefore appears to prepare the particle for the next step of the infectious cycle.

The capsids of RNA viruses such as MS2 are great models for studying protein self-assembly because they are made almost entirely of multiple copies of a single coat protein (CP). Although CP is the minimal repeating unit of the capsid, previous studies have shown that CP exists as a homodimer (CP2) even in an acid-disassembled system, indicating that CP2 is an obligate dimer. Here, we investigate the molecular basis of this obligate dimerization using coarse-grained structure-based models and molecular dynamics simulations. We find that, unlike monomeric proteins of similar size, CP populates a single partially folded ensemble whose "foldedness" is sensitive to denaturing conditions. In contrast, CP2 folds similarly to single-domain proteins populating only the folded and the unfolded ensembles, separated by a prominent folding free energy barrier. Several intramonomer contacts form early, but the CP2 folding barrier is crossed only when the intermonomer contacts are made. A dissection of the structure of CP2 through mutant folding simulations shows that the folding barrier arises both from the topology of CP and the interface contacts of CP2. Together, our results show that CP2 is an obligate dimer because of kinetic stability, that is, dimerization induces a folding barrier and that makes it difficult for proteins in the dimer minimum to partially unfold and access the monomeric state without completely unfolding. We discuss the advantages of this obligate dimerization in the context of dimer design and virus stability.

Rapid and sustained disinfection of surfaces is necessary to check the spread of pathogenic microbes. The current study proposes a method of synthesis and use of copper nanoparticles (CuNPs) for contact disinfection of pathogenic microorganisms. Polyphenol stabilized CuNPs were synthesized by successive reductive disassembly and reassembly of copper phenolic complexes. Morphological and compositional characterization by transmission electron microscope (TEM), selected area diffraction and electron energy loss spectroscopy revealed monodispersed spherical (ϕ5-8 nm) CuNPs with coexisting Cu, Cu(I) and Cu (II) phases. Various commercial grade porous and non-porous substrates, such as, glass, stainless steel, cloth, plastic and silk were coated with the nanoparticles. Complete disinfection of 107copies of surrogate enveloped and non-enveloped viruses: bacteriophage MS2, SUSP2, phi6; and gram negative as well as gram positive bacteria:Escherichia coliandStaphylococcus aureuswas achieved on most substrates within minutes. Structural cell damage was further analytically confirmed by TEM. The formulation was well retained on woven cloth surfaces even after repeated washing, thereby revealing its promising potential for use in biosafe clothing. In the face of the current pandemic, the nanomaterials developed are also of commercial utility as an eco-friendly, mass producible alternative to bleach and alcohol based public space sanitizers used today.

We found that ultraviolet-A (UV-A) light exposure resulted in a modest reduction in recovery of methicillin-resistant Staphylococcus aureus (MRSA), Candida auris, bacteriophage MS2, and bacteriophage Phi X174, but not Clostridioides difficile spores, on steel disk carriers. Four hours of UV-A exposure from a ceiling light fixture resulted in a significant reduction in pathogenic microorganisms recovered from in-use medical equipment. These findings suggest that UV-A could be useful as a means to provide continuous low-level decontamination of surfaces in health care facilities.

Virus-like particle (VLP) conjugates are being developed for biomedical applications; however, there is a lack of quantitative analytical methods to measure the extent of conjugation and modification of VLP based therapeutics. Charge detection mass spectrometry (CDMS) can measure mass distributions for large and heterogeneous complexes and is emerging as a valuable tool in the analysis of biologics. In this study, CDMS is used to characterize the stoichiometry and population distribution of antibodies covalently conjugated to the surface of a bacteriophage MS2 VLP. Initial CDMS analysis of the unconjugated MS2 particles suggested that they had packaged a broad distribution of exogenous genomic material. We developed procedures to remove the undesired genomic material from the VLP preparation and observed that, for the samples where the genomic fragments were removed, the antibody coupling reaction efficiency increased by almost a factor of 2. This meant there were (1) fewer VLPs with no antibodies bound, which is an important consideration for the efficacy of a targeted therapeutic and (2) fewer antibodies were wasted during the coupling reaction. CDMS could be employed in a similar manner as a tool to characterize coupling reaction product distributions and precursors and help inform the development of the next generation of conjugate-based therapies.

AIMS: (1) To develop an analytical method for recovery and quantification of bacteriophage MS2 – as a surrogate for foot and mouth disease virus (FMDV) – from complex porous surfaces, with and without the presence of laboratory-developed agricultural grime; (2) To evaluate, with a 4-log dynamic range, the virucidal activity of common biocides for their ability to decontaminate surfaces, and hence remediate facilities, following a foreign animal disease contamination incident. METHODS AND RESULTS: An analytical method was developed and optimized for MS2 recovery from simulated agricultural surfaces. The addition of Dey-Engley neutralizing broth to an extraction buffer improved MS2 viability in liquid extracts, with optimal analytical holding times determined as < 8h to ≤24h, depending on matrix. The recovery of MS2 from surface materials decreased in order: non-porous reference material > grimed porous materials > non-grimed porous materials. In disinfectant testing, two spray applications of pAB were effective against MS2 (≥ 4-log reduction) on all operational-scale materials. 2% citric acid had limited effectiveness, with ≥ 4-log reduction observed on a selected subset of grimed concrete samples. CONCLUSIONS: Decontamination efficacy test results can be affected by surface characteristics, extraction buffer composition, analytical holding time, and surface-specific organism survivability. Efficacy should be evaluated using a test method that reflects the environmental characteristics of the intended application. SIGNIFICANCE AND IMPACT OF THE STUDY: The results of this study demonstrate the importance of analytical method verification tests for disinfectant testing prior to application in complex environments.

Summarization: Nanoparticles (NPs) are used in numerous applications and have been observed to accumulate in natural water bodies, including aquifers where they can interact with suspended colloids and viruses. This study examines the attachment of bacteriophage MS2 onto titanium dioxide (TiO 2 ) anatase NPs using three different MS2 concentrations. Batch experiments, were conducted at room temperature to investigate the effect of ambient light and the presence of quartz sand on MS2 and TiO 2 NPs heteroaggregation. Appropriate attachment isotherms were determined. Extended DLVO (XDLVO) theory was used to quantify the various interaction energy profiles. The results of batch experiments demonstrated that MS2 attachment onto TiO 2 NPs was favored in the presence of sand under ambient light, while under dark conditions no clear trend was observed. Estimated XDLVO interaction energy profiles indicated that hydrophobic interactions may play a major role and influence the aggregation and heteroaggregation of MS2 and TiO 2 NPs, as well as the simultaneous attachment of MS2 and TiO 2 NPs onto quartz sand. Παρουσιάστηκε στο: Colloids and Surfaces B: Biointerfaces

Cryo-electron microscopy and single-particle image analysis are frequently used methods for macromolecular structure determination. Conventional single-particle analysis, however, usually takes advantage of inherent sample symmetries which assist in the calculation of the structure of interest (such as viruses). Many viruses assemble an icosahedral capsid and often icosahedral symmetry is applied during structure determination. Symmetry imposition, however, results in the loss of asymmetric features of the virus. Here, we provide a brief overview of the methods used to investigate non-symmetric capsid features. These include the recently developed focussed classification as well as more conventional methods which simply do not impose any symmetry. Asymmetric single-particle image analysis can reveal novel aspects of virus structure. For example, the VP4 capsid spike of rotavirus is only present at partial occupancy, the bacteriophage MS2 capsid contains a single copy of a maturation protein and some viruses also encode portals or portal-like assemblies for the packaging and/or release of their genome upon infection. Advances in single-particle image reconstruction methods now permit novel discoveries from previous single-particle data sets which are expanding our understanding of fundamental aspects of virus biology such as viral entry and egress.

Bacteriophage MS2 was employed for targeted delivery of an apoptosis-inducing agent, Tl+, into a tumor tissue. The targeted delivery was ensured by iRGD peptide, a ligand of integrins presumably located on the surface of endotheliocytes of the tumor tissue neovasculature and certain tumor cells. The synthesized peptide was conjugated to MS2 capsid proteins. Tl+ ions from TlNO3 penetrated the phage particles and tightly bound to phage RNA. Peptide-modified MS2 preparations filled with Tl+ caused cell death in two types of cultivated human breast cancer cells and effected necrosis of these tumor xenografts in mice. Neither peptide-conjugated bacteriophage MS2 without Tl+ nor the phage filled with Tl+ but without the peptide or the same phage with the non-conjugated peptide in solution produced such effects. The preparation exhibited no acute toxicity at a therapeutic dose.

The purpose of this study was to evaluate the efficacy of a waste stabilization pond (WSP) system to reduce pathogen contaminants in sludge. This included examining the factors that influence the fate and concentration of human pathogens and their indicators in two sludge layers. The decay rates of five study microorganisms were determined under in-situ conditions at a WSP. The background levels of fecal origin microorganisms were consistently detected (ranging: Escherichia coli 104 to 106, enterococci 101 to 103, F-specific bacteriophage (MS2) 101 to 103 and somatic coliphage 101 to 104 colony-forming units (CFU) mL−1, as well as 101 to 102 human adenovirus gene copies mL−1) in the primary facultative pond. Among microorganisms tested, the bacteria generally decayed faster than adenovirus and bacteriophage, particularly in the upper sludge layer. Due to the observed regrowth of E. coli, it may have a limited value as an indicator for pathogen removal in the wastewater stabilization ponds. The abundance of E. coli numbers within the pond biome followed changes in pond temperature over time. The results of the study suggest that viruses could survive for a long time, particularly in deeper layers (>1 metre) in the sludge, during winter months (T90 = 156 d). The presence of human pathogens in WSP sludge, in particular viruses, may be a barrier to its beneficial reuse in agriculture. The results indicate that additional treatment of sludge may be required to mitigate potential public health risks from reuse of sludge for agricultural purposes.

Although rare cancerous cells are considered as more objective indications for a precise early diagnosis of cancers, accurate counting of them still is a spirited challenge. We reported a signal multiplication strategy by constructing element-tagged viruslike nanoparticles (VLNPs) with a precise number of atoms for a membrane biomarker mediated higher sensitive cell counting using inductively coupled plasma mass spectrometry (ICPMS). Typical bacteriophage MS2 was exemplified to demonstrate the effectiveness of the element-tagged VLNPs as signal multipliers. Dibenzylcyclooctyne-poly(ethylene glycol)-folate (DBCO-PEG-FA) and DOTA-Eu complex tag modified (FA-PEG)69-MS2-(DOTA-Eu)965 targeted the folate receptor (FR) on KB cells as low as subzeptomole FRs could be quantified by 153Eu-species unspecific isotope dilution ICPMS, allowing us to be able to count at least 5 KB cells. While more than 2197 KB cells were needed to give a significant ICPMS signal using FA-PEG-DOTA-Eu, demonstrating more than 2 orders of magnitude signal multiplication and resulting in total 4.0 × 108 times signal amplification relative to one KB cell. We believe that such a signal multiplication strategy can be expanded to quantify and count other membrane biomarkers and their host cells using various VLNPs modified with different kinds and precise numbers of elements and guiding groups. In this way, prescribed multiples of signal amplification can be realized for a more accurate ICPMS-based quantitative bioanalysis because targeted molecules/cells in a complicated biological system might exist in orders of magnitude wide concentration range.

Sunlight inactivation is important for disinfection of viruses in sunlit waters. As such, attempts have been made to predict the endogenous photoinactivation rate of bacteriophage MS2 using biological weighting functions, which describe microorganism sensitivity to sunlight inactivation as a function of wavelength. In this study, four biological weighting function models were compared to assess their ability to predict endogenous inactivation rates (kendo) of MS2. Previously-published and newly-collected datasets consisting of an incident irradiance spectrum (used as an input to the model) and a measured inactivation rate (kobs) were used for model validation and comparison. kendo values predicted by each model were compared with measured kobs to evaluate the ability of each biological weighting function to predict endogenous sunlight inactivation rates. A model previously developed by Mattle et al. (Env. Sci. Technol. 49, 334–342) over-predicted inactivation rates, whereas the other three models – a model from Fisher et al. (Env. Sci. Technol. 45, 9249–9255), a new model developed in this study, and a modification of the model by Mattle et al. (developed as part of this study) – were better able to estimate inactivation rates. The biological relevance of the spectral shape of each biological weighting function is discussed.

Abstract Viral pandemic outbreaks cause a significant burden on global health as well as healthcare expenditure. The use of antiviral agents not only reduces the spread of viral pathogens but also diminishes the likelihood of them causing infection. The antiviral properties of novel copper-silver and copper-zinc intermetallic nanoparticles against Escherichia coli bacteriophage MS2 (RNA virus) and Escherichia coli bacteriophage T4 (DNA virus) are presented. The intermetallic nanoparticles were spherical in shape and were between 90 and 120 nm. Antiviral activity was assessed at concentrations ranging from 0.05 to 2.0 wt/v% for 3 and 24 h using DNA and RNA virus model organisms. Both types of nanoparticles demonstrated strong potency towards RNA viruses (> 89% viral reduction), whilst copper-silver nanoparticles were slightly more toxic towards DNA viruses when compared to copper-zinc nanoparticles. Both nanoparticles were then incorporated into polymeric fibres (carrier) to investigate their antiviral effectiveness when composited into polymeric matrices. Fibres containing copper-silver nanoparticles exhibited favourable antiviral properties, with a viral reduction of 75% after 3 h of exposure. The excellent antiviral properties of the intermetallic nanoparticles reported in this study against both types of viruses together with their unique material properties can make them significant alternatives to conventional antiviral therapies and decontamination agents.

Background: Ultraviolet-C (UV-C) light devices are effective in reducing contamination on N95 filtering facepiece respirators. However, limited information is available on whether UV-C devices meet the Food and Drug Administration’s (FDA) microbiological requirements for Emergency Use Authorization (EUA) for respirator bioburden reduction. Methods: We tested the ability of 2 UV-C light boxes to achieve the 3-log10 microorganism reductions required for EUA for reuse by single users. Whole 3M 1860 or Moldex 1513 respirators were inoculated on the exterior facepiece, interior facepiece, and internal fibers with bacteriophage MS2 and/or 4 strains of bacteria and treated with UV-C cycles of 1 or 20 minutes. Colorimetric indicators were used to assess penetration of UV-C through the respirators. Results: For 1 UV-C box, a 20-minute treatment achieved the required bioburden reduction for Moldex 1513 but not 3M 1860 respirators. For the second UV-C box, a 1-minute treatment achieved the required bioburden reduction in 4 bacterial strains for the Moldex 1513 respirator. Colorimetric indicators demonstrated penetration of UV-C through all layers of the Moldex 1513 respirator but not the 3M 1860 respirator. Conclusions: Our findings demonstrate that UV-C box technologies can achieve bioburden reductions required by the FDA for EUA for single users but highlight the potential for variable efficacy for different types of respirators.

The pathogen concentration in human excreta needs to be managed appropriately, but a predictive approach has yet to be implemented due to a lack of kinetics models for pathogen inactivation that are available under varied environmental conditions. Our goals were to develop inactivation kinetics models of microorganisms applicable under varied environmental conditions of excreta matrices and to identify the appropriate indicators that can be monitored during disinfection processes. We conducted a systematic review targeting previous studies that presented time-course decay of a microorganism and environmental conditions of matrices. Defined as a function of measurable factors including treatment time, pH, temperature, ammonia concentration and moisture content, the kinetic model parameters were statistically estimated using hierarchical Bayesian modeling. The inactivation kinetics models were constructed for Escherichia coli, Salmonella, Enterococcus, Ascaris eggs, bacteriophage MS2, enterobacteria phage phiX174 and adenovirus. The inactivation rates of a microorganism were predicted using the established model. Ascaris eggs were identified as the most tolerant microorganisms, followed by bacteriophage MS2 and Enterococcus. Ammonia concentration, temperature and moisture content were the critical factors for the Ascaris inactivation. Our model predictions coincided with the current WHO guidelines. The developed inactivation kinetics models enable us to predict microbial concentration in excreta matrices under varied environmental conditions, which is essential for microbiological risk management in emerging resource recovery practices from human excreta.

Aims The efficacy of a novel photochemical method for generating chlorine dioxide (photoClO2 ) was evaluated against human noroviruses (HuNoV) surrogate, bacteriophage MS2, and Clostridium difficile endospores. Methods and results Chlorine dioxide was generated by mixing 1% sodium chlorite with 10 parts-per-million (ppm) Eosin Y and irradiating with a photo-activator-excitable light. PhotoClO2 efficacy was assessed against bacteriophage MS2 and C. difficile endospores in suspension, on hard surfaces and greenhouse conditions under soiled and unsoiled conditions. The estimated effective photoClO2 produced and consumed was 20·39 ± 0·16 ppm at a rate of 8·16 ppm per min in a 1% sodium chlorite solution. In suspension, MS2 phage was reduced by 3·35 and >5·10 log10 PFU per ml in 120 and 90 min, with and without soil, respectively. At the same time, when dried on stainless steel surface, MS2 phage was reduced by >4·53 log10 PFU per carrier in 30 min under both conditions. On the other hand, C. difficile endospores in suspension were reduced by 2·26 and 3·65 log10 CFU per ml in 120 min with and without soiling, respectively. However, on stainless steel surface, maximal reductions of the C. difficile endospores were 0·8 and 1·5 log10 CFU per carrier with and without soiling, respectively, and a maximal reduction of 2·97 log10 CFU per carrier under greenhouse conditions at 24 h. Conclusions Overall, photoClO2 showed promise as a technology to control HuNoV contamination on environmental surfaces but requires further optimization and testing against C. difficile endospores. Significance and impact of the study Results from this investigation will serve as a model for how to generate and quantify photoClO2 and how to appropriately evaluate this new class of disinfectants against environmentally resilient pathogens: viruses and bacterial endospores.

Viruses are infections species that infect a large spectrum of living systems. Although displaying a wide variety of shapes and sizes, they are all composed of nucleic acid encapsulated into a protein capsid. After virions enter the host cell, they replicate to produce multiple copies of themselves. They then lyse the host, releasing virions to infect new cells. The high proliferation rate of viruses is the underlying cause of their fast transmission among living species. Although many viruses are harmless, some of them are responsible for severe diseases such as AIDS, viral hepatitis, and flu. Traditionally, electron microscopy is used to identify and characterize viruses. This approach is time- and labor-consuming, which is problematic upon pandemic proliferation of previously unknown viruses, such as H1N1 and COVID-19. Herein, we demonstrate a novel diagnosis approach for label-free identification and structural characterization of individual viruses that is based on a combination of nanoscale Raman and infrared spectroscopy. Using atomic force microscopy-infrared (AFM-IR) spectroscopy, we were able to probe structural organization of the virions of Herpes Simplex Type 1 viruses and bacteriophage MS2. We also showed that tip-enhanced Raman spectroscopy (TERS) could be used to reveal protein secondary structure and amino acid composition of the virus surface. Our results show that AFM-IR and TERS provide different but complementary information about the structure of complex biological specimens. This structural information can be used for fast and reliable identification of viruses. This nanoscale bimodal imaging approach can be also used to investigate the origin of viral polymorphism and study mechanisms of virion assembly.

The inactivation of bacteriophage MS2 by sodium hypochlorite was investigated to understand the effect of solution chemistry on the disinfection efficacy in the presence of particles. Kaolinite and Microcystis aeruginosa (M. aeruginosa) were used as the models of inorganic and organic particles to simulate high turbidity and algal cells, respectively, in drinking water sources. In both particle-containing solutions, lower pH, the presence of cations (di-valent Ca2+) and natural organic matters (NOM) were regarded as the main factors to influence the aggregation and inactivation of MS2. The results showed that MS2 aggregated in all solutions at pH 3.0, protecting the inner viruses. At pH 7.0, the presence of Na+ cations (0–200 mmol/L) did not affect the inactivation efficacy of MS2, which always followed the order of particles-free ≈ kaolinite > M. aeruginosa. The inactivation efficacy of MS2 in the presence of Ca2+ cations followed the order of kaolinite > particles-free > M. aeruginosa at 0–50 mmol/L Ca2+ cations, while the inactivation efficacy remained almost constant in the range of 100–200 mmol/L Ca2+ cations. By contrast, kaolinite offered not enough protection to adsorbed MS2, but MS2 aggregation decreased disinfection efficacy at a high concentration of Ca2+ cations. Moreover, the presence of humic acid as NOM decreased the inactivation of MS2 more significantly than M. aeruginosa due to the more consumption of free chlorine from humic acids. Therefore, the co-existence of NOM and di-valent Ca2+ cations are potential challenges for the inactivation of viruses by sodium hypochlorite in safe drinking water.

Reverse osmosis (RO) membranes are commoditized and available for household water treatment (HWT) in areas where access to safe water is limited. The RO membranes for HWT (residential RO) are typically operated intermittently without a cleaning process. This suggests that a unique mechanism of membrane deterioration, membrane oxidation, one of the main causes of RO membrane deterioration in industrial settings (desalination and wastewater reclamation), is not involved. Furthermore, the intermittent operation provides repeated shear stress on the membrane surface. This study aimed to evaluate the impact of repeated pressurization on virus (bacteriophage MS2 and φX-174) removal by residential RO and to determine the location of integrity loss. We repeatedly pressurized and de-pressurized spiral-wound residential RO membranes for up to 10 000 cycles, while periodically evaluating virus removal. E. coli removal was also determined after 10 000 cycles. Moreover, these membranes were examined for virus and E. coli removal in a flat-sheet configuration. For the first 3000–4000 cycles, φX-174 removal was maintained at approximately 4 log10 (99.99%), and then dramatically decreased. After 10 000 cycles, even E. coli leaked from the membrane. The deterioration of virus removal in a flat-sheet configuration indicates integrity loss at the membrane surface. Therefore, repeated pressurization deteriorated the virus removal performance of residential RO. The number of times that the RO membrane can be pressurized should be included as a criterion to determine the frequency of membrane replacement in residential RO.

Background: Contaminated surfaces are a potential source for spread of respiratory viruses including SARS-CoV-2. Ultraviolet-C (UV-C) light is effective against RNA and DNA viruses and could be useful for decontamination of high-touch fomites that are shared by multiple users. Methods : A modification of the American Society for Testing and Materials standard quantitative carrier disk test method (ASTM E-2197-11) was used to examine the effectiveness of ultraviolet-C (UV-C) light for rapid decontamination of plastic airport security bins inoculated at 3 sites with methicillin-resistant Staphylococcus aureus (MRSA) and bacteriophages MS2, PhiX174, and Phi6, an enveloped RNA virus used as a surrogate for coronaviruses. Three log 10 reductions on inoculated plastic bins were considered effective for decontamination. Results: UV-C light administered as 10-, 20-, or 30-second cycles in proximity to a plastic bin reduced contamination on each of the test sites, including vertical and horizontal surfaces. The 30-second cycle met criteria for decontamination of all 3 test sites for all the test organisms except bacteriophage MS2 which was reduced by greater than 2 log 10 PFU at each site. Conclusions: UV-C light is an attractive technology for rapid decontamination of airport security bins. Further work is needed to evaluate the utility of UV-C light in real-world settings and to develop methods to provide automated movement of bins through a UV-C decontamination process.

We recently reported the photodynamic inactivation (PDI) of bacteriophage MS2 with a photosensitiser- 5, 10, 15, 20-tetrakis (1-methyl-4-pyridinio) porphyrin- tetra- p-toluene sulfonate (TMPyP) in solution and concluded that the A-protein of the virus is the main target of inactivation. Here, we have extended these studies and carried out PDI of bacteriophage Qβ, bovine enterovirus 2 (BEV-2) and type 1 murine norovirus (MNV-1). The rate of inactivation observed was in the order MS2 > Qβ > MNV-1 > BEV-2. Data suggested that TMPyP-treatment could also target the viral genome as well as result in disintegration/disassembly of viral particles. Although emergence of viral drug resistance is a well-documented phenomenon, it was not possible to generate PDI-resistant MS2. However, emergence of a mutation in the lysis protein was detected after serial exposure to PDI.

A reactive electrochemical membrane (REM) system was developed with titanium suboxide microfiltration membrane serving as the filter and the anode, and was examined to inactivate Escherichia coli (E. coli) in water at various current densities. After passing through the membrane filter, the concentration of E. coli decreased from 6.46 log CFU/mL to 0.18 log CFU/mL. The REM operation and effects, including membrane pressure, anode potential, protein leakage, and cell morphology, were characterized under different treatment conditions. It was found that several mechanisms, including membrane filtration, external electrical field influence, and direct oxidation, functioned in concert to lead to bacteria removal and inactivation, and direct oxidation likely played the major role. As revealed by scanning electron microscope and extracellular protein analysis, high current density and voltage caused severe cell damage that resulted in partial or complete cell disintegration. The removal of a model virus, bacteriophage MS2, was also investigated at the current density of 10 mA cm−2 and achieved 6.74 log reduction compared to the original concentration (1011 PFU/mL). In addition to illustration of mechanisms, this study may provide a potentially promising approach that is suitable for decentralized treatment to meet dispersed water disinfection needs.

We investigated virus removal and the formation of disinfection by-products by a combined ozonation, coagulation, and ceramic membrane filtration process (O3 + PACl + CMF) for water reclamation. Pilot-scale continuous operation used secondary effluent as test water, and virus removal performance was tested using F-specific bacteriophage MS2. The rate of increase of transmembrane pressure decreased from 29 to 1 kPa/d with increasing ozone dosage from 0 to 6 mg/L. Ozonation reduced reversible fouling, and residual O3 reduced irreversible fouling. Ozonation increased MS2 removal from 2.1- to 6.8-log with increasing specific ozone dosage from 0.16 to 1.29 mg-O3/mg-C. In PACl + CMF, however, MS2 removal decreased from 8.4- to 6.7-log. Lab-scale batch experiments revealed that increasing ozone dosage tended to inhibit MS2 coagulation. The inhibition was attributed to the negative effect of ozonation on particle destabilization, making it harder for the coagulant to neutralize surface charge in ozonated water. The formation of polar compounds such as formaldehyde during ozonation seems to increase negative charge. Although ozonation formed small amounts of N-nitrosodimethylamine (NDMA), it reduced their formation potential, possibly reducing the final concentration of NDMA in reclaimed water. Therefore, O3 + PACl + CMF not only improved operational performance, but also produced reclaimed water with reduced public health risks associated with viruses and NDMA.

Human noroviruses (HuNoVs) outbreaks have been linked to contaminated fresh-cut produce. Propolis has been used as a folk medicine because of its extensive bioactivity and antimicrobial properties. The aim of this study was to investigate the antiviral properties of propolis water extracts (PWE) and propolis ethanol extracts (PEE) against noroviruses, and its application in fresh-cut produce. Extraction yields, total phenolic content, total flavonoid, and soluble carbohydrate content of the propolis extracts were measured to estimate their antiviral compounds. The HuNoV GII.4 and its surrogates (murine norovirus MNV-1 and bacteriophage MS2) were used to evaluate the anti-noroviral activities of propolis extracts. The polyphenol-rich extract (PEE) showed better antiviral activities than PWE. Time-of-addition experiments and TEM images suggested that PEE denatured the viral capsid protein and likely prevented adsorption and internalization of viruses into cells. The application of PEE for natural preservative in fresh juices was also analyzed. After 30 min of treatment of the four different juices, 500 μg/mL PEE reduced MNV-1 and MS2 titers by > 3.0 log10. Therefore, the PEE containing high amounts of polyphenol improved the safety of fresh juices and could be potential candidates for the development of novel natural antiviral preservative for fresh juices.

The presence of enteric viruses on fresh berries poses a health risk to consumers. After berries are picked, a washing step can reduce microbial counts on fruit surfaces to increase their safety. This study compared the effectiveness of tap water, commercial solutions of fruit and vegetable sanitizer composed of peroxyacetic acid and hydrogen peroxide (PAA) and neutral electrolyzed oxidizing water (NEW) for washing fresh blueberries that had been inoculated with either murine norovirus (MNV-1), bacteriophage MS2, bovine rotavirus (boRV) or hepatitis A virus (HAV). Initially, inactivation levels were measured after blueberries were washed for 1, 3, 5, 10 and 20 min. Subsequently, the effect of combining up to three sequential 1-min washes was evaluated. Washes with water lasting up to 20 min reduced MNV-1, MS2 and HAV populations by 1–2 log10 (PFU) and boRV by less than 4 log10. Increasing washing time did not have a significant effect, except where NEW washes were increased by up to 3 min for MS2 and by 10 min for boRV, with viral titers decreasing by > 4 log10 and >5 log10, respectively. Viral counts were further reduced through additional washes as follows: reduction of MS2 with water, of MNV-1 with NEW and HAV with PAA. PAA achieved significant and rapid inactivation of boRV (>6 log10 PFU after 1 min), MNV-1 (>4 log10 after 10 min) and MS2 (>3 log10 after 3 min). HAV was moderately affected ( 4 log10 after 1 min), MS2 (>4 log10 after 3 min) and HAV (>4 log10 after 1 min). For boRV, washing time needed to be increased to 10 min or three sequential washes were needed to reach >5 log10 reduction. These results highlight the efficiency of PAA at reducing infectivity of MNV-1, MS2 bacteriophage and boRV, and NEW to inactivate the four viruses.

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.

Surfactant Modified Zeolite (SMZ) represents a versatile, cost-effective permeable reactive material, capable of treating multiple classes of contaminants. The potential for HDTMA-Br, a cationic surfactant commonly used to modify zeolite, to desorb from the zeolite surface has been identified as a potential issue for the ongoing use of SMZ in water remediation contexts. This paper investigates the toxicity of HDTMA-Br towards enteric virus surrogates, F-RNA bacteriophage MS2 and E. coli, Bacillus subtilis, and soil microflora. The concentration of surfactant desorbing from SMZ was quantified through a bioassay using E. coli. Results showed HDTMA-Br concentrations of ≥10-5M were toxic to MS2, ≥10-4M were toxic to E. coli and ≥10-6M were toxic to B. subtilis. No toxic relationship was established between HDTMA-Br and soil microflora. Desorption of ≥10-4M of HDTMA-Br was shown for the two SMZ samples under the mixing conditions used. Effects of this surfactant on total soil microflora were ambiguous since no toxic relationship could be established, however, HDTMA-Br, at concentrations desorbing from SMZ, were shown to impact the soil bacterium B. subtilis. Further research is required to determine the effect of this surfactant on microbial populations and species diversity in soils.

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.

This study examined the effect of 4 thermal processing methods on inactivation of male-specific bacteriophage MS2 in cow milk. MS2 was used as a viral surrogate of enteric viruses in this inactivation study. Milk samples were spiked at 10, 103, 105, and 107 PFU/ml with a milk fat content of 1.5, 2.5 and 3% weight per volume. We found that with higher milk fat content, survival of MS2 increased in the samples spiked with 105 or 107 PFU/ml, indicating that increasing the milk fat content had a significant effect on the survival of MS2 (p

Virus inactivation by nanoparticles (NPs) is hypothesized to affect virus fate and transport in the subsurface. This study examines the interactions of viruses with titanium dioxide (TiO2) anatase NPs, which is a good disinfectant with unique physiochemical properties. The bacteriophage MS2 was used as a model virus. A series of batch experiments of MS2 inactivation by TiO2 NPs were conducted at room temperature (25°C), in the presence and absence of quartz sand, with and without ambient light. Three sets of experiments were performed in phosphate buffered saline solution (PBS) and one in distilled deionized water (ddH2O). The virus inactivation experimental data were satisfactorily fitted with a pseudo-first order expression with a time dependent rate coefficient. Quartz sand was shown to affect MS2 inactivation by TiO2 NPs both in the presence and absence of ambient light, because, under the experimental conditions of this study, the quartz sand offers a protection to the attached MS2 against inactivation. Moreover, it was shown that low TiO2 concentration (10mg/L) affected only slightly MS2 inactivation with and without ambient light. Furthermore, PBS hindered MS2 inactivation by TiO2 NPs.

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 (0–50 ng μl −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, but the benefit provided by background RNA was source dependent. At a given target concentration, similar RT efficiencies were achieved with gene-specific primers and random hexamers, but the latter required much higher concentrations. With 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 for this systematic variability, but the assay accuracy depended critically on the length of the standard RNA extending to the 3' end of the qPCR target site. Our results shed some light on previous contradictory conclusions in the literature, and provide insights that may aid in the design of RT-qPCR assays and the design of synthetic RNA standards when full-length material is not available.

Portable equipment and other small devices used in health care are a potential source for dissemination of pathogens. We demonstrated that a high-level disinfection cabinet using ultrasonic submicron droplets of peracetic acid and hydrogen peroxide was effective against methicillin-resistant Staphylococcus aureus, Clostridiodes difficile spores, the nonenveloped virus bacteriophage MS2, and Candida auris on steel disk carriers. The device also eliminated Candida auris inoculated on real-world items of portable equipment.

Affinity selection using phage-display technologies is a powerful tool for identifying the peptide epitopes of monoclonal antibodies. Coupling affinity selection with deep sequencing technologies allows for the broad assessment of selectant populations. Here, we describe a method for using a phage-display platform to assess antibody specificity in human serum. We describe the method with reference to the bacteriophage MS2 virus-like particle (VLP) platform, but it can be adapted to other phage-display technologies as well.

The self-assembly of viral capsids is an essential step to the formation of infectious viruses. Elucidating the kinetic mechanisms of how a capsid or virus-like particle assembles could advance our knowledge about the viral lifecycle, as well as the general principles in self-assembly of biomaterials. However, current understanding of capsid assembly remains incomplete for many viruses due to the fact that the transient intermediates along the assembling pathways are experimentally difficult to be detected. In this paper, we constructed a new multiscale computational framework to simulate the self-assembly of virus like particles. We applied our method to the coat proteins of Bacteriophage MS2 as a specific model system. This virus-like particle of Bacteriophage MS2 has a unique feature that its 90 sequence-identical dimers can be classified into two structurally various groups: one is the symmetric CC dimer; the other is the asymmetric AB dimer. The homotypic interactions between AB dimers result in a 5-fold symmetric contact, while the heterotypic interactions between AB and CC dimers result in 6-fold symmetric contact. We found that the assembly can be described as a physical process of phase transition that is regulated by various factors such as concentration and specific stoichiometry between AB and CC dimers. Our simulations also demonstrate that heterotypic and homotypic interfaces play distinctive roles in modulating the assembling kinetics. The interaction between AB and CC dimers is much more dynamic than the interaction between two AB dimers. We therefore suggest that the alternate growth of viral capsid through the heterotypic dimer interactions dominates the assembling pathways. This is, to the best of our knowledge, the first multiscale model to simulate the assembling process of coat proteins in Bacteriophage MS2. The generality of this approach opens the door to its further applications in assembly of other viral capsids, virus-like particles and novel drug delivery systems.

Enteric viruses (viruses that infect the gastrointestinal tract) are responsible for most water-transmitted diseases. They are shed in high concentrations in the feces of infected individuals, persist for an extended period of time in water, and are highly infective. Exposure to contaminated water directly (through ingestion) or indirectly (for example, through hand-water contacts followed by hand-to-mouth contacts) increases the risk of virus transmission. The work described herein provides a quantitative model for estimating human-pathogenic virus retention on skin following contact with contaminated water. The work will be important in refining the contribution of indirect transmission of virus to risks associated with water-related activities., Indirect exposure to waterborne viruses increases the risk of infection, especially among children with frequent hand-to-mouth contacts. Here, we quantified the transfer of one bacteriophage (MS2) and two enteric viruses (adenovirus and coxsackievirus) from liquid to skin. MS2, a commonly used enteric virus surrogate, was used to compare virus transfer rates in a volunteer trial to those obtained using human cadaver skin and synthetic skin. MS2 transfer to volunteer skin was similar to transfer to cadaver skin but significantly different from transfer to synthetic skin. The transfer of MS2, adenovirus, and coxsackievirus to cadaver skin was modeled using measurements for viruses attaching to the skin (adsorbed) and viruses in liquid residual on skin (unadsorbed). We find virus transfer per surface area is a function of the concentration of virus in the liquid and the film thickness of liquid retained on the skin and is estimable using a linear model. Notably, the amount of MS2 adsorbed on the skin was on average 5 times higher than the amount of adenovirus and 4 times higher than the amount of coxsackievirus. Quantification of pathogenic virus retention to skin would thus be overestimated using MS2 adsorption data. This study provides models of virus transfer useful for risk assessments of water-related activities, demonstrates significant differences in the transfer of pathogenic virus and MS2, and suggests cadaver skin as an alternative testing system for studying interactions between viruses and skin. IMPORTANCE Enteric viruses (viruses that infect the gastrointestinal tract) are responsible for most water-transmitted diseases. They are shed in high concentrations in the feces of infected individuals, persist for an extended period of time in water, and are highly infective. Exposure to contaminated water directly (through ingestion) or indirectly (for example, through hand-water contacts followed by hand-to-mouth contacts) increases the risk of virus transmission. The work described herein provides a quantitative model for estimating human-pathogenic virus retention on skin following contact with contaminated water. The work will be important in refining the contribution of indirect transmission of virus to risks associated with water-related activities.

Recently, ultraviolet light-emitting diodes (UV-LEDs) have emerged as a new UV source, bringing flexibility for various UV wavelength combinations due to their unique feature of wavelength diversity. In this study, we investigated inactivation mechanisms of representative microorganisms at different wavelength combinations using UV-LEDs. Two types of indicator microorganisms were examined, namely Escherichia coli (E. coli) as a representative bacteria and bacteriophage MS2 as a representative virus. Different inactivation effects were observed, and the results for UVA pretreatment followed by UVC inactivation were particularly interesting. While a substantial shoulder in the E. coli UVC inactivation curve was observed, this was reduced by UVA pretreatment (365 nm) at 17 J/cm2. Further, 52 J/cm2 UVA eliminated the shoulder in the fluence-response curves, resulting in improved UVC (265 nm) inactivation of E. coli by over two orders of magnitude. No inactivation improvement was observed for MS2. Moreover, UVA pretreatment eliminated photoreactivation of E. coli but did not affect dark repair. Detailed investigation of inactivation mechanisms revealed that hydroxyl radicals (•OH) played a significant role in the effects of UVA pretreatment. This study demonstrated that •OH radicals were generated inside E. coli cells during UVA pretreatment, which accounted for the subsequent effects on E. coli. The impact of UVA pretreatment on E. coli inactivation and reactivation was mainly due to increased levels of •OH radicals in E. coli cells, impairing cell functions such as DNA self-repair.

Potable groundwater contamination by human enteric viruses poses serious health risks. Our understanding of virus subsurface transport has largely depended on studying bacteriophages as surrogates. Few studies have compared the transport behaviour of enteric viruses, especially norovirus, with phage surrogates. We conducted laboratory column experiments to investigate norovirus and bacteriophage MS2 (MS2) filtration in alluvial sand, and rotavirus, adenovirus and MS2 filtration in alluvial gravel aquifer media in 2 mM NaCl (pH 6.6–6.9) with pore velocities of 4.6–5.4 m/day. The data were analysed using colloid filtration theory and HYDRUS-1D 2-site attachment-detachment modelling. Norovirus removal was somewhat lower than MS2 removal in alluvial sand. The removal of rotavirus and adenovirus was markedly greater than MS2 removal in alluvial gravel. These findings concurred with the log10 reduction values, mass recoveries, attachment efficiencies and irreversible deposition rate constants. The modelling results suggested that the MS2 detachment rates were in the same order of magnitude as norovirus, but they were 1 order of magnitude faster than those of rotavirus and adenovirus. The attachment of viruses and MS2 was largely reversible with faster detachment than attachment rates, favouring free virus transport. These findings highlight the risk associated with continual virus transport through subsurface media if viruses are not inactivated and remobilising previously attached viruses could trigger contamination events. Thus, virus attachment reversibility should be considered in virus transport predictions in subsurface media. Further research is needed to compare surrogates with enteric viruses, especially norovirus, regarding their transport behaviours under different experimental conditions.

Highlights • Anaerobic membrane bioreactor effluent was treated for non-potable reuse. • Photon shielding by HS− inhibited UV inactivation of MS2 bacteriophage. • Hydrogen peroxide oxidized sulfides to sulfate at pH ≥ 8. • 180–225 mJ cm−2 UV doses achieved 5-log MS2 inactivation post-H2O2. • Anaerobic reuse was competitive with aerobic reuse at, Anaerobic membrane bioreactors reduce the energy cost of wastewater treatment and meet filtration requirements for non-potable reuse. However, sulfides (H2S/HS−) formed during anaerobic treatment exert a high chlorine demand and inhibit UV disinfection by photon shielding at 254 nm. This study evaluated the feasibility of hydrogen peroxide (H2O2) for sulfide oxidation, UV disinfection for inactivation of MS2 bacteriophage, and chlorine to provide a residual for distribution. H2O2 treatment at pH ≥ 8 favored sulfide oxidation to sulfate in 30 min at a 4:1 H2O2:sulfide stoichiometry. Compared to a 6:1 H2O2:sulfide molar ratio, treatment of anaerobic effluent with 0.5 mM sulfides with a 4:1 H2O2:sulfide molar ratio would increase the applied UV fluence needed for 5-log MS2 inactivation from 180 mJ cm−2 to 225 mJ cm−2. However, the lower H2O2 dose reduced the dose of chlorine needed to quench residual H2O2 and provide a residual for distribution. Treatment at the 4:1 H2O2:sulfide molar ratio was favored, because the cost savings in H2O2 and chlorine reagents outweighed the energy savings associated with UV treatment. However, H2O2/UV/chlorine treatment of anaerobic effluent was cost-competitive with conventional treatment of aerobic effluent for non-potable reuse only for < 285 µM sulfides., Graphical abstract Image, graphical abstract

Packaging of the genome into a protein capsid and its subsequent delivery into a host cell are two fundamental processes in the life cycle of a virus. Unlike double-stranded DNA viruses, which pump their genome into a preformed capsid, single-stranded RNA (ssRNA) viruses, such as bacteriophage MS2, co-assemble their capsid with the genome; however, the structural basis of this co-assembly is poorly understood. MS2 infects Escherichia coli via the host 'sex pilus' (F-pilus); it was the first fully sequenced organism and is a model system for studies of translational gene regulation, RNA-protein interactions, and RNA virus assembly. Its positive-sense ssRNA genome of 3,569 bases is enclosed in a capsid with one maturation protein monomer and 89 coat protein dimers arranged in a T = 3 icosahedral lattice. The maturation protein is responsible for attaching the virus to an F-pilus and delivering the viral genome into the host during infection, but how the genome is organized and delivered is not known. Here we describe the MS2 structure at 3.6 Å resolution, determined by electron-counting cryo-electron microscopy (cryoEM) and asymmetric reconstruction. We traced approximately 80% of the backbone of the viral genome, built atomic models for 16 RNA stem-loops, and identified three conserved motifs of RNA-coat protein interactions among 15 of these stem-loops with diverse sequences. The stem-loop at the 3' end of the genome interacts extensively with the maturation protein, which, with just a six-helix bundle and a six-stranded β-sheet, forms a genome-delivery apparatus and joins 89 coat protein dimers to form a capsid. This atomic description of genome-capsid interactions in a spherical ssRNA virus provides insight into genome delivery via the host sex pilus and mechanisms underlying ssRNA-capsid co-assembly, and inspires speculation about the links between nucleoprotein complexes and the origins of viruses.

RNA carries the genetic instructions for many viruses to replicate in their host cells. The photochemical reactions that take place in RNA and affect viral infectivity in natural and engineered environments, however, remain poorly understood. We exposed RNA oligomer segments from the genome of bacteriophage MS2 to UV254, simulated sunlight, and singlet oxygen (1O2) and analyzed the oligomer reaction kinetics with reverse transcription quantitative PCR (RT-qPCR) and quantitative matrix-assisted laser desorption–ionization time-of-flight (MALDI-TOF) mass spectrometry (MS). Following UV254 exposure, quantitative MALDI-TOF-MS detected significantly more RNA modifications than did RT-qPCR, suggesting that certain chemical modifications in the RNA were not detected by the reverse transcriptase enzyme. In contrast, MALDI-TOF-MS tracked as much 1O2-induced RNA damage as RT-qPCR. After 5 h of simulated sunlight exposure (5100 J/m2 UVB and 1.2 × 105 J/m2 UVA), neither MALDI-TOF-MS nor RT-qPCR detected significant d...

MS2 inactivation by UV irradiance was investigated with the focus on how the disinfection efficacy is influenced by bacteriophage MS2 aggregation and adsorption to particles in solutions with different compositions. Kaolinite and Microcystis aeruginosa were used as model inorganic and organic particles, respectively. In the absence of model particles, MS2 aggregates formed in either 1mM NaCl at pH=3 or 50-200mM ionic strength CaCl2 solutions at pH=7 led to a decrease in the MS2 inactivation efficacy because the virions located inside the aggregate were protected from the UV irradiation. In the presence of kaolinite and Microcystis aeruginosa, MS2 adsorbed onto the particles in either 1mM NaCl at pH=3 or 50-200mM CaCl2 solutions at pH=7. In contrast to MS2 aggregates formed without the presence of particles, more MS2 virions adsorbed on these particles were exposed to UV irradiation to allow an increase in MS2 inactivation. In either 1mM NaCl at pH from 4 to 8 or 2-200mM NaCl solutions at pH=7, the absence of MS2 aggregation and adsorption onto the model particles explained why MS2 inactivation was not influenced by pH, ionic strength, and the presence of model particles in these conditions. The influence of virus adsorption and aggregation on the UV disinfection efficiency found in this research suggests the necessity of accounting for particles and cation composition in virus inactivation for drinking water.

© 2016 American Chemical Society. Virus-like particles are used to encapsulate drugs, imaging agents, enzymes, and other biologically active molecules in order to enhance their function. However, the size of most virus-like particles is inflexible, precluding the design of appropriately sized containers for different applications. Here, we describe a chromatographic selection for virus-like particle assembly. Using this selection, we identified a single amino acid substitution to the coat protein of bacteriophage MS2 that mediates a uniform switch in particle geometry from T = 3 to T = 1 icosahedral symmetry. The resulting smaller particle retains the ability to be disassembled and reassembled in vitro and to be chemically modified to load cargo into its interior cavity. The pair of 27 and 17 nm MS2 particles will allow direct examination of the effect of size on function in established applications of virus-like particles, including drug delivery and imaging.

The spread of virus-induced infectious diseases through airborne routes of transmission is a global concern for economic and medical reasons. To study virus transmission, it is essential to have an effective aerosol collector such as the growth tube collector (GTC) system that utilizes water-based condensation for collecting virus-containing aerosols. In this work, we characterized the GTC system using bacteriophage MS2 as a surrogate for a small RNA virus. We investigated using RNA extraction and reverse transcription- polymerase chain reaction (RT-PCR) to study the total virus collection efficiency of the GTC system. Plaque assays were also used to enumerate viable viruses collected by the GTC system compared to that by a commercially available apparatus, the SKC® Biosampler. The plaque assay counts were used to enumerate viable viruses whereas RT-PCR provides a total virus count, including those viruses inactivated during collection. The effects of relative humidity (RH) and other conditions on collection efficiency were also investigated. Our results suggest that the GTC has a collection efficiency for viable viruses between 0.24 and 1.8% and a total virus collection efficiency between 18.3 and 79.0%, which is 1-2 orders of magnitude higher than that of the SKC® Biosampler. Moreover, higher RH significantly increases both the viable and total collection efficiency of the GTC, while its effect on the collection efficiency of the SKC® Biosampler is not significant.