Phi X174 Research Articles

Advances in basic and clinical immunology in 2012

Basic and clinical immunology articles published in the Journal in 2012 were mostly related to the expanding area of primary immunodeficiencies (PIDs). Novel forms of PID were identified by using whole-exome sequencing or after careful examination of flow cytometric data, as in the reports of lymphocyte-specific protein tyrosine kinase, CD27, and CD21 deficiencies. Absent IgG and IgA memory B cells were described in patients with hyper-IgE syndrome, which is consistent with defective antibody response and suggests a potential benefit of immunoglobulin replacement. Impaired production of antibodies to polysaccharide antigens by the human B-cell subset analog to murine B-1 cells was reported in a child with selective polysaccharide antibody deficiency. Increased production of inflammatory cytokines by monocyte-derived cells on Toll-like receptor activation was reported in patients with X-linked agammaglobulinemia, underscoring the important role of Bruton tyrosine kinase in modulation of inflammation. The mechanisms explaining susceptibility to yeast infections and development of chronic mucocutaneous candidiasis were extensively studied. Universal newborn screening for T-cell deficiencies is being implemented in several states, resulting in the diagnosis of a higher number of immunodeficient newborns than previously estimated. The use of laboratory testing to distinguish PIDs from HIV infection was clarified. In the management of PIDs, refinement of indication and strategies to hematopoietic stem cell transplantation resulted in improved outcomes. The use of anti-IL-6 mAbs showed promise as an alternative treatment in patients with Schnitzler syndrome.

The Duke University Medical Center (DUMC) Experience with Common Variable Immune Deficiency (CVID)

RationaleCVID is a heterogeneous group of disorders, characterized by humoral immune deficiency and associated with cellular defects with variable presentation, making diagnosis and management uniquely challenging. We sought to describe the DUMC experience with patients referred from around the region and managed for CVID.MethodsAll patient encounters entered in the DUMC Health System from January 2005 until January 2012 were screened for a diagnosis code consistent with CVID. Patient characteristics including comorbid disease conditions, infectious complications, and immunologic parameters were recorded and analyzed.ResultsOur search revealed 283 individuals recorded as having CVID. A review of their records revealed 103 of these cases met the standard criteria for CVID. Median immunoglobulin levels were IgG 206 mg/dL, IgA 5 mg/dL, IgM 20 mg/dL. 38 % were 18 years or younger at the time of diagnosis. 94% had significant comorbid disease that included autoimmune disease, malignancy, lymphoproliferative disease, chronic lung disease, chronic GI disease and/or mood disorder. Cytopenias were common with leukopenia (23%), thrombocytopenia (44%), anemia (49%), and neutropenia (24%) observed. There was a statistically significant correlation between diagnosis at 18 years or younger and the presence of pancytopenia (p= <0.0001). 9 patients underwent immunization with bacteriophage ΦX174, all of whom demonstrated abnormal antibody response.ConclusionsWe present data on 103 patients with CVID seen at a major academic medical center. The diagnosis of CVID was incorrect in 36% of the identified cases and the majority of our patients had significant comorbid disease, highlighting some of the challenges associated with diagnosing and managing CVID. RationaleCVID is a heterogeneous group of disorders, characterized by humoral immune deficiency and associated with cellular defects with variable presentation, making diagnosis and management uniquely challenging. We sought to describe the DUMC experience with patients referred from around the region and managed for CVID. CVID is a heterogeneous group of disorders, characterized by humoral immune deficiency and associated with cellular defects with variable presentation, making diagnosis and management uniquely challenging. We sought to describe the DUMC experience with patients referred from around the region and managed for CVID. MethodsAll patient encounters entered in the DUMC Health System from January 2005 until January 2012 were screened for a diagnosis code consistent with CVID. Patient characteristics including comorbid disease conditions, infectious complications, and immunologic parameters were recorded and analyzed. All patient encounters entered in the DUMC Health System from January 2005 until January 2012 were screened for a diagnosis code consistent with CVID. Patient characteristics including comorbid disease conditions, infectious complications, and immunologic parameters were recorded and analyzed. ResultsOur search revealed 283 individuals recorded as having CVID. A review of their records revealed 103 of these cases met the standard criteria for CVID. Median immunoglobulin levels were IgG 206 mg/dL, IgA 5 mg/dL, IgM 20 mg/dL. 38 % were 18 years or younger at the time of diagnosis. 94% had significant comorbid disease that included autoimmune disease, malignancy, lymphoproliferative disease, chronic lung disease, chronic GI disease and/or mood disorder. Cytopenias were common with leukopenia (23%), thrombocytopenia (44%), anemia (49%), and neutropenia (24%) observed. There was a statistically significant correlation between diagnosis at 18 years or younger and the presence of pancytopenia (p= <0.0001). 9 patients underwent immunization with bacteriophage ΦX174, all of whom demonstrated abnormal antibody response. Our search revealed 283 individuals recorded as having CVID. A review of their records revealed 103 of these cases met the standard criteria for CVID. Median immunoglobulin levels were IgG 206 mg/dL, IgA 5 mg/dL, IgM 20 mg/dL. 38 % were 18 years or younger at the time of diagnosis. 94% had significant comorbid disease that included autoimmune disease, malignancy, lymphoproliferative disease, chronic lung disease, chronic GI disease and/or mood disorder. Cytopenias were common with leukopenia (23%), thrombocytopenia (44%), anemia (49%), and neutropenia (24%) observed. There was a statistically significant correlation between diagnosis at 18 years or younger and the presence of pancytopenia (p= <0.0001). 9 patients underwent immunization with bacteriophage ΦX174, all of whom demonstrated abnormal antibody response. ConclusionsWe present data on 103 patients with CVID seen at a major academic medical center. The diagnosis of CVID was incorrect in 36% of the identified cases and the majority of our patients had significant comorbid disease, highlighting some of the challenges associated with diagnosing and managing CVID. We present data on 103 patients with CVID seen at a major academic medical center. The diagnosis of CVID was incorrect in 36% of the identified cases and the majority of our patients had significant comorbid disease, highlighting some of the challenges associated with diagnosing and managing CVID.

Improved Virus Removal in Ceramic Depth Filters Modified with MgO

Ceramic filters, working on the depth filtration principle, are known to improve drinking water quality by removing human pathogenic microorganisms from contaminated water. However, these microfilters show no sufficient barrier for viruses having diameters down to 20 nm. Recently, it was shown that the addition of positively charged materials, for example, iron oxyhydroxide, can improve virus removal by adsorption mechanisms. In this work, we modified a common ceramic filter based on diatomaceous earth by introducing a novel virus adsorbent material, magnesium oxyhydroxide, into the filter matrix. Such filters showed an improved removal of about 4-log in regard to bacteriophages MS2 and PhiX174. This is explained with the electrostatic enhanced adsorption approach that is the favorable adsorption of negatively charged viruses onto positively charged patches in an otherwise negatively charged filter matrix. Furthermore, we provide theoretical evidence applying calculations according to Derjaguin-Landau-Verwey-Overbeek theory to strengthen our experimental results. However, modified filters showed a significant variance in virus removal efficiency over the course of long-term filtration experiments with virus removal increasing with filter operation time (or filter aging). This is explained by transformational changes of MgO in the filter upon contact with water. It also demonstrates that filter history is of great concern when filters working on the adsorption principles are evaluated in regard to their retention performance as their surface characteristics may alter with use.

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NTP can also inactivate viruses. In the previous research, critical damages for the inactivation of λ phage were done on coat proteins and M13 phage on DNA. These results were obtained with wet phage sample [1]. We also tried to use dry sample. We used bacteriophage φX174 which is resistant to drying. When the bacteriophage is inactivated by NTP, the damage should exist either on viral nucleic acids or coat proteins or both. It is possible to extract the nucleic acids from the bacteriophage so that the damage to the DNA can be separately analyzed to exclude the effect of the proteins. The DNA extracted from the plasma-exposed phage can be assayed its plaque forming activity by transfection. The coat proteins were analysed by SDS-PAGE. Using these assays, the damages of viral DNA and coat proteins have been determined separately. The results for the dry φX174 phage inactivated by the exposure to DBD showed both DNA and coat proteins were damaged and the critical damage were done to the coat proteins. This is similar to the result of the wet sample. We also report a single-molecule-based analysis of strand breakages on large DNA molecules induced by the plasma exposure. Single-molecule observation of DNA that involved molecular combing was used to measure the length of individual DNA molecules. The measured DNA length showed that plasma exposure caused a marked change in length of DNA molecules. The rate of plasma-induced strand breakage on large random-coiled DNA molecules was determined using a simple mathematical model. The measured rate shows good relation with the plasma exposure time, and could be used for safety evaluation of the plasma treated water. References

Adaptive regulatory substitutions affect multiple stages in the life cycle of the bacteriophage φX174.

BackgroundPreviously, we showed that adaptive substitutions in one of the three promoters of the bacteriophage ϕX174 improved fitness at high-temperature by decreasing transcript levels three- to four-fold. To understand how such an extreme change in gene expression might lead to an almost two-fold increase in fitness at the adaptive temperature, we focused on stages in the life cycle of the phage that occur before and after the initiation of transcription. For both the ancestral strain and two single-substitution strains with down-regulated transcription, we measured seven phenotypic components of fitness (attachment, ejection, eclipse, virion assembly, latent period, lysis rate and burst size) during a single cycle of infection at each of two temperatures. The lower temperature, 37°C, is the optimal temperature at which phages are cultivated in the lab; the higher temperature, 42°C, exerts strong selection and is the condition under which these substitutions arose in evolution experiments. We augmented this study by developing an individual-based stochastic model of this same life cycle to explore potential explanations for our empirical results.ResultsOf the seven fitness parameters, three showed significant differences between strains that carried an adaptive substitution and the ancestor, indicating the presence of pleiotropy in regulatory evolution. 1) Eclipse was longer in the adaptive strains at both the optimal and high-temperature environments. 2) Lysis rate was greater in the adaptive strains at the high temperature. 3) Burst size for the mutants was double that of the ancestor at the high temperature, but half that at the lower temperature. Simulation results suggest that eclipse length and latent period variance can explain differences in burst sizes and fitness between the mutant and ancestral strains.ConclusionsDown-regulating transcription affects several steps in the phage life cycle, and all of these occur after the initiation of transcription. We attribute the apparent tradeoff between delayed progeny production and faster progeny release to improved host resource utilization at high temperature.

Effect of Long-Term Freezing and Freeze–Thaw Cycles on Indigenous and Inoculated Microorganisms in Dewatered Blackwater

Wastewater treatment in many Arctic regions is inadequate, even nonexisting. Natural freezing of wastewater in those areas may be beneficial for reduction of microorganisms. The aim of this study was to investigate the effect of long-term freezing, and repeated freezing and thawing, on indigenous coliforms, fecal streptococci, and antibiotic-resistant (AR) bacteria, and inoculated Salmonella Enteriditis and E. coli bacteriophage ΦX174 in dewatered blackwater. At the end of the long-term freezing experiment (10 months), an MPN recovery study was done, including the microbial groups that had shown the largest reduction, using tryptone soy broth at incubation temperatures of 10 and 20 °C overnight for the coliforms and AR bacteria, and buffered peptone water at incubation temperature of 37 °C for 18-20 h for Salmonella. Fecal streptococci were more resistant to long-term freezing than the coliform group. Total number of AR bacteria decreased slowly but constantly over the 10-month freezing period. Salmonella rapidly decreased and were nondetectable within a week but exhibited some recovery after 10 months of freezing, whereas limited or no recovery of coliforms and AR-bacteria was detected. Bacteriophages showed limited reduction during the long-term freezing. Repeated freezing and thawing increased the reduction of all tested microbial groups markedly.

Cotransport of clay colloids and viruses in water saturated porous media

This study examines the cotransport of clay colloids and viruses in laboratory packed columns. Bacteriophages MS2 and ΦX174 were used as model viruses, kaolinite (kGa-1b) and montmorillonite (STx-1b) as model clay colloids, and glass beads as model packing material. The combined and synergistic effects of clay colloids and pore water velocity on virus transport and retention in porous media were examined at three pore water velocities (0.38, 0.74, and 1.21cm/min). The results indicated that the mass recovery of viruses and clay colloids decreased as the pore water velocity decreased; whereas, for the cotransport experiments no clear trend was observed. Temporal moments of the breakthrough concentrations suggested that the presence of clays significantly influenced virus transport and irreversible deposition onto glass beads. Mass recovery values for both viruses, calculated based on total virus concentration in the effluent, were reduced compared to those in the absence of clays. The transport of both suspended and attached onto suspended clay-particles viruses was retarded, compared to the tracer, only at the highest pore water velocity. Moreover both clay colloids were shown to hinder virus transport at the highest pore water velocity. At the lower velocities MS2 transport was hindered and ΦX174 transport was facilitated with the exception of U=0.74cm/min in the presence of KGa-1b. Both MS2 and ΦX174 were attached in greater amounts onto KGa-1b than STx-1b. Also, MS2 exhibited greater affinity than ΦX174 for both clays.

Study on the removal efficiency of UF membranes using bacteriophages in bench-scale and semi-technical scale

To determine the removal efficiency of ultrafiltration (UF) membranes for nano-particles in the size range of viruses the state of the art uses challenge tests with virus-spiked water. This work focuses on bench-scale and semi-technical scale experiments. Different experimental parameters influencing the removal efficiency of the tested UF membrane modules were analyzed and evaluated for bench- and semi-technical scale experiments. Organic matter in the water matrix highly influenced the removal of the tested bacteriophages MS2 and phiX174. Less membrane fouling (low ΔTMP) led to a reduced phage reduction. Increased flux positively affected phage removal in natural waters. The tested bacteriophages MS2 and phiX174 revealed different removal properties. MS2, which is widely used as a model organism to determine virus removal efficiencies of membranes, mostly showed a better removal than phiX174 for the natural water qualities tested. It seems that MS2 is possibly a less conservative surrogate for human enteric virus removal than phiX174. In bench-scale experiments log removal values (LRV) for MS2 of 2.5-6.0 and of 2.5-4.5 for phiX174 were obtained for the examined range of parameters. Phage removal obtained with differently fabricated semi-technical modules was quite variable for comparable parameter settings, indicating that module fabrication can lead to differing results. Potting temperature and module size were identified as influencing factors. In conclusion, careful attention has to be paid to the choice of experimental settings and module potting when using bench-scale or semi-technical scale experiments for UF membrane challenge tests.

High virus retention mediated by zirconia microtubes with tailored porosity

In this study the fabrication of zirconia microtubes for virus filtration using a sequence of slurry preparation, extrusion process, debinding and final sintering is presented. The sintered zirconia microtubes are characterised by microstructural analysis including Hg intrusion porosimetry, BET analysis and three-point bending tests. Zirconia microtubes sintered at 1050 °C are found to provide membrane properties featuring an open porosity of 50.7% with pore sizes in the range of 10–40 nm, a specific surface area of 23.2 m 2 /g and an average bending strength of 32.9 MPa, being suitable for virus filtration. Based on these mesoporous microtubes water permeate fluxes of 45 L/m 2 h are provided in dead-end filtration mode. A virus retention of 99.9999% (log reduction value of 6) for small bacteriophages MS2 and PhiX174 which served as surrogates for human pathogenic viruses is achieved in this study demonstrating a stable ceramic membrane with high viral retention properties.

Mg/Al layered double hydroxide for bacteriophage removal in aqueous solution

The objective of this study was to investigate the removal of bacteriophages in Mg/Al layered double hydroxide (LDH). Batch experiments were performed with bacteriophage MS2 in a powder form of Mg/Al LDH under various LDH doses. Column experiments were also performed under flow-through condition with bacteriophages MS2 and phiX174 in Mg/Al LDH immobilized on sand surfaces. Batch tests demonstrated that the powder form of Mg/Al LDH was effective in removing MS2 with the removal capacity of 2.2 × 10(8) plaque forming unit (pfu)/g under the given experimental conditions (LDH dose = 2 g/L; initial MS2 concentration = 4.61 × 10(5) pfu/mL). Column experiments showed that the log removal of phiX174 was 4.40 in columns containing 100% Mg/Al LDH-coated sand while it was 0.05 in 100% quartz sand. These findings indicated that Mg/Al LDH-coated sand was effective in removing bacteriophages compared with sand. A more than 4 log removal (=5.44) of MS2 was achieved in 100% Mg/Al LDH-coated sand. This study demonstrates the potential application of Mg/Al LDH for virus removal in water treatment.

Minimal requirements for inhibition of MraY by lysis protein E from bacteriophage ΦX174.

The DNA phage ΦX174 encodes the integral membrane protein E whose expression leads to host cell lysis by inhibition of the peptidoglycan synthesis enzyme MraY. Here we use mutagenesis to characterize the molecular details of the E lysis mechanism. We find that a minimal 18-residue region with the modified wild-type sequences of the conserved transmembrane helix of E is sufficient to lyse host cells and that specific residues within and at the boundaries of this helix are important for activity. This suggests that positioning of the helix in the membrane is critical for interactions with MraY. We further characterize the interaction site of the transmembrane helix with MraY demonstrating E forms a stable complex with MraY. Triggering cell lysis by peptidoglycan synthesis inhibition is a traditional route for antimicrobial strategies. Understanding the mechanism of bacterial cell lysis by E will provide insights into new antimicrobial strategies using re-engineered E peptides.

ParticleCall: A particle filter for base calling in next-generation sequencing systems

BackgroundNext-generation sequencing systems are capable of rapid and cost-effective DNA sequencing, thus enabling routine sequencing tasks and taking us one step closer to personalized medicine. Accuracy and lengths of their reads, however, are yet to surpass those provided by the conventional Sanger sequencing method. This motivates the search for computationally efficient algorithms capable of reliable and accurate detection of the order of nucleotides in short DNA fragments from the acquired data.ResultsIn this paper, we consider Illumina’s sequencing-by-synthesis platform which relies on reversible terminator chemistry and describe the acquired signal by reformulating its mathematical model as a Hidden Markov Model. Relying on this model and sequential Monte Carlo methods, we develop a parameter estimation and base calling scheme called ParticleCall. ParticleCall is tested on a data set obtained by sequencing phiX174 bacteriophage using Illumina’s Genome Analyzer II. The results show that the developed base calling scheme is significantly more computationally efficient than the best performing unsupervised method currently available, while achieving the same accuracy.ConclusionsThe proposed ParticleCall provides more accurate calls than the Illumina’s base calling algorithm, Bustard. At the same time, ParticleCall is significantly more computationally efficient than other recent schemes with similar performance, rendering it more feasible for high-throughput sequencing data analysis. Improvement of base calling accuracy will have immediate beneficial effects on the performance of downstream applications such as SNP and genotype calling.ParticleCall is freely available at https://sourceforge.net/projects/particlecall.

Virus inactivation in the presence of quartz sand under static and dynamic batch conditions at different temperatures

Virus inactivation is one of the most important factors that controls virus fate and transport in the subsurface. In this study the inactivation of viruses in the presence of quartz sand was examined. The bacteriophages MS2 and ΦX174 were used as model viruses. Experiments were performed at 4°C and 20°C, under constant controlled conditions, to investigate the effect of virus type, temperature, sand size, and initial virus concentration on virus inactivation. The experimental virus inactivation data were satisfactorily represented by a pseudo-first order expression with time-dependent rate coefficients. Furthermore, the results indicated that virus inactivation was substantially affected by the ambient temperature and initial virus concentration. The inactivation rate of MS2 was shown to be greater than that of ΦX174. However, the greatest inactivation was observed for MS2 without the presence of sand, at 20°C. Sand surfaces offered protection against inactivation especially under static conditions. However, no obvious relationship between sand particle size and virus inactivation could be established from the experimental data. Moreover, the inactivation rates were shown to increase with decreasing virus concentration.

Nucleoside Analogue Mutagenesis of a Single-Stranded DNA Virus: Evolution and Resistance

It has been well established that chemical mutagenesis has adverse fitness effects in RNA viruses, often leading to population extinction. This is mainly a consequence of the high RNA virus spontaneous mutation rates, which situate them close to the extinction threshold. Single-stranded DNA viruses are the fastest-mutating DNA-based systems, with per-nucleotide mutation rates close to those of some RNA viruses, but chemical mutagenesis has been much less studied in this type of viruses. Here, we serially passaged bacteriophage X174 in the presence of the nucleoside analogue 5-fluorouracil (5-FU). We found that 5-FU was unable to trigger population extinction for the range of concentrations tested, but it negatively affected viral adaptability. The phage evolved partial drug resistance, and parallel nucleotide substitutions appearing in independently evolved lines were identified as candidate resistance mutations. Using site-directed mutagenesis, two single-nucleotide substitutions in the lysis protein E (T572C and A781G) were shown to be selectively advantageous in the presence of 5-FU. In RNA viruses, base analogue resistance is often mediated by changes in the viral polymerase, but this mechanism is not possible for X174 and other single-stranded DNA viruses because they do not encode their own polymerase. In addition to increasing mutation rates, 5-FU produces a wide variety of cytotoxic effects at the levels of replication, transcription, and translation. We found that substitutions T572C and A781G lost their ability to confer 5-FU resistance after cells were supplemented with deoxythymidine, suggesting that their mechanism of action is at the DNA level. We hypothesize that regulation of lysis time may allow the virus to optimize progeny size in cells showing defects in DNA synthesis.

Construction of Semisynthetic DNA–Protein Conjugates with Phi X174 Gene-A* Protein

DNA-protein conjugates have frequently been used as versatile molecular tools for a variety of applications in biotechnology to harness synergistic effects of DNA and protein functions. With applications for DNA-protein conjugates growing, easy-to-use and economical methods for the synthesis of DNA-protein conjugates are required. In this study, we developed a method for site-specific labeling of single-stranded DNA (ssDNA) to a recombinant protein of interest (POI) through the Gene-A* protein (Gene-A*) from bacteriophage phi X174, without any chemical modifications of ssDNA. Gene-A* protein is an enzyme that site-selectively cleaves an oligodeoxyribonucleotide (ODN) containing a Gene-A* recognition sequence, at which point a tyrosine residue of Gene-A* is bonded to the 5'-phosphoryl group of the cleavage site via a stable phosphotyrosine linkage. Here, we constructed three kinds of recombinant proteins fused to Gene-A*: N-terminally Gene-A*-fused enhanced green fluorescent protein (EGFP), C-terminally Gene-A*-fused EGFP, and N-terminally Gene-A*-fused firefly luciferase (FLuc). The reaction yields of DNA-protein conjugation catalyzed by the Gene-A* moiety reached 80-90% in the three proteins, and kinetic study revealed that the reaction achieved a steady state after 10 min. Moreover, dot blot analyses were performed to evaluate the hybridization and aptamer-forming ability of ssDNA conjugated to the Gene-A* moiety of a recombinant Gene-A*-FLuc protein. This study demonstrated that a strategy using recombinant proteins fused to Gene-A* could offer a versatile, rapid, easy-to-use, and economical platform for producing DNA-protein conjugates.

THE DISTRIBUTION OF MUTATIONAL FITNESS EFFECTS OF PHAGE φX174 ON DIFFERENT HOSTS

Adaptation depends greatly on the distribution of mutation fitness effects (DMFE), but the phenotypic expression of mutations is often environment dependent. The environments faced by multihost pathogens are mostly governed by their hosts and therefore measuring the DMFE on multiple hosts can inform on the likelihood of short-term establishment and longer term adaptation of emerging pathogens. We explored this by measuring the growth rate of 36 mutants of the lytic bacteriophage φX174 on two host backgrounds, Escherichia coli (EcC) and Salmonella typhimurium (StGal). The DMFE showed higher mean and variance on EcC than on StGal. Most mutations were either deleterious or neutral on both hosts, but a greater proportion of mutations were deleterious on StGal. We identified two mutations with beneficial fitness effects on EcC that were neutral on StGal. Host-specific differences in fitness were associated with particular functional classes of genes involved in the initial stages of infection in accordance with previous studies of host specificity. Overall, there was a positive correlation between the effects of mutations on each host, suggesting that most new mutations will have general, rather than host-specific fitness effects. We consider these results in light of simple fitness landscape models of adaptation and discuss the relevance of context-dependent DMFE for multihost pathogens.

Construction of a Salmonella Gallinarum ghost as a novel inactivated vaccine candidate and its protective efficacy against fowl typhoid in chickens

In order to develop a novel, safe and immunogenic fowl typhoid (FT) vaccine candidate, a Salmonella Gallinarum ghost with controlled expression of the bacteriophage PhiX174 lysis gene E was constructed using pMMP99 plasmid in this study. The formation of the Salmonella Gallinarum ghost with tunnel formation and loss of cytoplasmic contents was observed by scanning electron microscopy and transmission electron microscopy. No viable cells were detectable 24 h 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 Salmonella Gallinarum ghost vaccine was tested in chickens that were divided into four groups: group A (non-immunized control), group B (orally immunized), group C (subcutaneously immunized) and group D (intramuscularly immunized). The birds were immunized at day 7 of age. None of the immunized animals showed any adverse reactions such as abnormal behavior, mortality, or signs of FT such as anorexia, depression, or diarrhea. These birds were subsequently challenged with a virulent Salmonella Gallinarum strain at 3 weeks post-immunization (wpi). Significant protection against the virulent challenge was observed in all immunized groups based on mortality and post-mortem lesions compared to the non-immunized control group. In addition, immunization with the Salmonella Gallinarum ghosts induced significantly high systemic IgG response in all immunized groups. Among the groups, orally-vaccinated group B showed significantly higher levels of secreted IgA. A potent antigen-specific lymphocyte activation response along with significantly increased percentages of CD4+ and CD8+ T lymphocytes found in all immunized groups clearly indicate the induction of cellular immune responses. Overall, these findings suggest that the newly constructed Salmonella Gallinarum ghost appears to be a safe, highly immunogenic, and efficient non-living bacterial vaccine candidate that protects against FT.

Differential Transcription of Bacteriophage φX174 Genes at 37°C and 42°C

To investigate how high temperature affects viral transcription, the absolute amounts of mRNA for six bacteriophage φX174 genes were compared at 37°C and 42°C using Q-PCR. At 37°C, mRNA levels for all genes were consistent with previous studies, but at 42°C mRNA levels for four genes were significantly different from levels at 37°C. Transcript levels were higher for genes B and D; the promoter before gene B appears to be up-regulated at high temperature. Levels for genes F and G were reduced at high temperature, possibly due to increased efficiency of the transcription termination signal immediately upstream of gene F. These functional changes in φX174 gene regulation at high temperature have not been described previously. Studies of phage evolution at high temperatures indicate that this difference in transcript levels is subject to adaptation.

Phenotypic evidence of ultra-highly diluted homeopathic remedies acting at gene expression level: a novel probe on experimental phage infectivity in bacteria

To explore if some ultra-highly diluted homeopathic remedies claimed to have antiviral effects can demonstrate any discernible action in the bacteria Escherichia coli through modulating infectivity potentials of the bacteriophage φX174 DNA. φX174 was selected because of its known host specificity to E. coli and its constitutive expression of lytic gene E when inside the bacterial host. We deployed the "bacteriophage assay system" by "top layer agar plating" method of plaque-counting for evaluation of efficacy of the homeopathic remedies in rendering the bacteria's protective ability against the attack of φX174. The plaque number in the agar-plated Petri dishes, either containing the phage-bacteria mixture subjected to one of the diluted homeopathic drugs under test (1% volume ratio; Belladonna 30C, Rhus Tox 30C, Arnica 30C) or the succussed 1% "alcoholic vehicle" of the drug was recorded. The plaques represented the bacterial colony actually infected and lysed by φX174. Conversely, we subjected φX174 to the homeopathic drug treatment before allowing them to interact with the bacteria to ascertain if the drug itself had any direct effect on the infective potential of the phage DNA entering into the bacterial cell. Each homeopathic remedy showed a significant decrease in plaque number on pretreated bacteria (1 h prior to infection) with respect to untreated and placebo-treated controls; there was only an insignificant change in the plaque number when φX174 was pretreated with the drugs. As φX174 starts lytic cycle when inside the bacterial cell, the loss of plaque number would mean that either the lytic gene E in many was repressed or the entire phage DNA was annihilated by the bacterial gene product (restriction enzymes) known to be regulated by a cluster of genes. This provides phenotypic evidence for the ability of ultra-highly diluted homeopathic remedies to regulate expression of certain gene(s) depending on need of the organism.

Photoinactivation of virus on iron-oxide coated sand: Enhancing inactivation in sunlit waters

Adsorption onto iron oxides can enhance the removal of waterborne viruses in constructed wetlands and soils. If reversible adsorption is not coupled with inactivation, however, infective viruses may be released when changes in solution conditions cause desorption. The goals of this study were to investigate the release of infective bacteriophages MS2 and ΦX174 (two human viral indicators) after adsorption onto an iron oxide coated sand (IOCS), and to promote viral inactivation by exploiting the photoreactive properties of the IOCS. The iron oxide coating greatly enhanced viral adsorption (adsorption densities up to ∼109 infective viruses/g IOCS) onto the sand, but had no affect on infectivity. Viruses that were adsorbed onto IOCS under control conditions (pH 7.5, 10 mM Tris, 1250 μS/cm) were released into solution in an infective state with increases in pH and humic acid concentrations. The exposure of IOCS-adsorbed MS2 to sunlight irradiation caused significant inactivation via a photocatalytic mechanism in both buffered solutions and in wastewater samples (4.9 log10 and 3.3 log10 inactivation after 24-h exposure, respectively). Unlike MS2, ΦX174 inactivation was not enhanced by photocatalysis. In summary, IOCS enhanced the separation of viruses from the water column, and additionally provided a photocatalytic mechanism to promote inactivation of one of the surrogates studied. These qualities make it an attractive option for improving viral control strategies in constructed wetlands.