Identification of nurse shark VNAR single-domain antibodies targeting the spike S2 subunit of SARS-CoV-2.

SARS-CoV-2 is the etiological agent of the COVID-19 pandemic. Antibody-based therapeutics targeting the spike protein, specifically the S1 subunit or the receptor binding domain (RBD) of SARS-CoV-2, have gained attention due to their clinical efficacy in treating patients diagnosed with COVID-19. An alternative to conventional antibody therapeutics is the use of shark new antigen variable receptor domain (VNAR ) antibodies. VNAR s are small (<15 kDa) and can reach deep into the pockets or grooves of the target antigen. Here, we have isolated 53 VNAR s that bind to the S2 subunit by phage panning from a naïve nurse shark VNAR phage display library constructed in our laboratory. Among those binders, S2A9 showed the best neutralization activity against the original pseudotyped SARS-CoV-2 virus. Several binders, including S2A9, showed cross-reactivity against S2 subunits from other ß coronaviruses. Furthermore, S2A9 showed neutralization activity against all variants of concern (VOCs) from alpha to omicron (including BA1, BA2, BA4, and BA5) in both pseudovirus and live virus neutralization assays. Our findings suggest that S2A9 could be a promising lead molecule for the development of broadly neutralizing antibodies against SARS-CoV-2 and emerging variants. The nurse shark VNAR phage library offers a novel platform that can be used to rapidly isolate single-domain antibodies against emerging viral pathogens.

Potential Applications of Thermophilic Bacteriophages in One Health.

Bacteriophages have a wide range of applications such as combating antibiotic resistance, preventing food contamination for food safety, and as biomarkers to indirectly assess the quality of water. Additionally, bacteriophage components (endolysins and coat proteins) have a lot of applications in food processing, vaccine design, and the delivery of cargo to the body. Therefore, bacteriophages/components have a multitude of applications in human, plant/veterinary, and environmental health (One Health). Despite their versatility, bacteriophage/component use is mostly limited to temperatures within 4-40 °C. This limits their applications (e.g., in food processing conditions, pasteurization, and vaccine design). Advances in thermophilic bacteriophage research have uncovered novel thermophilic endolysins (e.g., ΦGVE2 amidase and MMPphg) that can be used in food processing and in veterinary medicine. The endolysins are thermostable at temperatures > 65 °C and have broad antimicrobial activities. In addition to thermophilic endolysins, enzymes (DNA polymerase and ligases) derived from thermophages have different applications in molecular biology/biotechnology: to generate DNA libraries and develop diagnostics for human and animal pathogens. Furthermore, coat proteins from thermophages are being explored to develop virus-like particle platforms with versatile applications in human and animal health. Overall, bacteriophages, especially those that are thermophilic, have a plethora of applications in One Health.

Potential Use of a Combined Bacteriophage-Probiotic Sanitation System to Control Microbial Contamination and AMR in Healthcare Settings: A Pre-Post Intervention Study.

Microbial contamination in the hospital environment is a major concern for public health, since it significantly contributes to the onset of healthcare-associated infections (HAIs), which are further complicated by the alarming level of antimicrobial resistance (AMR) of HAI-associated pathogens. Chemical disinfection to control bioburden has a temporary effect and can favor the selection of resistant pathogens, as observed during the COVID-19 pandemic. Instead, probiotic-based sanitation (probiotic cleaning hygiene system, PCHS) was reported to stably abate pathogens, AMR, and HAIs. PCHS action is not rapid nor specific, being based on competitive exclusion, but the addition of lytic bacteriophages that quickly and specifically kill selected bacteria was shown to improve PCHS effectiveness. This study aimed to investigate the effect of such combined probiotic-phage sanitation (PCHSφ) in two Italian hospitals, targeting staphylococcal contamination. The results showed that PCHSφ could provide a significantly higher removal of staphylococci, including resistant strains, compared with disinfectants (-76%, p < 0.05) and PCHS alone (-50%, p < 0.05). Extraordinary sporadic chlorine disinfection appeared compatible with PCHSφ, while frequent routine chlorine usage inactivated the probiotic/phage components, preventing PCHSφ action. The collected data highlight the potential of a biological sanitation for better control of the infectious risk in healthcare facilities, without worsening pollution and AMR concerns.

Transfer of Phi6 bacteriophage between human skin and surfaces common to consumer-facing environments.

AIMS: This study aimed to determine the extent of Phi6 (Φ6) transfer between skin and surfaces relevant to consumer-facing environments based on inoculum matrix, surface type and contact time. METHODS AND RESULTS: Φ6 transfer rates were determined from skin-to-fomite and fomite-to-skin influenced by inoculum matrix (artificial saliva and tripartite), surface type (aluminium, plastic, stainless steel, touchscreen, vinyl and wood) and contact time (5 and 10 s). Significant differences in estimated means were observed based on surface type (both transfer directions), inoculum matrix (skin-to-fomite) and contact time (both transfer directions). During a sequential transfer experiment from fomite-to-skin, the maximum number of consecutive transfer events observed was 3.33 ± 1.19, 2.33 ± 1.20 and 1.67 ± 1.21 for plastic, touchscreen and vinyl, respectively. CONCLUSIONS: Contact time significantly impacted Φ6 transfer rates, which may be attributed to skin absorption dynamics. Surface type should be considered for assessing Φ6 transfer rates. SIGNIFICANCE AND IMPACT OF THE STUDY: Although the persistence of Φ6 on fomites has been characterized, limited data are available regarding the transfer of Φ6 among skin and fomites. Determining Φ6 transfer rates for surfaces in consumer-facing environments based on these factors is needed to better inform future virus transmission mitigation strategies.

Viral Preservation with Protein-Supplemented Nebulizing Media in Aerosols.

The outbreak of SARS-CoV-2 has emphasized the need for a deeper understanding of infectivity, spread, and treatment of airborne viruses. Bacteriophages (phages) serve as ideal surrogates for respiratory pathogenic viruses thanks to their high tractability and the structural similarities tailless phages bear to viral pathogens. However, the aerosolization of enveloped SARS-CoV-2 surrogate phi6 usually results in a >3-log10 reduction in viability, limiting its usefulness as a surrogate for aerosolized coronavirus in "real world" contexts, such as a sneeze or cough. Recent work has shown that saliva or artificial saliva greatly improves the stability of viruses in aerosols and microdroplets relative to standard dilution/storage buffers like suspension medium (SM) buffer. These findings led us to investigate whether we could formulate media that preserves the viability of phi6 and other phages in artificially derived aerosols. Results indicate that SM buffer supplemented with bovine serum albumin (BSA) significantly improves the recovery of airborne phi6, MS2, and 80α and outperforms commercially formulated artificial saliva. Particle sizing and acoustic particle trapping data indicate that BSA supplementation dose-dependently improves viral survivability by reducing the extent of particle evaporation. These data suggest that our viral preservation medium may facilitate a lower-cost alternative to artificial saliva for future applied aerobiology studies. IMPORTANCE We have identified common and inexpensive lab reagents that confer increased aerosol survivability on phi6 and other phages. Our results suggest that soluble protein is a key protective component in nebulizing medium. Protein supplementation likely reduces exposure of the phage to the air-water interface by reducing the extent of particle evaporation. These findings will be useful for applications in which researchers wish to improve the survivability of these (and likely other) aerosolized viruses to better approximate highly transmissible airborne viruses like SARS-CoV-2.

Durability and Surface Oxidation States of Antiviral Nano-Columnar Copper Thin Films.

Antiviral coatings that inactivate a broad spectrum of viruses are important in combating the evolution and emergence of viruses. In this study, nano-columnar Cu thin films have been proposed, inspired by cicada wings (which exhibit mechano-bactericidal activity). Nano-columnar thin films of Cu and its oxides were fabricated by the sputtering method, and their antiviral activities were evaluated against envelope-type bacteriophage Φ6 and non-envelope-type bacteriophage Qß. Among all of the fabricated films, Cu thin films showed the highest antiviral activity. The infectious activity of the bacteriophages was reduced by 5 orders of magnitude within 30 min by the Cu thin films, by 3 orders of magnitude by the Cu2O thin films, and by less than 1 order of magnitude by the CuO thin films. After exposure to ambient air for 1 month, the antiviral activity of the Cu2O thin film decreased by 1 order of magnitude; the Cu thin films consistently maintained a higher antiviral activity than the Cu2O thin films. Subsequently, the surface oxidation states of the thin films were analyzed by X-ray photoelectron spectroscopy; Cu thin films exhibited slower oxidation to the CuO than Cu2O thin films. This oxidation resistance could be a characteristic property of nanostructured Cu fabricated by the sputtering method. Finally, the antiviral activity of the nano-columnar Cu thin films against infectious viruses in humans was demonstrated by the binding inhibition of the SARS-CoV-2 spike protein to the angiotensin-converting enzyme 2 receptor within 10 min.

Isolation, characterization, therapeutic potency, and genomic analysis of a novel bacteriophage vB_KshKPC-M against carbapenemase-producing Klebsiella pneumoniae strains (CRKP) isolated from Ventilator-associated pneumoniae (VAP) infection of COVID-19 patients.

BACKGROUND: Carbapenem-resistant Klebsiella pneumoniae (CRKP) is a significant clinical problem, given the lack of therapeutic options. The CRKP strains have emerged as an essential worldwide healthcare issue during the last 10 years. Global expansion of the CRKP has made it a significant public health hazard. We must consider to novel therapeutic techniques. Bacteriophages are potent restorative cases against infections with multiple drug-resistant bacteria. The Phages offer promising prospects for the treatment of CRKP infections. OBJECTIVE: In this study, a novel K. pneumoniae phage vB_KshKPC-M was isolated, characterized, and sequenced, which was able to infect and lyse Carbapenem-resistant K. pneumoniae host specifically. METHODS: One hundred clinical isolates of K. pneumoniae were collected from patients with COVID-19 associated with ventilator-associated acute pneumonia hospitalized at Shahid Beheshti Hospital, Kashan, Iran, from 2020 to 2021. Initially, all samples were cultured, and bacterial isolates identified by conventional biochemical tests, and then the ureD gene was used by PCR to confirm the isolates. The Antibiotic susceptibility test in the disc diffusion method and Minimum inhibitory concentrations for Colistin was done and interpreted according to guidelines. Phenotypic and molecular methods determined the Carbapenem resistance of isolates. The blaKPC, blaNDM, and blaOXA-23 genes were amplified for this detection. Biofilm determination of CRKP isolates was performed using a quantitative microtiter plate (MTP) method. The phage was isolated from wastewater during the summer season at a specific position from Beheshti Hospital (Kashan, Iran). The sample was processed and purified against the bacterial host, a CRKP strain isolated from a patient suffering from COVID-19 pneumoniae and resistance to Colistin with high potency for biofilm production. This isolate is called Kp100. The separated phages were diluted and titration by the double overlay agar plaque assay. The separate Phage is concentrated with 10% PEG and stored at -80 °C until use. The phage host range was identified by the spot test method. The purified phage morphology was determined using a transmission electron microscope. The phage stability tests (pH and temperature) were analyzed. The effect of cationic ions on phage adsorption was evaluated. The optimal titer of bacteriophage was determined to reduce the concentration of the CRKP strain. One-step growth assays were performed to identify the purified phage burst's latent cycle and size. The SDS-PAGE was used for phage proteins analysis. Phage DNA was extracted by chloroform technique, and the whole genome of lytic phage was sequenced using Illumina HiSeq technology (Illumina, San Diego, CA). For quality assurance and preprocessing, such as trimming, Geneious Prime 2021.2.2 and Spades 3.9.0. The whole genome sequence of the lytic phage is linked to the GenBank database accession number. RASTtk-v1.073 was used to predict and annotate the ORFs. Prediction of ORF was performed using PHASTER software. ResFinder is used to assess the presence of antimicrobial resistance and virulence genes in the genome. The tRNAs can-SE v2.0.6 is used to determine the presence of tRNA in the genome. Linear genome comparisons of phages and visualization of coding regions were performed using Easyfig 2.2.3 and Mauve 2.4.0. Phage lifestyles were predicted using the program PHACTS. Phylogenetic analysis and amino acid sequences of phage core proteins, such as the major capsid protein. Phylogenies were reconstructed using the Neighbor-Joining method with 1000 bootstrap repeat. HHpred software was used to predict depolymerase. In this study, GraphPad Prism version 9.1 was used for the statistical analysis. Student's t-test was used to compare the sets and the control sets, and the significance level was set at P ≤ 0.05. RESULTS: Phage vB_KshKPC-M is assigned to the Siphoviridae, order Caudovirales. It was identified as a linear double-stranded DNA phage of 54,378 bp with 50.08% G + C content, had a relatively broad host range (97.7%), a short latency of 20 min, and a high burst size of 260 PFU/cell, and was maintained stable at different pH (3-11) and temperature (45-65 °C). The vB_KshKPC-M genome contains 91 open-reading frames. No tRNA, antibiotic resistance, toxin, virulence-related genes, or lysogen-forming gene clusters were detected in the phage genome. Comparative genomic analysis revealed that phage vB_KshKPC-M has sequence similarity to the Klebsiella phages, phage 13 (NC_049844.1), phage Sushi (NC_028774.1), phage vB_KpnD_PeteCarol (OL539448.1) and phage PWKp14 (MZ634345.1). CONCLUSION: The broad host range and antibacterial activity make it a promising candidate for future phage therapy applications. The isolated phage was able to lyse most of the antibiotic-resistant clinical isolates. Therefore, this phage can be used alone or as a phage mixture in future studies to control and inhibit respiratory infections caused by these bacteria, especially in treating respiratory infections caused by resistant strains in sick patients.

Mechanical tuning of virus-like particles.

HYPOTHESIS: Virus-like particles (VLPs) are promising scaffolds for developing mucosal vaccines. For their optimal performance, in addition to design parameters from an immunological perspective, biophysical properties may need to be considered. EXPERIMENTS: We investigated the mechanical properties of VLPs scaffolded on the coat protein of Acinetobacter phage AP205 using atomic force microscopy and small angle X-ray scattering. FINDINGS: Investigations showed that AP205 VLP is a tough nanoshell of stiffness 93 ± 23 pN/nm and elastic modulus 0.11 GPa. However, its mechanical properties are modulated by attaching muco-inert polyethylene glycol to 46 ± 10 pN/nm and 0.05 GPa. Addition of antigenic peptides derived from SARS-CoV2 spike protein by genetic fusion increased the stiffness to 146 ± 54 pN/nm although the elastic modulus remained unchanged. These results, which are interpreted in terms of shell thickness and coat protein net charge variations, demonstrate that surface conjugation can induce appreciable changes in the biophysical properties of VLP-scaffolded vaccines.

Analysis of Bacteriophage Behavior of a Human RNA Virus, SARS-CoV-2, through the Integrated Approach of Immunofluorescence Microscopy, Proteomics and D-Amino Acid Quantification.

SARS-CoV-2, one of the human RNA viruses, is widely studied around the world. Significant efforts have been made to understand its molecular mechanisms of action and how it interacts with epithelial cells and the human microbiome since it has also been observed in gut microbiome bacteria. Many studies emphasize the importance of surface immunity and also that the mucosal system is critical in the interaction of the pathogen with the cells of the oral, nasal, pharyngeal, and intestinal epithelium. Recent studies have shown how bacteria in the human gut microbiome produce toxins capable of altering the classical mechanisms of interaction of viruses with surface cells. This paper presents a simple approach to highlight the initial behavior of a novel pathogen, SARS-CoV-2, on the human microbiome. The immunofluorescence microscopy technique can be combined with spectral counting performed at mass spectrometry of viral peptides in bacterial cultures, along with identification of the presence of D-amino acids within viral peptides in bacterial cultures and in patients' blood. This approach makes it possible to establish the possible expression or increase of viral RNA viruses in general and SARS-CoV-2, as discussed in this study, and to determine whether or not the microbiome is involved in the pathogenetic mechanisms of the viruses. This novel combined approach can provide information more rapidly, avoiding the biases of virological diagnosis and identifying whether a virus can interact with, bind to, and infect bacteria and epithelial cells. Understanding whether some viruses have bacteriophagic behavior allows vaccine therapies to be focused either toward certain toxins produced by bacteria in the microbiome or toward finding inert or symbiotic viral mutations with the human microbiome. This new knowledge opens a scenario on a possible future vaccine: the probiotics vaccine, engineered with the right resistance to viruses that attach to both the epithelium human surface and gut microbiome bacteria.

Antimicrobial and Virucidal Potential of Morpholinium-Based Ionic Liquids.

Witnessed by the ongoing spread of antimicrobial resistant bacteria as well as the recent global pandemic of the SARS-CoV-2 virus, the development of new disinfection strategies is of great importance, and novel substance classes as effective antimicrobials and virucides are urgently needed. Ionic liquids (ILs), low-melting salts, have been already recognized as efficient antimicrobial agents with prospects for antiviral potential. In this study, we examined the antiviral activity of 12 morpholinium based herbicidal ionic liquids with a tripartite test system, including enzyme inhibition tests, virucidal activity determination against five model viruses and activity against five bacterial species. The antimicrobial and enzymatic tests confirmed that the inhibiting activity of ILs corresponds with the number of long alkyl side chains and that [Dec2Mor]+ based ILs are promising candidates as novel antimicrobials. The virucidal tests showed that ILs antiviral activity depends on the type and structure of the virus, revealing enveloped Phi6 phage as highly susceptible to the ILs action, while the non-enveloped phages PRD1 and MS2 proved completely resistant to ionic liquids. Furthermore, a comparison of results obtained for P100 and P001 phages demonstrated for the first time that the susceptibility of viruses to ionic liquids can be dependent on differences in the phage tail structure.

Identify the Virus-like Models for COVID-19 as Bio-Threats: Combining Phage Display, Spectral Detection and Algorithms Analysis.

The rapid identification and recognition of COVID-19 have been challenging since its outbreak. Multiple methods were developed to realize fast monitoring early to prevent and control the pandemic. In addition, it is difficult and unrealistic to apply the actual virus to study and research because of the highly infectious and pathogenic SARS-CoV-2. In this study, the virus-like models were designed and produced to replace the original virus as bio-threats. Three-dimensional excitation-emission matrix fluorescence and Raman spectroscopy were employed for differentiation and recognition among the produced bio-threats and other viruses, proteins, and bacteria. Combined with PCA and LDA analysis, the identification of the models for SARS-CoV-2 was achieved, reaching a correction of 88.9% and 96.3% after cross-validation, respectively. This idea might provide a possible pattern for detecting and controlling SARS-CoV-2 from the perspective of combining optics and algorithms, which could be applied in the early-warning system against COVID-19 or other bio-threats in the future.

The Breadth of Bacteriophages Contributing to the Development of the Phage-Based Vaccines for COVID-19: An Ideal Platform to Design the Multiplex Vaccine.

Phages are highly ubiquitous biological agents, which means they are ideal tools for molecular biology and recombinant DNA technology. The development of a phage display technology was a turning point in the design of phage-based vaccines. Phages are now recognized as universal adjuvant-free nanovaccine platforms. Phages are well-suited for vaccine design owing to their high stability in harsh conditions and simple and inexpensive large-scale production. The aim of this review is to summarize the overall breadth of the antiviral therapeutic perspective of phages contributing to the development of phage-based vaccines for COVID-19. We show that phage vaccines induce a strong and specific humoral response by targeted phage particles carrying the epitopes of SARS-CoV-2. Further, the engineering of the T4 bacteriophage by CRISPR (clustered regularly interspaced short palindromic repeats) presents phage vaccines as a valuable platform with potential capabilities of genetic plasticity, intrinsic immunogenicity, and stability.

Efficacy in Galleria mellonella Larvae and Application Potential Assessment of a New Bacteriophage BUCT700 Extensively Lyse Stenotrophomonas maltophilia.

In recent years, Stenotrophomonas maltophilia (S. maltophilia) has become an important pathogen of clinically acquired infections accompanied by high pathogenicity and high mortality. Moreover, infections caused by multidrug-resistant S. maltophilia have emerged as a serious challenge in clinical practice. Bacteriophages are considered a promising alternative for the treatment of S. maltophilia infections due to their unique antibacterial mechanism and superior bactericidal ability compared with traditional antibiotic agents. Here, we reported a new phage BUCT700 that has a double-stranded DNA genome of 43,214 bp with 70% GC content. A total of 55 ORFs and no virulence or antimicrobial resistance genes were annotated in the genome of phage BUCT700. Phage BUCT700 has a broad host range (28/43) and can lyse multiple ST types of clinical S. maltophilia (21/33). Furthermore, bacteriophage BUCT700 used the Type IV fimbrial biogenesis protein PilX as an adsorption receptor. In the stability test, phage BUCT700 showed excellent thermal stability (4 to 60°C) and pH tolerance (pH = 4 to 12). Moreover, phage BUCT700 was able to maintain a high titer during long-term storage. The adsorption curve and one-step growth curve showed that phage BUCT700 could rapidly adsorb to the surface of S. maltophilia and produce a significant number of phage virions. In vivo, BUCT700 significantly increased the survival rate of S. maltophilia-infected Galleria mellonella (G. mellonella) larvae from 0% to 100% within 72 h, especially in the prophylactic model. In conclusion, these findings indicate that phage BUCT700 has promising potential for clinical application either as a prophylactic or therapeutic agent. IMPORTANCE The risk of Stenotrophomonas maltophilia infections mediated by the medical devices is exacerbated with an increase in the number of ICU patients during the Corona Virus Disease 2019 (COVID-19) epidemic. Complications caused by S. maltophilia infections could complicate the state of an illness, greatly extending the length of hospitalization and increasing the financial burden. Phage therapy might be a potential and promising alternative for clinical treatment of multidrug-resistant bacterial infections. Here, we investigated the protective effects of phage BUCT700 as prophylactic and therapeutic agents in Galleria mellonella models of infection, respectively. This study demonstrates that phage therapy can provide protection in targeting S. maltophilia-related infection, especially as prophylaxis.

Smartphone-read phage lateral flow assay for point-of-care detection of infection.

The COVID-19 pandemic has highlighted the urgent need for sensitive, affordable, and widely accessible testing at the point of care. Here we demonstrate a new, universal LFA platform technology using M13 phage conjugated with antibodies and HRP enzymes that offers high analytical sensitivity and excellent performance in a complex clinical matrix. We also report its complete integration into a sensitive chemiluminescence-based smartphone-readable lateral flow assay for the detection of SARS-CoV-2 nucleoprotein. We screened 84 anti-nucleoprotein monoclonal antibody pairs in phage LFA and identified an antibody pair that gave an LoD of 25 pg mL-1 nucleoprotein in nasal swab extract using a FluorChem gel documentation system and 100 pg mL-1 when the test was imaged and analyzed by an in-house-developed smartphone reader. The smartphone-read LFA signals for positive clinical samples tested (N = 15, with known Ct) were statistically different (p < 0.001) from signals for negative clinical samples (N = 11). The phage LFA technology combined with smartphone chemiluminescence imaging can enable the timely development of ultrasensitive, affordable point-of-care testing platforms for SARS-CoV-2 and beyond.

Development and Characterization of Phage Display-Derived Monoclonal Antibodies to the S2 Domain of Spike Proteins of Wild-Type SARS-CoV-2 and Multiple Variants.

The rapid emergence of new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants has resulted in the ongoing global coronavirus disease 2019 (COVID-19) pandemic. Thus, the rapid development of a platform to detect a broad range of SARS-CoV-2 variants is essential for successful COVID-19 management. In this study, four SARS-CoV-2 spike protein-specific single-chain variable fragments (scFvs) were isolated from a synthetic antibody library using phage display technology. Following the conversion of these scFvs into monoclonal antibodies (mAbs) (K104.1-K104.4) and production and purification of the mAbs, the antibody pair (K104.1 and K104.2) that exhibited the highest binding affinity (K104.1 and K104.2, 1.3 nM and 1.9 nM) was selected. Biochemical analyses revealed that this antibody pair specifically bound to different sites on the S2 subunit of the spike protein. Furthermore, we developed a highly sensitive sandwich immunoassay using this antibody pair that accurately and quantitatively detected the spike proteins of wild-type SARS-CoV-2 and multiple variants, including Alpha, Beta, Gamma, Delta, Kappa, and Omicron, in the picomolar range. Conclusively, the novel phage display-derived mAbs we have developed may be useful for the rapid and efficient detection of the fast-evolving SARS-CoV-2.

Virus inactivation by sequential ultraviolet-chlorine disinfection: Synergistic effect and mechanism.

The COVID-19 outbreak has raised concerns about the efficacy of the disinfection process followed in water treatment plants in preventing the spread of viruses. Ultraviolet (UV) and chlorine multi-barrier disinfection processes are commonly used in water treatment plants; however, their effects on virus inactivation are still unclear. In this study, the effects of different disinfection processes (i.e., UV, free chlorine, and their combination) on waterborne viruses were analyzed using bacteriophage surrogates (i.e., MS2 and PR772) as alternative indicators. The results showed that the inactivation rates of PR772 by either UV or free chlorine disinfection were higher than those of MS2. PR772 was approximately 1.5 times more sensitive to UV disinfection and 8.4 times more sensitive to chlorine disinfection than MS2. Sequential UV-chlorine disinfection had a synergistic effect on virus inactivation, which was enhanced by an increase in the UV dose. As compared with single free chlorine disinfection, UV irradiation at 40 mJ cm-2 enhanced MS2 and PR772 inactivation significantly with a 2.7-fold (MS2) and a 1.7-fold (PR772) increase in the inactivation rate constants on subsequent chlorination in phosphate buffered saline. The synergistic effect was also observed in real wastewater samples, in which the MS2 inactivation rate increased 1.4-fold on subsequent chlorination following UV irradiation at 40 mJ cm-2. The mechanism of the synergistic effect of sequential UV-chlorine disinfection was determined via sodium dodecyl sulfate-polyacrylamide gel electrophoresis, using MS2 as an indicator. The results showed that the synergistic effect was due to damage to MS2 surface proteins caused by previous UV disinfection, which enhanced the sensitivity of MS2 to chlorination. This study provides a feasible approach for the efficient inactivation of viruses in water supply and drainage.

Autoantibody discovery across monogenic, acquired, and COVID-19-associated autoimmunity with scalable PhIP-seq.

Phage immunoprecipitation sequencing (PhIP-seq) allows for unbiased, proteome-wide autoantibody discovery across a variety of disease settings, with identification of disease-specific autoantigens providing new insight into previously poorly understood forms of immune dysregulation. Despite several successful implementations of PhIP-seq for autoantigen discovery, including our previous work (Vazquez et al., 2020), current protocols are inherently difficult to scale to accommodate large cohorts of cases and importantly, healthy controls. Here, we develop and validate a high throughput extension of PhIP-seq in various etiologies of autoimmune and inflammatory diseases, including APS1, IPEX, RAG1/2 deficiency, Kawasaki disease (KD), multisystem inflammatory syndrome in children (MIS-C), and finally, mild and severe forms of COVID-19. We demonstrate that these scaled datasets enable machine-learning approaches that result in robust prediction of disease status, as well as the ability to detect both known and novel autoantigens, such as prodynorphin (PDYN) in APS1 patients, and intestinally expressed proteins BEST4 and BTNL8 in IPEX patients. Remarkably, BEST4 antibodies were also found in two patients with RAG1/2 deficiency, one of whom had very early onset IBD. Scaled PhIP-seq examination of both MIS-C and KD demonstrated rare, overlapping antigens, including CGNL1, as well as several strongly enriched putative pneumonia-associated antigens in severe COVID-19, including the endosomal protein EEA1. Together, scaled PhIP-seq provides a valuable tool for broadly assessing both rare and common autoantigen overlap between autoimmune diseases of varying origins and etiologies.

Current Status of Phage Therapy against Infectious Diseases and Potential Application beyond Infectious Diseases.

Intestinal microbiota plays a key role in regulating the pathogenesis of human disease and maintaining health. Many diseases, mainly induced by bacteria, are on the rise due to the emergence of antibiotic-resistant strains. Intestinal microorganisms include organisms such as bacteria, viruses, and fungi. They play an important role in maintaining human health. Among these microorganisms, phages are the main members of intestinal viromes. In particular, the viral fraction, composed essentially of phages, affects homeostasis by exerting selective pressure on bacterial communities living in the intestinal tract. In recent years, with the widespread use and even abuse of antibacterial drugs, more and more drug-resistant bacteria have been found, and they show a trend of high drug resistance and multidrug resistance. Therefore, it has also become increasingly difficult to treat serious bacterial infections. Phages, a natural antibacterial agent with strong specificity and rapid proliferation, have come back to the field of vision of clinicians and scholars. In this study, the current state of research on intestinal phages was discussed, with an exploration of the impact of phage therapy against infectious diseases, as well as potential application beyond infectious diseases.

Bacteriophage Cocktails in the Post-COVID Rehabilitation.

Increasing evidence suggests that gut dysbiosis is associated with coronavirus disease 2019 (COVID-19) infection and may persist long after disease resolution. The excessive use of antimicrobials in patients with COVID-19 can lead to additional destruction of the microbiota, as well as to the growth and spread of antimicrobial resistance. The problem of bacterial resistance to antibiotics encourages the search for alternative methods of limiting bacterial growth and restoring the normal balance of the microbiota in the human body. Bacteriophages are promising candidates as potential regulators of the microbiota. In the present study, two complex phage cocktails targeting multiple bacterial species were used in the rehabilitation of thirty patients after COVID-19, and the effectiveness of the bacteriophages against the clinical strain of Klebsiella pneumoniae was evaluated for the first time using real-time visualization on a 3D Cell Explorer microscope. Application of phage cocktails for two weeks showed safety and the absence of adverse effects. An almost threefold statistically significant decrease in the anaerobic imbalance ratio, together with an erythrocyte sedimentation rate (ESR), was detected. This work will serve as a starting point for a broader and more detailed study of the use of phages and their effects on the microbiome.

The gut virome in health and disease: new insights and associations.

PURPOSE OF REVIEW: Recent years have seen great strides made in the field of viral metagenomics. Many studies have reported alterations in the virome in different disease states. The vast majority of the human intestinal virome consists of bacteriophages, viruses that infect bacteria. The dynamic relationship between gut bacterial populations and bacteriophages is influenced by environmental factors that also impact host health and disease. In this review, we focus on studies highlighting the dynamics of the gut virome and fluctuations associated with disease states. RECENT FINDINGS: Novel correlations have been identified between the human gut virome and diseases such as obesity, necrotizing enterocolitis and severe acute respiratory syndrome coronavirus 2 infection. Further associations between the virome and cognition, diet and geography highlight the complexity of factors that can influence the dynamic relationship between gut bacteria, bacteriophages and health. SUMMARY: Here, we highlight some novel associations between the virome and health that will be the foundation for future studies in this field. The future development of microbiome-based interventions, identification of biomarkers, and novel therapeutics will require a thorough understanding of the gut virome and its dynamics.