Massively parallel dissection of RNA in RNA-protein interactions in vivo.

Many of the biological functions performed by RNA are mediated by RNA-binding proteins (RBPs), and understanding the molecular basis of these interactions is fundamental to biology. Here, we present massively parallel RNA assay combined with immunoprecipitation (MPRNA-IP) for in vivo high-throughput dissection of RNA-protein interactions and describe statistical models for identifying RNA domains and parsing the structural contributions of RNA. By using custom pools of tens of thousands of RNA sequences containing systematically designed truncations and mutations, MPRNA-IP is able to identify RNA domains, sequences, and secondary structures necessary and sufficient for protein binding in a single experiment. We show that this approach is successful for multiple RNAs of interest, including the long noncoding RNA NORAD, bacteriophage MS2 RNA, and human telomerase RNA, and we use it to interrogate the hitherto unknown sequence or structural RNA-binding preferences of the DNA-looping factor CTCF. By integrating systematic mutation analysis with crosslinking immunoprecipitation, MPRNA-IP provides a novel high-throughput way to elucidate RNA-based mechanisms behind RNA-protein interactions in vivo.

The dsRNA isolated from Escherichia coli infected with the MS2 bacteriophage induces the production of interferons.

Viral infections pose a significant threat to public health, and the production of interferons represents one of the most critical antiviral innate immune responses of the host. Consequently, the screening and identification of compounds or reagents that induce interferon production are of paramount importance. This study commenced with the cultivation of host bacterium 15,597, followed by the infection of Escherichia coli with the MS2 bacteriophage. Utilizing the J2 capture technique, a class of dsRNA mixtures (MS2+15,597) was isolated from the E. coli infected with the MS2 bacteriophage. Subsequent investigations were conducted on the immunostimulatory activity of the MS2+15,597 mixture. The results indicated that the dsRNA mixtures (MS2+15,597) extracted from E. coli infected with the MS2 bacteriophage possess the capability to activate innate immunity, thereby inducing the production of interferon-ß. These dsRNA mixtures can activate the RIG-I and TLR3 pattern recognition receptors, stimulating the expression of interferon stimulatory factors 3/7, which in turn triggers the NF-κB signaling pathway, culminating in the cellular production of interferon-ß to achieve antiviral effects. This study offers novel insights and strategies for the development of broad-spectrum antiviral drugs, potentially providing new modalities for future antiviral therapies.

Photostability and photodynamic antimicrobial profile of dye extracts from four (4) plants: prospects for eco-friendly low-cost food disinfection and topical biomedical applications.

In this study, photostability and photodynamic antimicrobial performance of dye extracts from Hibiscus sabdariffa (HS) calyces, Sorghum bicolor (SB) leaf sheaths, Lawsonia inermis (LI) leaves and Curcuma longa (CL) roots were investigated in Acetate-HCl (AH) Buffer (pH 4.6), Tris Base-HCl (TBH) Buffer (pH 8.6), distilled water (dH2O), and Phosphate Buffer Saline (PBS, pH 7.2) using Bacillus subtilis as model for gram positive bacteria, Escherichia coli as model for gram negative bacteria, phage MS2 as model for non-envelope viruses and phage phi6 as model for envelope viruses including SARS CoV-2 which is the causative agent of COVID-19. Our results showed that the photostability of the dye extracts is in the decreasing order of LI > CL > SB > HS. The dye extract-HS is photostable in dH2O but bleaches in buffers-AH, TBH and PBS. The rate of bleaching is higher in AH compared to in TBH and PBS. The bleaching and buffers affected the photodynamic and non-photodynamic antimicrobial activity of the dye extracts. The photodynamic antibacterial activity of the dye extracts is in the decreasing order of CL > HS > LI > SB while the non-photodynamic antibacterial activity is in the decreasing order of LI > CL > HS > SB. The non-photodynamic antiviral activity pattern observed is the same as that of non-photodynamic antibacterial activity observed. However, the photodynamic antiviral activity of the dye extracts is in the decreasing order of CL > LI > HS > SB. Given their performance, the dye extracts maybe mostly suitable for environmental applications including fresh produce and food disinfection, sanitation of hands and contact surfaces where water can serve as diluent for the extracts and the microenvironment is free of salts.

Heat inactivation of aqueous viable norovirus and MS2 bacteriophage.

AIMS: This study aimed to compare the heat inactivation kinetics of viable human norovirus with the surrogate, MS2 bacteriophage as well as assess the decay of the RNA signal. METHODS AND RESULTS: Human intestinal enteroids were used to analyze the heat inactivation kinetics of viable human norovirus compared to the surrogate MS2 bacteriophage, which was cultured using a plaque assay. Norovirus decay rates were 0.22 min-1, 0.68 min-1, and 1.11 min-1 for 50°C, 60°C, and 70°C, respectively, and MS2 bacteriophage decay rates were 0.0065 min-1, 0.045 min-1, and 0.16 min-1 for 50°C, 60°C, and 70°C, respectively. Norovirus had significantly higher decay rates than MS2 bacteriophage at all tested temperatures (P = .002-.007). No decrease of RNA titers as measured by reverse transcription-PCR for both human norovirus and MS2 bacteriophage over time was observed, indicating molecular methods do not accurately depict viable human norovirus after heat inactivation and treatment efficiency is underestimated. CONCLUSIONS: Overall, our data demonstrate that MS2 bacteriophage is a conservative surrogate to measure heat inactivation and potentially overestimates the infectious risk of norovirus. Furthermore, this study corroborates that measuring viral RNA titers, as evaluated by PCR methods, does not correlate with the persistence of viable norovirus under heat inactivation.

Evaluation of woodchips-amended biosand filter for nitrate and MS2 bacteriophage reduction.

In this study, two types of woodchip-amended biosand filters (Filter A sand: woodchip = 33%: 67% versus Filter B sand: woodchip = 50%: 50%, by volume) were constructed, and their abilities to remove MS2 bacteriophage and nitrate were investigated. The results indicated that Filter A and Filter B could reduce nitrate up to 40 and 36%, respectively, indicating that the nitrate reduction increased with the increase in woodchip proportion. The study underscores a positive correlation between nitrate reduction and proportional increase in woodchip content, implying the potential for fine-tuning nitrate removal by varying sand-woodchip compositions. W-BSFs could remove MS2 bacteriophage to 1.91-log10 (98.8%) by Filter A and 1.88-log10 (98.7%) by Filter B over 39 weeks. The difference in sand-woodchip proportion did not significantly impact the MS2 reduction, demonstrating that a single W-BSF can maintain its virus removal performance fairly well over a long-term period. These results indicated that the nitrate reduction could be adjusted by varying sand-woodchip contents without impacting virus removal performance. Microbial community analysis indicated that the nitrate removal by the W-BSFs could be attributed to the denitrifying bacteria, such as the family Streptomycetaceae, the genera Pseudomonas, and Bacillus, and relative abundances of the phylum Nitrospirae.

Ozone and photodynamic inactivation of norovirus surrogate bacteriophage MS2 in fresh Brazilian berries and surfaces.

This study assessed the efficacy of ozone (bubble diffusion in water; 6.25 ppm) and photodynamic inactivation (PDT) using curcumin (75 µM) as photosensitizer (LED emission 430-470 nm; 33.6 mW/cm2 irradiance; 16.1, 20.2, and 24.2 J/cm2 light dose) against the Norovirus surrogate bacteriophage MS2 in Brazilian berries (black mulberry and pitanga) and surfaces (glass and stainless steel). Contaminated berries and surfaces were immersed in ozonized water or exposed to PDT-curcumin for different time intervals. Transmission electron microscopy was used to assess the effects of the treatments on MS2 viral particles. The MS2 inactivation by ozone and PDT-curcumin varied with the fruit and the surface tested. Ozone reduced the MS2 titer up to 3.6 log PFU/g in black mulberry and 4.1 log PFU/g in pitanga. On surfaces, the MS2 reduction by ozone reached 3.6 and 4.8 log PFU/cm2 on glass and stainless steel, respectively. PDT-curcumin reduced the MS2 3.2 and 4.8 log PFU/g in black mulberry and pitanga and 2.7 and 3.3 log PFU/cm2 on glass and stainless steel, respectively. MS2 particles were disintegrated by exposure of MS2 to ozone and PDT-curcumin on pitanga. Results can contribute to establishing effective practices for controlling NoV in fruits and surfaces, estimated based on MS2 bacteriophage behavior.

Efficacy of Grignard Pure to Inactivate Airborne Phage MS2, a Common SARS-CoV-2 Surrogate.

Grignard Pure (GP) is a unique and proprietary blend of triethylene glycol (TEG) and inert ingredients designed for continuous antimicrobial treatment of air. TEG has been designated as a ″Safer Chemical" by the US EPA. GP has already received approval from the US EPA under its Section 18 Public Health Emergency Exemption program for use in seven states. This study characterizes the efficacy of GP for inactivating MS2 bacteriophage─a nonenveloped virus widely used as a surrogate for SARS-CoV-2. Experiments measured the decrease in airborne viable MS2 concentration in the presence of different concentrations of GP from 60 to 90 min, accounting for both natural die-off and settling of MS2. Experiments were conducted both by introducing GP aerosol into air containing MS2 and by introducing airborne MS2 into air containing GP aerosol. GP is consistently able to rapidly reduce viable MS2 bacteriophage concentration by 2-3 logs at GP concentrations of 0.04-0.5 mg/m3 (corresponding to TEG concentrations of 0.025 to 0.287 mg/m3). Related GP efficacy experiments by the US EPA, as well as GP (TEG) safety and toxicology, are also discussed.

Adsorption of Respiratory Syncytial Virus, Rhinovirus, SARS-CoV-2, and F+ Bacteriophage MS2 RNA onto Wastewater Solids from Raw Wastewater.

Despite the widespread adoption of wastewater surveillance, more research is needed to understand the fate and transport of viral genetic markers in wastewater. This information is essential for optimizing monitoring strategies and interpreting wastewater surveillance data. In this study, we examined the solid-liquid partitioning behavior of four viruses in wastewater: SARS-CoV-2, respiratory syncytial virus (RSV), rhinovirus (RV), and F+ coliphage/MS2. We used two approaches: (1) laboratory partitioning experiments using lab-grown viruses and (2) distribution experiments using endogenous viruses in raw wastewater. Partition experiments were conducted at 4 and 22 °C. Wastewater samples were spiked with varying concentrations of each virus, solids and liquids were separated via centrifugation, and viral RNA concentrations were quantified using reverse-transcription-digital droplet PCR (RT-ddPCR). For the distribution experiments, wastewater samples were collected from six wastewater treatment plants and processed without spiking exogenous viruses; viral RNA concentrations were measured in wastewater solids and liquids. In both experiments, RNA concentrations were higher in the solid fraction than the liquid fraction by approximately 3-4 orders of magnitude. Partition coefficients (KF) ranged from 2000-270,000 mL·g-1 across viruses and temperature conditions. Distribution coefficients (Kd) were consistent with results from partitioning experiments. Further research is needed to understand how virus and wastewater characteristics might influence the partitioning of viral genetic markers in wastewater.

Antiviral activity of copper contact surfaces against MS2 coliphage and hepatitis a virus.

AIMS: Viral diseases can be indirectly transmitted by contaminated non-food contact surfaces to final food products by cross-contamination. The interaction of metal surfaces and viruses, MS2 coliphage and hepatitis A virus (HAV), was investigated for strategy development in decreasing this transmission risk. METHODS AND RESULTS: MS2 deposited onto stainless-steel surface was stable but inactivated at 0.95 log10 PFU min-1 on 99.9% copper surfaces. Greater copper-inactivation of MS2 was observed in (a) simple media (phosphate buffered saline, PBS) than protein-rich media (beef extract buffer), and (b) acidic than pH ≥ 6.8 environments. Among food matrices (strawberry juices and beef broth), the greatest MS2 inactivation by copper occurred in filtered strawberry juice at pH 3.5. At a reduction of 0.17 log10 PFU min-1, HAV survived longer than MS2 on copper by FRhK-4 cell infectivity assay. CONCLUSIONS: The inactivation of virus on copper surfaces was greater in acidic viral surrounding environments and in simple PBS medium. In the same 99% PBS medium, MS2 may not be an appropriate surrogate for HAV when assessing viral inactivation on copper surfaces.

In situ structures of the genome and genome-delivery apparatus in a single-stranded RNA virus.

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 ß-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.

ssRNA phage penetration triggers detachment of the F-pilus.

Although the F-specific ssRNA phage MS2 has long had paradigm status, little is known about penetration of the genomic RNA (gRNA) into the cell. The phage initially binds to the F-pilus using its maturation protein (Mat), and then the Mat-bound gRNA is released from the viral capsid and somehow crosses the bacterial envelope into the cytoplasm. To address the mechanics of this process, we fluorescently labeled the ssRNA phage MS2 to track F-pilus dynamics during infection. We discovered that ssRNA phage infection triggers the release of F-pili from host cells, and that higher multiplicity of infection (MOI) correlates with detachment of longer F-pili. We also report that entry of gRNA into the host cytoplasm requires the F-plasmid-encoded coupling protein, TraD, which is located at the cytoplasmic entrance of the F-encoded type IV secretion system (T4SS). However, TraD is not essential for pilus detachment, indicating that detachment is triggered by an early step of MS2 engagement with the F-pilus or T4SS. We propose a multistep model in which the ssRNA phage binds to the F-pilus and through pilus retraction engages with the distal end of the T4SS channel at the cell surface. Continued pilus retraction pulls the Mat-gRNA complex out of the virion into the T4SS channel, causing a torsional stress that breaks the mature F-pilus at the cell surface. We propose that phage-induced disruptions of F-pilus dynamics provides a selective advantage for infecting phages and thus may be prevalent among the phages specific for retractile pili.

Synonymous modification results in high-fidelity gene expression of repetitive protein and nucleotide sequences.

Repetitive nucleotide or amino acid sequences are often engineered into probes and biosensors to achieve functional readouts and robust signal amplification. However, these repeated sequences are notoriously prone to aberrant deletion and degradation, impacting the ability to correctly detect and interpret biological functions. Here, we introduce a facile and generalizable approach to solve this often unappreciated problem by modifying the nucleotide sequences of the target mRNA to make them nonrepetitive but still functional ("synonymous"). We first demonstrated the procedure by designing a cassette of synonymous MS2 RNA motifs and tandem coat proteins for RNA imaging and showed a dramatic improvement in signal and reproducibility in single-RNA detection in live cells. The same approach was extended to enhancing the stability of engineered fluorescent biosensors containing a fluorescent resonance energy transfer (FRET) pair of fluorescent proteins on which a great majority of systems thus far in the field are based. Using the synonymous modification to FRET biosensors, we achieved correct expression of full-length sensors, eliminating the aberrant truncation products that often were assumed to be due to nonspecific proteolytic cleavages. Importantly, the biological interpretations of the sensor are significantly different when a correct, full-length biosensor is expressed. Thus, we show here a useful and generally applicable method to maintain the integrity of expressed genes, critical for the correct interpretation of probe readouts.

Evaluating the impact of ultraviolet C exposure conditions on coliphage MS2 inactivation on surfaces.

The COVID-19 pandemic has raised interest in using devices that generate ultraviolet C (UVC) radiation as an alternative approach for reducing or eliminating microorganisms on surfaces. Studies investigating the efficacy of UVC radiation against pathogens use a wide range of laboratory methods and experimental conditions that can make cross-comparison of results and extrapolation of findings to real-world settings difficult. Here, we use three different UVC-generating sources - a broad-spectrum pulsed xenon light, a continuous light-emitting diode (LED), and a low-pressure mercury vapour lamp - to evaluate the impact of different experimental conditions on UVC efficacy against the coliphage MS2 on surfaces. We find that a nonlinear dose-response relationship exists for all three light sources, meaning that linear extrapolation of doses resulting in a 1-log10 (90%) reduction does not accurately predict the dose required for higher (e.g. 3-log10 or 99.9%) log10 reductions. In addition, our results show that the inoculum characteristics and underlying substrate play an important role in determining UVC efficacy. Variations in microscopic surface topography may shield MS2 from UVC radiation to different degrees, which impacts UVC device efficacy. These findings are important to consider in comparing results from different UVC studies and in estimating device performance in field conditions.

Inactivation of MS2 bacteriophage on copper film deployed in high touch areas of a public transport system.

Although SARS-CoV-2 is primarily an airborne risk, the COVID-19 pandemic also highlighted the need for self-disinfection surfaces that could withstand the demand of high occupant densities characteristic of public transportation systems. The aim of this study was to evaluate the durability and antiviral activity of a copper film deployed for 90 days in two high touch locations within an active metropolitan bus and railcar. The antiviral efficacy of this copper film after being deployed in transit vehicles for 90 days (deployed copper film) was then compared to new (unused) copper film to determine if frequent touches and cleaning protocols could decrease the efficacy of the copper films. Deployed copper film, new copper film, and aluminium foil (positive control) coupons were inoculated with ~1 × 106 MS2 virus particles, allowed a contact time of either 5- or 10-min, and analysed for residual viral infectiousness. On both new and deployed copper films, MS2 was completely inactivated (≥5 log reduction) at both time points. These results suggest that the copper film may provide the durability demanded by high touch public spaces while maintaining the antiviral activity necessary to reduce exposure risk and viral transmission via surfaces in public transportation settings.

Transfer of MS2 bacteriophage from surfaces to raspberry and pitanga fruits and virus survival in response to sanitization, frozen storage and preservation technologies.

This study assessed the norovirus (NoV) surrogate bacteriophage MS2 transfer from stainless steel, glass and low-density polypropylene surfaces to raspberry and pitanga fruits. The effect of sodium hypochlorite (100 ppm, 1 min) on MS2 survival on whole fruits, the MS2 survival in sanitized fruits and derived pulps during frozen storage, and in response to preservation technologies (heat, organic acids and salts) was also assessed. The highest (p < 0.05) viral transfer (%) was observed from glass and stainless steel (∼90%) to raspberry, and from glass and polypropylene (∼75%) to pitanga, after 60 min of contact. Sodium hypochlorite reduced (p < 0.05) MS2 titer by 3.5 and 3.8 log PFU/g in raspberry and pitanga, respectively. MS2 decreased (p < 0.05) up to 1.4 log PFU/g in frozen stored sanitized fruits (whole fruits and pulps) after 15 days, with no further changes after 30 days. Thermal treatments reduced MS2 titer (p < 0.05) in both fruit pulps. MS2 inactivation was higher in pitanga pulp. The addition of ascorbic acid, citric acid, sodium benzoate, or sodium metabisulfite had little effect (<1 log PFU/g) on MS2 concentration in either fruit. These results may inform NoV risk management practice in processing and handling of fruits.

Measurements of the self-assembly kinetics of individual viral capsids around their RNA genome.

Self-assembly is widely used by biological systems to build functional nanostructures, such as the protein capsids of RNA viruses. But because assembly is a collective phenomenon involving many weakly interacting subunits and a broad range of timescales, measurements of the assembly pathways have been elusive. We use interferometric scattering microscopy to measure the assembly kinetics of individual MS2 bacteriophage capsids around MS2 RNA. By recording how many coat proteins bind to each of many individual RNA strands, we find that assembly proceeds by nucleation followed by monotonic growth. Our measurements reveal the assembly pathways in quantitative detail and also show their failure modes. We use these results to critically examine models of the assembly process.

Visualizing a viral genome with contrast variation small angle X-ray scattering.

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.

Bacteriophage MS2 displays unreported capsid variability assembling T = 4 and mixed capsids.

Bacteriophage MS2 is a positive-sense, single-stranded RNA virus encapsulated in an asymmetric T = 3 pseudo-icosahedral capsid. It infects Escherichia coli through the F-pilus, in which it binds through a maturation protein incorporated into its capsid. Cryogenic electron microscopy has previously shown that its genome is highly ordered within virions, and that it regulates the assembly process of the capsid. In this study, we have assembled recombinant MS2 capsids with non-genomic RNA containing the capsid incorporation sequence, and investigated the structures formed, revealing that T = 3, T = 4 and mixed capsids between these two triangulation numbers are generated, and resolving structures of T = 3 and T = 4 capsids to 4 Å and 6 Å respectively. We conclude that the basic MS2 capsid can form a mix of T = 3 and T = 4 structures, supporting a role for the ordered genome in favouring the formation of functional T = 3 virions.

Miniaturizing Wet Scrubbers for Aerosolized Droplet Capture.

Aerosols dispersed and transmitted through the air (e.g., particulate matter pollution and bioaerosols) are ubiquitous and one of the leading causes of adverse health effects and disease transmission. A variety of sampling methods (e.g., filters, cyclones, and impactors) have been developed to assess personal exposures. However, a gap still remains in the accessibility and ease-of-use of these technologies for people without experience or training in collecting airborne samples. Additionally, wet scrubbers (large non-portable industrial systems) utilize liquid sprays to remove aerosols from the air; the goal is to "scrub" (i.e., clean) the exhaust of industrial smokestacks, not collect the aerosols for analysis. Inspired by wet scrubbers, we developed a device fundamentally different from existing portable air samplers by using aerosolized microdroplets to capture aerosols in personal spaces (e.g., homes, offices, and schools). Our aerosol-sampling device is the size of a small teapot, can be operated without specialized training, and features a winding flow path in a supersaturated relative humidity environment, enabling droplet growth. The integrated open mesofluidic channels shuttle coalesced droplets to a collection chamber for subsequent sample analysis. Here, we present the experimental demonstration of aerosol capture in water droplets. An iterative study optimized the non-linear flow manipulating baffles and enabled an 83% retention of the aerosolized microdroplets in the confined volume of our device. As a proof-of-concept for aerosol capture into a liquid medium, 0.5-3 µm model particles were used to evaluate aerosol capture efficiency. Finally, we demonstrate that the device can capture and keep a bioaerosol (bacteriophage MS2) viable for downstream analysis.

Transfer Rate of Enveloped and Nonenveloped Viruses between Fingerpads and Surfaces.

Fomites can represent a reservoir for pathogens, which may be subsequently transferred from surfaces to skin. In this study, we aim to understand how different factors (including virus type, surface type, time since last hand wash, and direction of transfer) affect virus transfer rates, defined as the fraction of virus transferred, between fingerpads and fomites. To determine this, 360 transfer events were performed with 20 volunteers using Phi6 (a surrogate for enveloped viruses), MS2 (a surrogate for nonenveloped viruses), and three clean surfaces (stainless steel, painted wood, and plastic). Considering all transfer events (all surfaces and both transfer directions combined), the mean transfer rates of Phi6 and MS2 were 0.17 and 0.26, respectively. Transfer of MS2 was significantly higher than that of Phi6 (P < 0.05). Surface type was a significant factor that affected the transfer rate of Phi6: Phi6 is more easily transferred to and from stainless steel and plastic than to and from painted wood. Direction of transfer was a significant factor affecting MS2 transfer rates: MS2 is more easily transferred from surfaces to fingerpads than from fingerpads to surfaces. Data from these virus transfer events, and subsequent transfer rate distributions, provide information that can be used to refine quantitative microbial risk assessments. This study provides a large-scale data set of transfer events with a surrogate for enveloped viruses, which extends the reach of the study to the role of fomites in the transmission of human enveloped viruses like influenza and SARS-CoV-2. IMPORTANCE This study created a large-scale data set for the transfer of enveloped viruses between skin and surfaces. The data set produced by this study provides information on modeling the distribution of enveloped and nonenveloped virus transfer rates, which can aid in the implementation of risk assessment models in the future. Additionally, enveloped and nonenveloped viruses were applied to experimental surfaces in an equivalent matrix to avoid matrix effects, so results between different viral species can be directly compared without confounding effects of different matrices. Our results indicating how virus type, surface type, time since last hand wash, and direction of transfer affect virus transfer rates can be used in decision-making processes to lower the risk of viral infection from transmission through fomites.