Phi X174 Research Articles

Delayed Lysis Confers Resistance to the Nucleoside Analogue 5-Fluorouracil and Alleviates Mutation Accumulation in the Single-Stranded DNA Bacteriophage ϕX174

Rates of spontaneous mutation determine viral fitness and adaptability. In RNA viruses, treatment with mutagenic nucleoside analogues selects for polymerase variants with increased fidelity, showing that viral mutation rates can be adjusted in response to imposed selective pressures. However, this type of resistance is not possible in viruses that do not encode their own polymerases, such as single-stranded DNA viruses. We previously showed that serial passaging of bacteriophage ϕX174 in the presence of the nucleoside analogue 5-fluorouracil (5-FU) favored substitutions in the lysis protein E (P. Domingo-Calap, M. Pereira-Gomez, and R. Sanjuán, J. Virol. 86:: 9640-9646, 2012, doi:10.1128/JVI.00613-12). Here, we found that approximately half (6/12) of the amino acid replacements in the N-terminal region of this protein led to delayed lysis, and two of these changes (V2A and D8A) also conferred partial resistance to 5-FU. By delaying lysis, the V2A and D8A substitutions allowed the virus to increase the burst size per cell in the presence of 5-FU. Furthermore, these substitutions tended to alleviate drug-induced mutagenesis by reducing the number of rounds of copying required for population growth, revealing a new mechanism of resistance. This form of mutation rate regulation may also be utilized by other viruses whose replication mode is similar to that of bacteriophage ϕX174. Many viruses display high rates of spontaneous mutations due to defects in proofreading or postreplicative repair, allowing them to rapidly adapt to changing environments. Viral mutation rates may have been optimized to achieve high adaptability without incurring an excessive genetic load. Supporting this, RNA viruses subjected to chemical mutagenesis treatments have been shown to evolve higher-fidelity polymerases. However, many viruses cannot modulate replication fidelity because they do not encode their own polymerase. Here, we show a new mechanism for regulating viral mutation rates. We found that, under mutagenic conditions, the single-stranded bacteriophage ϕX174 evolved delayed lysis, and that this allowed the virus to increase the amount of progeny produced per cell. As a result, the viral population was amplified in fewer infection cycles, reducing the chances for mutation appearance.

Photoreactivation of bacteriophages after UV disinfection: Role of genome structure and impacts of UV source

The UV inactivation kinetics of bacteriophages MS2, PhiX174, T1 and PRD1 and the potential of bacterial UV repair mechanisms to reactivate these bacteriophages is described here. The selected bacteriophages represent a range of genome size, single and double stranded genomes, circular and linear organization and RNA and DNA. Bacteriophages were exposed to UV irradiation from two different collimated beam UV irradiation sources (medium-pressure (MP) mercury lamps and low-pressure (LP) mercury lamps) and assayed during which host-phage cultures were exposed to photoreactivating light for 6 h, then incubated overnight at 37 °C in the dark. Dark controls following UV exposure were performed in parallel. UV inactivation kinetics (using dark controls) showed that circular ssDNA phage (PhiX174) was the most sensitive and linear ssRNA phage (MS2) was the more resistant phage. No photoreactivation was observed for MS2 (RNA phage) and the highest photoreactivation was observed for PRD1. In the case of PRD1, the dose required for 4-log reduction (dark control) was around 35 mJ/cm2, with a similar dose observed for both UV sources (MP and LP). When the photoreactivation step was added, the dose required for 4-log reduction using LP lamps was 103 mJ/cm2 and for MP lamps was 60 mJ/cm2. Genome organization differences between bacteriophages play an important role in resistance to UV inactivation and potential photoreactivation mediated by bacterial host mechanisms. The use of photoreactivation during the assay of PRD1 creates a more conservative surrogate for potential use in UV challenge testing.

Virus attachment onto quartz sand: Role of grain size and temperature

Virus transport in groundwater is controlled mainly by attachment onto the solid matrix and inactivation. Therefore, understanding how the various parameters affect virus attachment can lead to improved virus transport predictions and better health risk evaluations. This study is focused on the attachment of viruses onto quartz sand under batch experimental conditions. The bacteriophages ΦX174 and MS2 were used as model viruses. Three different sand grain sizes were employed for the static and dynamic experiments. The batch sorption experiments were performed under static conditions at 4°C and 20°C and dynamic conditions at 4°C. The experimental data were adequately described by the Freundlich isotherm. It was shown that temperature significantly affects virus attachment under static conditions. The attachment of both MS2 and ΦX174 onto quartz sand was greater at 20°C than 4°C. Higher virus attachment was observed under dynamic than static conditions, and in all cases, the affinity of MS2 for quartz sand was greater than that of ΦX174. Furthermore, in most of the cases considered, bacteriophage attachment was shown to decrease with increasing quartz sand size.

Kingdom-Agnostic Metagenomics and the Importance of Complete Characterization of Enteric Microbial Communities

Advanced sequencing techniques have shown that bacteria are not the only complex and important microbes in the human intestine. Nonbacterial organisms, particularly the virome and the mycobiome, are important regulators of intestinal immunity and inflammation. The virome is mucosal and systemic; it can alter the host response to bacteria and interact with host genes and bacteria to contribute to disease pathogenesis. The human mycobiome is also complex and can contribute to intestinal inflammation. We review what has recently been learned about the nonbacterial and nonarchaeal microbes in the gastrointestinal tract, discussing their potential effects on health and disease and analytical approaches for their study. Studies of associations between the microbiome and intestinal pathology should incorporate kingdom-agnostic approaches if we are to fully understand intestinal health and disease.

Antiviral properties of silver nanoparticles on a magnetic hybrid colloid.

Silver nanoparticles (AgNPs) are considered to be a potentially useful tool for controlling various pathogens. However, there are concerns about the release of AgNPs into environmental media, as they may generate adverse human health and ecological effects. In this study, we developed and evaluated a novel micrometer-sized magnetic hybrid colloid (MHC) decorated with variously sized AgNPs (AgNP-MHCs). After being applied for disinfection, these particles can be easily recovered from environmental media using their magnetic properties and remain effective for inactivating viral pathogens. We evaluated the efficacy of AgNP-MHCs for inactivating bacteriophage ΦX174, murine norovirus (MNV), and adenovirus serotype 2 (AdV2). These target viruses were exposed to AgNP-MHCs for 1, 3, and 6 h at 25°C and then analyzed by plaque assay and real-time TaqMan PCR. The AgNP-MHCs were exposed to a wide range of pH levels and to tap and surface water to assess their antiviral effects under different environmental conditions. Among the three types of AgNP-MHCs tested, Ag30-MHCs displayed the highest efficacy for inactivating the viruses. The ΦX174 and MNV were reduced by more than 2 log10 after exposure to 4.6 × 10(9) Ag30-MHCs/ml for 1 h. These results indicated that the AgNP-MHCs could be used to inactivate viral pathogens with minimum chance of potential release into environment.

Mechanistic modeling of viral filtration

A simple model based on changes in filter resistance and active area of the membrane has been used to model viral filtration. Viral particles have been modeled as colloidal particles disregarding any specific interaction and considering only passive transport in the system. The model is based on the assumption that purely steric interactions determine the ratio of concentration of viral particles inside the pore to concentration in solution at the pore mouth. Viral particles rejected by the membrane form a layer of high concentration near the membrane and this layer offers additional resistance to filtration. The membrane flux has been calculated by applying Darcy׳s law. The overall model involves use of six unknown parameters to account for cake formation, nature of virus, interaction between the virus and the membrane, and pore size. The breakthrough of the model virus, bacteriophage ϕX-174, through normal-flow virus filters using commercial process fluids has been chosen as the system used for model validation. The model has been fitted to the time profile of flux and the log reduction value (LRV) of viral particles across the different types of commercially available filters. The model will be useful when performing studies using scale down models for correlating LRV to flux decline. The model also provides us insights into the underlying mechanisms behind viral clearance achieved from the various commercially available viral filters.

Diagnostic Immunization With Bacteriophage ΦX 174 In Patients With Common Variable Immunodeficiency/Hypogammaglobulinemia

The use of the T cell-dependent neoantigen bacteriophage ΦX 174 has been described since the 1960s as a method to assess specific antibody response in patients with primary immune deficiencies. We reviewed a cohort of patients at Duke University Medical Center (DUMC) who received immunization with bacteriophage and report the clinical utility and safety of the immunization, as well as patient characteristics.

Generation of a safety enhanced Salmonella Gallinarum ghost using antibiotic resistance free plasmid and its potential as an effective inactivated vaccine candidate against fowl typhoid

A safety enhanced Salmonella Gallinarum (SG) ghost was constructed using an antibiotic resistance gene free plasmid and evaluated its potential as fowl typhoid (FT) vaccine candidate. The antibiotic resistance free pYA3342 plasmid possesses aspartate semialdehyde dehydrogenase gene which is complimentary to the deletion of the chromosomal asd gene in the bacterial host. This plasmid was incorporated with a ghost cassette containing the bacteriophage PhiX174 lysis gene E, designated as pJHL101. The plasmid pJHL101 was transformed into a two virulence genes-deleted SG. The SG ghosts with tunnel formation and loss of cytoplasmic contents were observed by scanning electron microscopy and transmission electron microscopy. The cell viability of the culture solution was decreased to 0% at 24h after the induction of gene E expression by an increase in temperature from 37°C to 42°C. The safety and protective efficacy of the SG ghost vaccine was further examined in chickens which were divided into three groups: group A (non-immunized control), group B (orally immunized), and group C (intramuscularly immunized). The birds were immunized at 7d of age. No clinical symptoms associated with FT such as anorexia, depression and greenish diarrhea were observed in the immunized chickens. Upon challenge with a virulent SG strain at 3 week post-immunization, the chickens immunized with the SG ghost via various routes were efficiently protected, as shown by significantly lower mortality and post-mortem lesions in comparison with control group. In addition, all the immunized chickens showed significantly higher antibody responses accompanied by a potent antigen-specific lymphocyte proliferative response along with significantly increased numbers of CD4+ and CD8+ T lymphocytes. Overall, our results provide a promising approach of generating SG ghosts using the antibiotic resistance free plasmid in order to prepare a non-living bacterial vaccine candidate which could be environmentally safe yet efficient to prevent FT in chickens.

Inactivation of F-specific bacteriophages during flocculation with polyaluminum chloride – A mechanistic study

Bacteriophages are often used as surrogates for enteric viruses in spiking experiments to determine the efficiencies of virus removal of certain water treatment measures, like e.g. flocculation or filtration steps. Such spiking experiments with bacteriophages are indispensable if the natural virus concentrations in the raw water of water treatment plants are too low to allow the determination of elimination levels over several orders of magnitude. In order to obtain reliable results from such spiking tests, it is essential that bacteriophages behave comparable to viruses and remain stable during the experiments. To test this, the influence of flocculation parameters on the bacteriophages MS2, Qβ and phiX174 was examined. Notably, the F-specific phages MS2 and Qβ were found to be inactivated in flocculation processes with polyaluminum chloride (PACl). In contrast, other aluminum coagulants like AlCl3 or Al2(SO4)3 did not show a comparable effect on MS2 in this study. In experiments testing the influence of different PACl species on MS2 and Qβ inactivation during flocculation, it could be shown that cationic dissolved PACl species (Al13) interacted with the MS2 surface and hereby reduced the surviving phage fraction to c/c0 values below 1*10−4 even at very low PACl concentrations of 7 μmol Al/L. Other inactivation mechanisms like the irreversible adsorption of phages to the floc structure or the damage of phage surfaces due to entrapment into the floc during coagulation and floc formation do not seem to contribute to the low surviving fraction found for both F-specific bacteriophages. Furthermore, no influence of phage agglomeration or pH drops during the flocculation process on phage inactivation could be observed. The somatic coliphage phiX174 in contrast did not show sensitivity to chemical stress and in accordance only slight interaction between Al13 and the phage surface was observed. Consequently, F-specific phages like MS2 should not be used as surrogate for viruses in flocculation experiments with PACl to determine the removal rates of viruses, as the results are influenced by a strong inactivation of the bacteriophages due to the experimental conditions.

Icosahedral bacteriophage ΦX174 forms a tail for DNA transport during infection

Prokaryotic viruses have evolved various mechanisms to transport their genomes across bacterial cell walls. Many bacteriophages use a tail to perform this function, whereas tail-less phages rely on host organelles. However, the tail-less, icosahedral, single-stranded DNA ΦX174-like coliphages do not fall into these well-defined infection processes. For these phages, DNA delivery requires a DNA pilot protein. Here we show that the ΦX174 pilot protein H oligomerizes to form a tube whose function is most probably to deliver the DNA genome across the host's periplasmic space to the cytoplasm. The 2.4 Å resolution crystal structure of the in vitro assembled H protein's central domain consists of a 170 Å-long α-helical barrel. The tube is constructed of ten α-helices with their amino termini arrayed in a right-handed super-helical coiled-coil and their carboxy termini arrayed in a left-handed super-helical coiled-coil. Genetic and biochemical studies demonstrate that the tube is essential for infectivity but does not affect in vivo virus assembly. Cryo-electron tomograms show that tubes span the periplasmic space and are present while the genome is being delivered into the host cell's cytoplasm. Both ends of the H protein contain transmembrane domains, which anchor the assembled tubes into the inner and outer cell membranes. The central channel of the H-protein tube is lined with amide and guanidinium side chains. This may be a general property of viral DNA conduits and is likely to be critical for efficient genome translocation into the host.

Transport and removal of bacteriophages MS2 and PhiX174 in steel slag-amended soils: column experiments and transport model analyses

The aim of this study was to investigate the removal of bacteriophages MS2 and PhiX174 in soils amended with converter furnace steel slag. Column experiments were performed to examine the bacteriophage removal in slag-amended (slag content: 0%, 25%, and 50%) loam soils. For comparison, column experiments were also conducted with Escherichia coli. In addition, chloride (Cl) was used as a conservative tracer to determine transport characteristics. Results showed mass recoveries of Cl of 98.6±3.5%, indicating that the experiments were conducted successfully. The mass recovery of MS2 was 86.7% in no slag (100% soil), decreasing to 0% in slag contents of 25% and 50%. The mass recovery of PhiX174 decreased from 87.8% to 51.5% with increasing slag content from 0% to 50%. In the case of E. coli, the mass recoveries decreased from 47.0% to 10.5% with increasing slag content from 0% to 50%. In the transport models analyses, the HYDRUS-1D code was used to quantify the sorption parameters from breakthrough curves. For the 100% soil column, a one-site kinetic sorption model was fitted to the data, whereas a two-site kinetic sorption model was fitted for slag-amended (25% and 50% slag) soil data. Results demonstrate that the addition of steel slag to soil enhances the removal of bacteriophages due to the presence of FeO in the steel slag. However, CaO could not contribute to the bacteriophage removal in our experimental conditions because the effluent pH (7.7–8.9) in slag-amended (25% and 50% slag) soils was not high enough to promote the bacteriophage inactivation.

Effects of a pressure release on virus retention with the Ultipor DV20 membrane

Recent data have shown that a temporary release in the transmembrane pressure can cause a significant increase in virus transmission through a number of commercial virus filters. The objective of this work was to study the effect of a pressure release on retention of the bacteriophage ΦX174 by the Ultipor DV20 membrane, with phage capture within the membrane visualized by confocal microscopy. Phage challenge tests showed a significant transient increase in phage transmission (typically by an order of magnitude) immediately after a pressure release. This transient increase was not due to any damage to the filter or to the presence of a back-flow (from permeate to feed). Confocal images demonstrated that the pressure release caused the migration of previously captured bacteriophage further into the depth of the filter. Data were analyzed using a modified internal polarization model, with the results providing important insights into the factors controlling virus retention during virus filtration.

Presence of bacteria in aqueous solution influences virus adsorption on nanoparticles

Virus contamination in wastewater is usually accompanied by the existence of various bacteria. Nanoparticles (NPs) have been shown to efficiently remove virus. In this study, bacterial cells, supernatants, and cultures were harvested separately from three strains at the culture ages of 6 and 24 h, corresponding to the log and stationary phases, respectively. The aim is to investigate how their presence affects virus adsorption on the three Fe and Al oxide NPs (α-Fe2O3, γ-Fe2O3-B, and Al2O3) and how these effects change with bacterial growth phase. Bacteriophage phiX174 was used as a virus model. Results showed that bacterial cells, supernatants, and cultures harvested at 6 h generally reduced virus adsorption by an average of 0.75±0.84, 7.7±9.0, and 10.3±8.6%, respectively, while those harvested at 24 h reduced virus adsorption by an average of 2.1±0.93, 21.5±6.6, and 24.6±6.9%, respectively. Among the NPs, α-Fe2O3 showed more sensitivity to bacteria than the other two, probably because of its relatively higher value of point of zero charge. It was found that cell-induced and supernatant-induced reductions were combined to achieve added results, in which the supernatants contributed much more than the cells, implying that the bacterial exudates might be more crucial in the reduced virus adsorption than the bacterial cells. These results strongly demonstrated that the bacteria-induced reduction in virus adsorption became more significant with culture age. It is suggested that studies conducted in the absence of bacteria may not accurately evaluate the potential of virus removal efficiency of the NPs in bacteria-containing environments.

Faecal sludge management with the larvae of the black soldier fly (Hermetia illucens) — From a hygiene aspect

Inadequate and lacking sanitation and wastewater treatment systems can lead to the spreading of diarrhoeal diseases. One contributing factor in the lack of such treatment systems is the lack of economic incentives for stakeholders throughout the service chain. However, the organic fraction of the waste is high in valuable plant nutrients and could be reused in agriculture and as animal feed. For example, grown larvae of the black soldier fly, Hermetia illucens L. (Diptera: Stratiomyidae), make an excellent protein source in animal feed, while the feeding activity of the larvae substantially reduces the dry mass of the treated material. This study examined the effect of black soldier fly larvae on the concentration of pathogenic microorganisms in human faeces and found a 6 log10 reduction in Salmonella spp. in human faeces in eight days, compared with a <2 log10 reduction in the control. No increased reduction was observed for Enterococcus spp., bacteriophage ΦX174 or Ascaris suum ova.

Effect of gamma irradiation on bacteriophages used as viral indicators

This study aimed to examine the susceptibility of indicator bacteriophages towards γ-radiation to evaluate their appropriateness as viral indicators for water quality control. The effects of γ-radiation on naturally occurring somatic coliphages, F-specific coliphages and Escherichia coli were examined in raw sewage and sewage sludge. As well, the effects of radiation on bacteriophages ΦX174 and MS2, and E. coli all grown in the laboratory and seeded in distilled water, autoclaved raw sewage and a 1% peptone solution were evaluated. The inactivation of E. coli was fairly similar in all matrices. In contrast, inactivation of bacteriophages was significantly greater in distilled water than in the other matrices. These results showed the great influence of the matrix characteristics on virus inactivation. Somatic coliphages in raw sewage and sewage sludge and ΦX174 in autoclaved sewage were inactivated similarly and were far more resistant than F-specific coliphages, MS2 and E. coli. As well, F-specific RNA bacteriophages in raw sewage and sewage sludge and MS2 in autoclaved sewage were inactivated similarly and were more resistant than E. coli. In contrast, MS2 was more susceptible to γ-radiation than E. coli in distilled water. Our results showed that ΦX174 is a suitable indicator for estimating virus inactivation by γ-irradiation and corroborate the use of somatic coliphages to survey the viral quality of treated water and sludges.

Selection Affects Genes Involved in Replication during Long-Term Evolution in Experimental Populations of the Bacteriophage φX174

Observing organisms that evolve in response to strong selection over very short time scales allows the determination of the molecular mechanisms underlying adaptation. Although dissecting these molecular mechanisms is expensive and time-consuming, general patterns can be detected from repeated experiments, illuminating the biological processes involved in evolutionary adaptation. The bacteriophage φX174 was grown for 50 days in replicate chemostats under two culture conditions: Escherichia coli C as host growing at 37°C and Salmonella typhimurium as host growing at 43.5°C. After 50 days, greater than 20 substitutions per chemostat had risen to detectable levels. Of the 97 substitutions, four occurred in all four chemostats, five arose in both culture conditions, eight arose in only the high temperature S. typhimurium chemostats, and seven arose only in the E. coli chemostats. The remaining substitutions were detected only in a single chemostat, however, almost half of these have been seen in other similar experiments. Our findings support previous studies that host recognition and capsid stability are two biological processes that are modified during adaptation to novel hosts and high temperature. Based upon the substitutions shared across both environments, it is apparent that genome replication and packaging are also affected during adaptation to the chemostat environment, rather than to temperature or host per se. This environment is characterized by a large number of phage and very few hosts, leading to competition among phage within the host. We conclude from these results that adaptation to a high density environment selects for changes in genome replication at both protein and DNA sequence levels.

The role of surface functionalization of colloidal alumina particles on their controlled interactions with viruses

Materials that interact in a controlled manner with viruses attract increasing interest in biotechnology, medicine, and environmental technology. Here, we show that virus–material interactions can be guided by intrinsic material surface chemistries, introduced by tailored surface functionalizations. For this purpose, colloidal alumina particles are surface functionalized with amino, carboxyl, phosphate, chloropropyl, and sulfonate groups in different surface concentrations and characterized in terms of elemental composition, electrokinetic, hydrophobic properties, and morphology. The interaction of the functionalized particles with hepatitis A virus and phages MS2 and PhiX174 is assessed by virus titer reduction after incubation with particles, activity of viruses conjugated to particles, and imaged by electron microscopy. Type and surface density of particle functional groups control the virus titer reduction between 0 and 99.999% (5 log values). For instance, high sulfonate surface concentrations (4.7 groups/nm2) inhibit attractive virus–material interactions and lead to complete virus recovery. Low sulfonate surface concentrations (1.2 groups/nm2), native alumina, and chloropropyl-functionalized particles induce strong virus-particle adsorption. The virus conformation and capsid amino acid composition further influence the virus–material interaction. Fundamental interrelations between material properties, virus properties, and the complex virus–material interaction are discussed and a versatile pool of surface functionalization strategies controlling virus–material interactions is presented.

The ABCs of Experimental Evolution

Microbial evolution is complex and is influenced by many sources of variation. Experimental evolution is no exception, although it is more controlled, easily replicated, and typically devoid of interactions between species. Mathematical modeling of the evolutionary process can help in understanding the underlying mechanisms that drive outcome of such experiments. These models can be complex and parameter rich, limiting their feasibility for statistical inference. In this paper, we introduce the use of Approximate Bayesian Computation (ABC) as a tool for statistical inference in the study of experimental evolution. ABC is a fast and simple method for fitting complex models to data. We utilize this method, coupled with a mechanistic model of experimental evolution, to study the evolution process of bacteriophage ϕX174 under benign selection pressure. Our results highlight three mutation-selection scenarios that could explain this process: high mutation/low selection pressure, low mutation/high selection pressure, and low mutation/low selection pressure, with posterior support of 19%, 9.5%, and 71.5% for each of these scenarios, respectively. Sequence data support the first candidate. Though surprising, this scenario was not improbable based on our analysis.

Virus inactivation by high frequency ultrasound in combination with visible light

In this study, the effects of high frequency ultrasound (US) and visible light (VL) on virus inactivation were investigated. The bacteriophages ΦX174 and MS2 were used as model viruses. The experiments were performed at room temperature at three different, relatively high US frequencies (i.e., 582, 862, and 1142kHz) with and without the use of VL, and different initial virus concentrations. The two bacteriophages were diluted in phosphate-buffered saline solution to a titer of 103–104pfu/mL. The experimental virus inactivation data were satisfactorily represented by a simple first-order kinetic expression. Virus inactivation was faster at the lower frequencies (582 and 862kHz). Furthermore, it was observed that MS2 was inactivated faster than ΦX174. The simultaneous use of US and VL was found to be more effective than US alone for MS2 inactivation, indicating the existence of a synergistic effect. However, the use of VL in conjunction with high frequency US hindered the inactivation of ΦX174.

Inactivation of bacteriophages by high levels of dissolved CO 2

We developed a system with high levels of dissolved CO 2 for water disinfection. Bacteriophages MS2, Qβ and ΦX174 were selected as the inactivation targets. A relatively mild inactivation effect was observed on MS2 and Qβ at different initial concentrations of dissolved CO 2 at 0.3 MPa in 20–30 min. When the pressure was increased to 0.6 MPa, the inactivation of MS2 and Qβ was differentially improved. However, this system was less effective for the inactivation of ΦX174. The capsid surface property is a probable reason for the low inactivation of ΦX174. The pH was not a key factor in the inactivation of bacteriophages; moreover, the results obtained using alternative gases (pressurized air and O 2) indicated that only CO 2 inactivated these bacteriophages. A comparison between the results of real time polymerase chain reaction (RT-PCR) and plaque assay showed that some RNA moved out from the capsid after treatment. Capsid damage by CO 2 expansion was the likely mechanism of inactivation with our method.