Structural Studies of Bacteriophage Φ6 and Its Transformations during Its Life Cycle.

From the first isolation of the cystovirus bacteriophage Φ6 from Pseudomonas syringae 50 years ago, we have progressed to a better understanding of the structure and transformations of many parts of the virion. The three-layered virion, encapsulating the tripartite double-stranded RNA (dsRNA) genome, breaches the cell envelope upon infection, generates its own transcripts, and coopts the bacterial machinery to produce its proteins. The generation of a new virion starts with a procapsid with a contracted shape, followed by the packaging of single-stranded RNA segments with concurrent expansion of the capsid, and finally replication to reconstitute the dsRNA genome. The outer two layers are then added, and the fully formed virion released by cell lysis. Most of the procapsid structure, composed of the proteins P1, P2, P4, and P7 is now known, as well as its transformations to the mature, packaged nucleocapsid. The outer two layers are less well-studied. One additional study investigated the binding of the host protein YajQ to the infecting nucleocapsid, where it enhances the transcription of the large RNA segment that codes for the capsid proteins. Finally, I relate the structural aspects of bacteriophage Φ6 to those of other dsRNA viruses, noting the similarities and differences.

Discovery and Classification of the φ6 Bacteriophage: An Historical Review.

The year 2023 marks the fiftieth anniversary of the discovery of the bacteriophage φ6. The review provides a look back on the initial discovery and classification of the lipid-containing and segmented double-stranded RNA (dsRNA) genome-containing bacteriophage-the first identified cystovirus. The historical discussion describes, for the most part, the first 10 years of the research employing contemporary mutation techniques, biochemical, and structural analysis to describe the basic outline of the virus replication mechanisms and structure. The physical nature of φ6 was initially controversial as it was the first bacteriophage found that contained segmented dsRNA, resulting in a series of early publications that defined the unusual genomic quality. The technology and methods utilized in the initial research (crude by current standards) meant that the first studies were quite time-consuming, hence the lengthy period covered by this review. Yet when the data were accepted, the relationship to the reoviruses was apparent, launching great interest in cystoviruses, research that continues to this day.

Antiviral Characterization of Advanced Materials: Use of Bacteriophage Phi 6 as Surrogate of Enveloped Viruses Such as SARS-CoV-2.

The bacteriophage phi 6 is a virus that belongs to a different Baltimore group than SARS-CoV-2 (group III instead of IV). However, it has a round-like shape and a lipid envelope like SARS-CoV-2, which render it very useful to be used as a surrogate of this infectious pathogen for biosafety reasons. Thus, recent antiviral studies have demonstrated that antiviral materials such as calcium alginate hydrogels, polyester-based fabrics coated with benzalkonium chloride (BAK), polyethylene terephthalate (PET) coated with BAK and polyester-based fabrics coated with cranberry extracts or solidified hand soap produce similar log reductions in viral titers of both types of enveloped viruses after similar viral contact times. Therefore, researchers with no access to biosafety level 3 facilities can perform antiviral tests of a broad range of biomaterials, composites, nanomaterials, nanocomposites, coatings and compounds against the bacteriophage phi 6 as a biosafe viral model of SARS-CoV-2. In fact, this bacteriophage has been used as a surrogate of SARS-CoV-2 to test a broad range of antiviral materials and compounds of different chemical natures (polymers, metals, alloys, ceramics, composites, etc.) and forms (films, coatings, nanomaterials, extracts, porous supports produced by additive manufacturing, etc.) during the current pandemic. Furthermore, this biosafe viral model has also been used as a surrogate of SARS-CoV-2 and other highly pathogenic enveloped viruses such as Ebola and influenza in a wide range of biotechnological applications.

Phi 6 Bacteriophage Inactivation by Metal Salts, Metal Powders, and Metal Surfaces.

The interaction of phages with abiotic environmental surfaces is usually an understudied field of phage ecology. In this study, we investigated the virucidal potential of different metal salts, metal and ceramic powders doped with Ag and Cu ions, and newly fabricated ceramic and metal surfaces against Phi6 bacteriophage. The new materials were fabricated by spark plasma sintering (SPS) and/or selective laser melting (SLM) techniques and had different surface free energies and infiltration features. We show that inactivation of Phi6 in solutions with Ag and Cu ions can be as effective as inactivation by pH, temperature, or UV. Adding powder to Ag and Cu ion solutions decreased their virucidal effect. The newly fabricated ceramic and metal surfaces showed very good virucidal activity. In particular, 45%TiO2 + 5%Ag + 45%ZrO2 + 5%Cu, in addition to virus adhesion, showed virucidal and infiltration properties. The results indicate that more than 99.99% of viruses deposited on the new ceramic surface were inactivated or irreversibly attached to it.

Non-Woven Infection Prevention Fabrics Coated with Biobased Cranberry Extracts Inactivate Enveloped Viruses Such as SARS-CoV-2 and Multidrug-Resistant Bacteria.

The Coronavirus Disease (COVID-19) pandemic is demanding the rapid action of the authorities and scientific community in order to find new antimicrobial solutions that could inactivate the pathogen SARS-CoV-2 that causes this disease. Gram-positive bacteria contribute to severe pneumonia associated with COVID-19, and their resistance to antibiotics is exponentially increasing. In this regard, non-woven fabrics are currently used for the fabrication of infection prevention clothing such as face masks, caps, scrubs, shirts, trousers, disposable gowns, overalls, hoods, aprons and shoe covers as protective tools against viral and bacterial infections. However, these non-woven fabrics are made of materials that do not exhibit intrinsic antimicrobial activity. Thus, we have here developed non-woven fabrics with antimicrobial coatings of cranberry extracts capable of inactivating enveloped viruses such as SARS-CoV-2 and the bacteriophage phi 6 (about 99% of viral inactivation in 1 min of viral contact), and two multidrug-resistant bacteria: the methicillin-resistant Staphylococcus aureus and the methicillin-resistant Staphylococcus epidermidis. The morphology, thermal and mechanical properties of the produced filters were characterized by optical and electron microscopy, differential scanning calorimetry, thermogravimetry and dynamic mechanical thermal analysis. The non-toxicity of these advanced technologies was ensured using a Caenorhabditis elegans in vivo model. These results open up a new prevention path using natural and biodegradable compounds for the fabrication of infection prevention clothing in the current COVID-19 pandemic and microbial resistant era.

Polyethylene Films Containing Plant Extracts in the Polymer Matrix as Antibacterial and Antiviral Materials.

Low density polyethylene (LDPE) films covered with active coatings containing mixtures of rosemary, raspberry, and pomegranate CO2 extracts were found to be active against selected bacterial strains that may extend the shelf life of food products. The coatings also offer antiviral activity, due to their influence on the activity of Φ6 bacteriophage, selected as a surrogate for SARS-CoV-2 particles. The mixture of these extracts could be incorporated into a polymer matrix to obtain a foil with antibacterial and antiviral properties. The initial goal of this work was to obtain active LDPE films containing a mixture of CO2 extracts of the aforementioned plants, incorporated into an LDPE matrix via an extrusion process. The second aim of this study was to demonstrate the antibacterial properties of the active films against Gram-positive and Gram-negative bacteria, and to determine the antiviral effect of the modified material on Φ6 bacteriophage. In addition, an analysis was made on the influence of the active mixture on the polymer physicochemical features, e.g., mechanical and thermal properties, as well as its color and transparency. The results of this research indicated that the LDPE film containing a mixture of raspberry, rosemary, and pomegranate CO2 extracts incorporated into an LDPE matrix inhibited the growth of Staphylococcus aureus. This film was also found to be active against Bacillus subtilis. This modified film did not inhibit the growth of Escherichia coli and Pseudomonas syringae cells; however, their number decreased significantly. The LDPE active film was also found to be active against Φ6 particles, meaning that the film had antiviral properties. The incorporation of the mixture of CO2 extracts into the polymer matrix affected its mechanical properties. It was observed that parameters describing mechanical properties decreased, although did not affect the transition of LDPE significantly. Additionally, the modified film exhibited barrier properties towards UV radiation. Modified PE/CO2 extracts films could be applied as a functional food packaging material with antibacterial and antiviral properties.

Ethanol Inactivation of Enveloped Viruses: Structural and Surface Chemistry Insights into Phi6.

Lipid-enveloped viruses, such as Ebola, influenza, or coronaviruses, are a major threat to human health. Ethanol is an efficient disinfectant that is widely used to inactivate these viruses and prevent their transmission. However, the interactions between ethanol and enveloped viruses leading to their inactivation are not yet fully understood. This study demonstrates the link between ethanol-induced viral inactivation and the nanostructural and chemical transformations of the model virus Phi6, an 85 nm diameter lipid-enveloped bacterial virus that is commonly used as surrogate for human pathogenic viruses. The virus morphology was investigated using small-angle X-ray scattering and dynamic light scattering and was related to its infectivity. The Phi6's surface chemistry was characterized by cryogenic X-ray photoelectron spectroscopy, and the modifications in protein structure were assessed by circular dichroism and fluorescence spectroscopy. Ethanol-triggered structural modifications were found in the lipid envelope, detaching from the protein capsid and forming coexisting nanostructures.

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.

UV Inactivation of SARS-CoV-2 across the UVC Spectrum: KrCl* Excimer, Mercury-Vapor, and Light-Emitting-Diode (LED) Sources.

Effective disinfection technology to combat severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can help reduce viral transmission during the ongoing COVID-19 global pandemic and in the future. UV devices emitting UVC irradiation (200 to 280 nm) have proven to be effective for virus disinfection, but limited information is available for SARS-CoV-2 due to the safety requirements of testing, which is limited to biosafety level 3 (BSL3) laboratories. In this study, inactivation of SARS-CoV-2 in thin-film buffered aqueous solution (pH 7.4) was determined across UVC irradiation wavelengths of 222 to 282 nm from krypton chloride (KrCl*) excimers, a low-pressure mercury-vapor lamp, and two UVC light-emitting diodes. Our results show that all tested UVC devices can effectively inactivate SARS-CoV-2, among which the KrCl* excimer had the best disinfection performance (i.e., highest inactivation rate). The inactivation rate constants of SARS-CoV-2 across wavelengths are similar to those for murine hepatitis virus (MHV) from our previous investigation, suggesting that MHV can serve as a reliable surrogate of SARS-CoV-2 with a lower BSL requirement (BSL2) during UV disinfection tests. This study provides fundamental information on UVC's action on SARS-CoV-2 and guidance for achieving reliable disinfection performance with UVC devices. IMPORTANCE UV light is an effective tool to help stem the spread of respiratory viruses and protect public health in commercial, public, transportation, and health care settings. For effective use of UV, there is a need to determine the efficiency of different UV wavelengths in killing pathogens, specifically SARS-CoV-2, to support efforts to control the ongoing COVID-19 global pandemic and future coronavirus-caused respiratory virus pandemics. We found that SARS-CoV-2 can be inactivated effectively using a broad range of UVC wavelengths, and 222 nm provided the best disinfection performance. Interestingly, 222-nm irradiation has been found to be safe for human exposure up to thresholds that are beyond those effective for inactivating viruses. Therefore, applying UV light from KrCl* excimers in public spaces can effectively help reduce viral aerosol or surface-based transmissions.

Antimicrobial Face Shield: Next Generation of Facial Protective Equipment against SARS-CoV-2 and Multidrug-Resistant Bacteria.

Transparent materials used for facial protection equipment provide protection against microbial infections caused by viruses and bacteria, including multidrug-resistant strains. However, transparent materials used for this type of application are made of materials that do not possess antimicrobial activity. They just avoid direct contact between the person and the biological agent. Therefore, healthy people can become infected through contact of the contaminated material surfaces and this equipment constitute an increasing source of infectious biological waste. Furthermore, infected people can transmit microbial infections easily because the protective equipment do not inactivate the microbial load generated while breathing, sneezing or coughing. In this regard, the goal of this work consisted of fabricating a transparent face shield with intrinsic antimicrobial activity that could provide extra-protection against infectious agents and reduce the generation of infectious waste. Thus, a single-use transparent antimicrobial face shield composed of polyethylene terephthalate and an antimicrobial coating of benzalkonium chloride has been developed for the next generation of facial protective equipment. The antimicrobial coating was analyzed by atomic force microscopy and field emission scanning electron microscopy with elemental analysis. This is the first facial transparent protective material capable of inactivating enveloped viruses such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in less than one minute of contact, and the methicillin-resistant Staphylococcus aureus and Staphylococcus epidermidis. Bacterial infections contribute to severe pneumonia associated with the SARS-CoV-2 infection, and their resistance to antibiotics is increasing. Our extra protective broad-spectrum antimicrobial composite material could also be applied for the fabrication of other facial protective tools such as such as goggles, helmets, plastic masks and space separation screens used for counters or vehicles. This low-cost technology would be very useful to combat the current pandemic and protect health care workers from multidrug-resistant infections in developed and underdeveloped countries.

Higher Concentrations of Bacterial Enveloped Virus Phi6 Can Protect the Virus from Environmental Decay.

Phage Phi6 is an enveloped virus considered a possible nonpathogenic surrogate for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and other viral pathogens in transmission studies. Larger input amounts of bacteriophage Phi6 are shown to delay and protect the phage from environmental decay, both when the phages are dried in plastic tubes and when they are stored in saline solution at 4°C. In contrast, when bacteriophage Phi6 is placed in LB (Luria-Bertani) growth medium (instead of saline) prior to placement on the plastic surface, the influence of the starting concentration on viral recovery is negligible. Protection is reflected in the phage half-lives at higher concentrations being longer than the half-lives at lower concentrations. Because experiments supporting the possibility of fomite transmission of SARS-CoV-2 and other viruses rely upon the survival of infectious virus following inoculation onto various surfaces, large initial amounts of input virus on a surface may generate artificially inflated survival times compared to realistic lower levels of virus that a subject would normally encounter. This is not only because there are extra half-lives to go through at higher concentrations but also because the half-lives themselves are extended at higher virus concentrations. It is important to design surface drying experiments for pathogens with realistic levels of input virus and to consider the role of the carrier and matrix if the results are to be clinically relevant. IMPORTANCE During the coronavirus disease 2019 (COVID-19) pandemic, much attention has been paid to the environmental decay of SARS-CoV-2 due to the proposed transmission of the virus via fomites. However, published experiments have commenced with inocula with very high virus titers, an experimental design not representative of real-life conditions. The study described here evaluated the impact of the initial virus titer on the environmental decay of an enveloped virus, using a nonpathogenic surrogate for the transmission of SARS-CoV-2, enveloped bacteriophage Phi6. We establish that higher concentrations of virus can protect the virus from environmental decay, depending on conditions. This has important implications for stability studies of SARS-CoV-2 and other viruses. Our results point to a limitation in the fundamental methodology that has been used to attribute fomite transmission for almost all respiratory viruses.

Survival of the enveloped bacteriophage Phi6 (a surrogate for SARS-CoV-2) in evaporated saliva microdroplets deposited on glass surfaces.

Survival of respiratory viral pathogens in expelled saliva microdroplets is central to their transmission, yet the factors that determine survival in such microdroplets are not well understood. Here we combine microscopy imaging with virus viability assays to study survival of three bacteriophages suggested as good models for respiratory pathogens: the enveloped Phi6 (a surrogate for SARS-CoV-2), and the non-enveloped PhiX174 and MS2. We measured virus viability in human saliva microdroplets, SM buffer, and water following deposition on glass surfaces at various relative humidities (RH). Saliva and water microdroplets dried out rapidly, within minutes, at all tested RH levels (23%, 43%, 57%, and 78%), while SM microdroplets remained hydrated at RH ≥ 57%. Generally, the survival of all three viruses in dry saliva microdroplets was significantly greater than those in SM buffer and water under all RH (except PhiX174 in water under 57% RH survived the best among 3 media). Thus, atmosphere RH and microdroplet hydration state are not sufficient to explain virus survival, indicating that the virus-suspended medium, and association with saliva components in particular, likely play a role in virus survival. Uncovering the exact properties and components that make saliva a favorable environment for the survival of viruses, in particular enveloped ones like Phi6, is thus of great importance for reducing transmission of viral respiratory pathogens including SARS-CoV-2.

Enzymatically synthesized 2'-fluoro-modified Dicer-substrate siRNA swarms against herpes simplex virus demonstrate enhanced antiviral efficacy and low cytotoxicity.

Chemical modifications of small interfering (si)RNAs are used to enhance their stability and potency, and to reduce possible off-target effects, including immunogenicity. We have earlier introduced highly effective antiviral siRNA swarms against herpes simplex virus (HSV), targeting 653 bp of the essential UL29 viral gene. Here, we report a method for enzymatic production and antiviral use of 2'-fluoro-modified siRNA swarms. Utilizing the RNA-dependent RNA polymerase from bacteriophage phi6, we produced 2'-F-siRNA swarms containing either all or a fraction of modified adenosine, cytidine or uridine residues in the antisense strand of the UL29 target. The siRNA containing modified pyrimidines demonstrated high resistance to RNase A and the antiviral potency of all the UL29-specific 2'-F-siRNA swarms was 100-fold in comparison with the unmodified counterpart, without additional cytotoxicity. Modest stimulation of innate immunity signaling, including induced expression of both type I and type III interferons, as well as interferon-stimulated gene 54, by 2'-F-cytidine and 2'-F-uridine modified siRNA swarms occurred at early time points after transfection while the 2'-F-adenosine-containing siRNA was similar to the unmodified antiviral siRNA swarm in this respect. The antiviral efficacy of the 2'-F-siRNA swarms and the elicited cellular innate responses did not correlate suggesting that innate immunity pathways do not significantly contribute to the observed enhanced antiviral activity of the modified siRNAs. The results support further applications of enzymatically produced siRNA molecules with incorporated adenosine nucleotides, carrying fluoro-modification on ribose C2' position, for further antiviral studies in vitro and in vivo.

Persistence of Bacteriophage Phi 6 on Porous and Nonporous Surfaces and the Potential for Its Use as an Ebola Virus or Coronavirus Surrogate.

The infection of health care workers during the 2013 to 2016 Ebola outbreak raised concerns about fomite transmission. In the wake of the coronavirus disease 2019 (COVID-19) pandemic, investigations are ongoing to determine the role of fomites in coronavirus transmission as well. The bacteriophage phi 6 has a phospholipid envelope and is commonly used in environmental studies as a surrogate for human enveloped viruses. The persistence of phi 6 was evaluated as a surrogate for Ebola virus (EBOV) and coronaviruses on porous and nonporous hospital surfaces. Phi 6 was suspended in a body fluid simulant and inoculated onto 1-cm2 coupons of steel, plastic, and two fabric curtain types. The coupons were placed at two controlled absolute humidity (AH) levels: a low AH of 3.0 g/m3 and a high AH of 14.4 g/m3 Phi 6 declined at a lower rate on all materials under low-AH conditions, with a decay rate of 0.06-log10 PFU/day to 0.11-log10 PFU/day, than under the higher AH conditions, with a decay rate of 0.65-log10 PFU/h to 1.42-log10 PFU/day. There was a significant difference in decay rates between porous and nonporous surfaces at both low AH (P < 0.0001) and high AH (P < 0.0001). Under these laboratory-simulated conditions, phi 6 was found to be a conservative surrogate for EBOV under low-AH conditions in that it persisted longer than Ebola virus in similar AH conditions. Additionally, some coronaviruses persist longer than phi 6 under similar conditions; therefore, phi 6 may not be a suitable surrogate for coronaviruses.IMPORTANCE Understanding the persistence of enveloped viruses helps inform infection control practices and procedures in health care facilities and community settings. These data convey to public health investigators that enveloped viruses can persist and remain infective on surfaces, thus demonstrating a potential risk for transmission. Under these laboratory-simulated Western indoor hospital conditions, we assessed the suitability of phi 6 as a surrogate for environmental persistence research related to enveloped viruses, including EBOV and coronaviruses.

Temperature controlled high-throughput magnetic tweezers show striking difference in activation energies of replicating viral RNA-dependent RNA polymerases.

RNA virus survival depends on efficient viral genome replication, which is performed by the viral RNA dependent RNA polymerase (RdRp). The recent development of high throughput magnetic tweezers has enabled the simultaneous observation of dozens of viral RdRp elongation traces on kilobases long templates, and this has shown that RdRp nucleotide addition kinetics is stochastically interrupted by rare pauses of 1-1000 s duration, of which the short-lived ones (1-10 s) are the temporal signature of a low fidelity catalytic pathway. We present a simple and precise temperature controlled system for magnetic tweezers to characterize the replication kinetics temperature dependence between 25°C and 45°C of RdRps from three RNA viruses, i.e. the double-stranded RNA bacteriophage Φ6, and the positive-sense single-stranded RNA poliovirus (PV) and human rhinovirus C (HRV-C). We found that Φ6 RdRp is largely temperature insensitive, while PV and HRV-C RdRps replication kinetics are activated by temperature. Furthermore, the activation energies we measured for PV RdRp catalytic state corroborate previous estimations from ensemble pre-steady state kinetic studies, further confirming the catalytic origin of the short pauses and their link to temperature independent RdRp fidelity. This work will enable future temperature controlled study of biomolecular complex at the single molecule level.

Next-generation sequencing of dsRNA is greatly improved by treatment with the inexpensive denaturing reagent DMSO.

dsRNA is the genetic material of important viruses and a key component of RNA interference-based immunity in eukaryotes. Previous studies have noted difficulties in determining the sequence of dsRNA molecules that have affected studies of immune function and estimates of viral diversity in nature. DMSO has been used to denature dsRNA prior to the reverse-transcription stage to improve reverse transcriptase PCR and Sanger sequencing. We systematically tested the utility of DMSO to improve the sequencing yield of a dsRNA virus (Φ6) in a short-read next-generation sequencing platform. DMSO treatment improved sequencing read recovery by over two orders of magnitude, even when RNA and cDNA concentrations were below the limit of detection. We also tested the effects of DMSO on a mock eukaryotic viral community and found that dsRNA virus reads increased with DMSO treatment. Furthermore, we provide evidence that DMSO treatment does not adversely affect recovery of reads from a ssRNA viral genome (influenza A/California/07/2009). We suggest that up to 50 % DMSO treatment be used prior to cDNA synthesis when samples of interest are composed of or may contain dsRNA.

Multiple liquid crystalline geometries of highly compacted nucleic acid in a dsRNA virus.

Characterizing the genome of mature virions is pivotal to understanding the highly dynamic processes of virus assembly and infection. Owing to the different cellular fates of DNA and RNA, the life cycles of double-stranded (ds)DNA and dsRNA viruses are dissimilar. In terms of nucleic acid packing, dsDNA viruses, which lack genome segmentation and intra-capsid transcriptional machinery, predominantly display single-spooled genome organizations1-8. Because the release of dsRNA into the cytoplasm triggers host defence mechanisms9, dsRNA viruses retain their genomes within a core particle that contains the enzymes required for RNA replication and transcription10-12. The genomes of dsRNA viruses vary greatly in the degree of segmentation. In members of the Reoviridae family, genomes consist of 10-12 segments and exhibit a non-spooled arrangement mediated by RNA-dependent RNA polymerases11-14. However, whether this arrangement is a general feature of dsRNA viruses remains unknown. Here, using cryo-electron microscopy to resolve the dsRNA genome structure of the tri-segmented bacteriophage ɸ6 of the Cystoviridae family, we show that dsRNA viruses can adopt a dsDNA-like single-spooled genome organization. We find that in this group of viruses, RNA-dependent RNA polymerases do not direct genome ordering, and the dsRNA can adopt multiple conformations. We build a model that encompasses 90% of the genome, and use this to quantify variation in the packing density and to characterize the different liquid crystalline geometries that are exhibited by the tightly compacted nucleic acid. Our results demonstrate that the canonical model for the packing of dsDNA can be extended to dsRNA viruses.

Microbial production of lipid-protein vesicles using enveloped bacteriophage phi6.

BACKGROUND: Cystoviruses have a phospholipid envelope around their nucleocapsid. Such a feature is unique among bacterial viruses (i.e., bacteriophages) and the mechanisms of virion envelopment within a bacterial host are largely unknown. The cystovirus Pseudomonas phage phi6 has an envelope that harbors five viral membrane proteins and phospholipids derived from the cytoplasmic membrane of its Gram-negative host. The phi6 major envelope protein P9 and the non-structural protein P12 are essential for the envelopment of its virions. Co-expression of P9 and P12 in a Pseudomonas host results in the formation of intracellular vesicles that are potential intermediates in the phi6 virion assembly pathway. This study evaluated the minimum requirements for the formation of phi6-specific vesicles and the possibility to localize P9-tagged heterologous proteins into such structures in Escherichia coli. RESULTS: Using transmission electron microscopy, we detected membranous structures in the cytoplasm of E. coli cells expressing P9. The density of the P9-specific membrane fraction was lower (approximately 1.13 g/cm3 in sucrose) than the densities of the bacterial cytoplasmic and outer membrane fractions. A P9-GFP fusion protein was used to study the targeting of heterologous proteins into P9 vesicles. Production of the GFP-tagged P9 vesicles required P12, which protected the fusion protein against proteolytic cleavage. Isolated vesicles contained predominantly P9-GFP, suggesting selective incorporation of P9-tagged fusion proteins into the vesicles. CONCLUSIONS: Our results demonstrate that the phi6 major envelope protein P9 can trigger formation of cytoplasmic membrane structures in E. coli in the absence of any other viral protein. Intracellular membrane structures are rare in bacteria, thus making them ideal chasses for cell-based vesicle production. The possibility to locate heterologous proteins into the P9-lipid vesicles facilitates the production of vesicular structures with novel properties. Such products have potential use in biotechnology and biomedicine.

A bioassay-based protocol for chemical neutralization of human faecal wastes treated by physico-chemical disinfection processes: A case study on benzalkonium chloride.

In situ physico-chemical disinfection of high risk faecal waste is both effective and widely used as a sanitation management strategy for infection prevention and control. Systematic tests where the performance of alternative physico-chemical disinfection methods is systematically compared and optimized must be based on reliable protocols. These protocol are currently not adequately addressing the neutralization related issues: the neutralization of the tested disinfectant after specified conditions of concentration and contact time (CT) is necessary to prevent continued disinfection after the intended contact time; moreover such neutralization is often necessary in practice and on a large scale to prevent adverse health and ecological impacts from remaining disinfectant after the target CT is achieved. Few studies adequately assess the extent of neutralization of the chemical disinfectant and are intended to optimize on-site disinfection practices for waste matrices posing high microbial risks. Hence, there is a need for effective and reproducible neutralization protocols in chemical disinfection trials and practice. Furthermore, for most of chemical disinfectants used in healthcare settings there is no practical methodology to reliably and conveniently measure the residual disinfectant concentration after its neutralization and also determine the optimum concentration of the neutralizer. Because some neutralizing compounds can themselves be toxic to the test microorganisms, it is necessary to optimize neutralization procedures in disinfection experiments for the development of infection control practices using accepted positive control microbes. In the presented work, a stepwise bioassay-based protocol using representative faecal indicator microbes is described for optimizing chemical disinfection and subsequent disinfectant neutralization of any infectious faecal waste matrix. The example described is for the quaternary ammonium compound benzalkonium chloride and its recommended chemical neutralizer in a high strength human faecal waste matrix.

Existing Host Range Mutations Constrain Further Emergence of RNA Viruses.

RNA viruses are capable of rapid host shifting, typically due to a point mutation that confers expanded host range. As additional point mutations are necessary for further expansions, epistasis among host range mutations can potentially affect the mutational neighborhood and frequency of niche expansion. We mapped the mutational neighborhood of host range expansion using three genotypes of the double-stranded RNA (dsRNA) bacteriophage φ6 (wild type and two isogenic host range mutants) on the novel host Pseudomonas syringae pv. atrofaciens. Both Sanger sequencing of 50 P. syringae pv. atrofaciens mutant clones for each genotype and population Illumina sequencing revealed the same high-frequency mutations allowing infection of P. syringae pv. atrofaciens. Wild-type φ6 had at least nine different ways of mutating to enter the novel host, eight of which are in p3 (host attachment protein gene), and 13/50 clones had unchanged p3 genes. However, the two isogenic mutants had dramatically restricted neighborhoods: only one or two mutations, all in p3. Deep sequencing revealed that wild-type clones without mutations in p3 likely had changes in p12 (morphogenic protein), a region that was not polymorphic for the two isogenic host range mutants. Sanger sequencing confirmed that 10/13 of the wild-type φ6 clones had nonsynonymous mutations in p12, and 2 others had point mutations in p9 and p5. None of these genes had previously been associated with host range expansion in φ6. We demonstrate, for the first time, epistatic constraint in an RNA virus due to host range mutations themselves, which has implications for models of serial host range expansion.IMPORTANCE RNA viruses mutate rapidly and frequently expand their host ranges to infect novel hosts, leading to serial host shifts. Using an RNA bacteriophage model system (Pseudomonas phage φ6), we studied the impact of preexisting host range mutations on another host range expansion. Results from both clonal Sanger and Illumina sequencing show that extant host range mutations dramatically narrow the neighborhood of potential host range mutations compared to that of wild-type φ6. This research suggests that serial host-shifting viruses may follow a small number of molecular paths to enter additional novel hosts. We also identified new genes involved in φ6 host range expansion, expanding our knowledge of this important model system in experimental evolution.