Genomic Analyses of Major SARS-CoV-2 Variants Predicting Multiple Regions of Pathogenic and Transmissive Importance.

The rapid evolution of SARS-CoV-2 has fueled its global proliferation since its discovery in 2019, with several notable variants having been responsible for increases in cases of coronavirus disease 2019 (COVID-19). Analyses of codon bias and usage in these variants between phylogenetic clades or lineages may grant insights into the evolution of SARS-CoV-2 and identify target codons indicative of evolutionary or mutative trends that may prove useful in tracking or defending oneself against emerging strains. We processed a cohort of 120 SARS-CoV-2 genome sequences through a statistical and bioinformatic pipeline to identify codons presenting evidence of selective pressure as well as codon coevolution. We report the identification of two codon sites in the orf8 and N genes demonstrating such evidence with real-world impacts on pathogenicity and transmissivity.

Genomic and Proteomic Analysis of Six Vi01-like Phages Reveals Wide Host Range and Multiple Tail Spike Proteins.

Enterobacteriaceae is a large family of Gram-negative bacteria composed of many pathogens, including Salmonella and Shigella. Here, we characterize six bacteriophages that infect Enterobacteriaceae, which were isolated from wastewater plants in the Wasatch front (Utah, United States). These phages are highly similar to the Kuttervirus vB_SenM_Vi01 (Vi01), which was isolated using wastewater from Kiel, Germany. The phages vary little in genome size and are between 157 kb and 164 kb, which is consistent with the sizes of other phages in the Vi01-like phage family. These six phages were characterized through genomic and proteomic comparison, mass spectrometry, and both laboratory and clinical host range studies. While their proteomes are largely unstudied, mass spectrometry analysis confirmed the production of five hypothetical proteins, several of which unveiled a potential operon that suggests a ferritin-mediated entry system on the Vi01-like phage family tail. However, no dependence on this pathway was observed for the single host tested herein. While unable to infect every genus of Enterobacteriaceae tested, these phages are extraordinarily broad ranged, with several demonstrating the ability to infect Salmonella enterica and Citrobacter freundii strains with generally high efficiency, as well as several clinical Salmonella enterica isolates, most likely due to their multiple tail fibers.

Complete genome sequences of five Ackermannviridae that infect Enterobacteriaceae hosts.

This announcement contains the whole genome sequences of five Ackermannviridae that infect members of the Enterobacteriaceae family of bacteria. Four of the five phages were isolated using Salmonella enterica serovar Typhimurium as a bacterial host: AR2819, Sajous1, SilasIsHot, and FrontPhageNews. ChubbyThor was isolated using Shigella boydii.

Genome sequences of 13 Bacillus anthracis Sterne bacteriophages isolated from soil in the western United States.

Bacillus anthracis, classified as a Tier 1 Select Agent by the Centers for Disease Control and Prevention (CDC), is the causative agent of anthrax in both humans and livestock. Herein, we report the full genome sequences of 13 bacteriophages that infect B. anthracis Sterne. These phages are grouped into four clusters and are similar to previously described Bacillus phages.

Diversity and conservation of the genome architecture of phages infecting the Alphaproteobacteria.

IMPORTANCE: This study reports the results of the largest analysis of genome sequences from phages that infect the Alphaproteobacteria class of bacterial hosts. We analyzed over 100 whole genome sequences of phages to construct dotplots, categorize them into genetically distinct clusters, generate a bootstrapped phylogenetic tree, compute protein orthologs, and predict packaging strategies. We determined that the phage sequences primarily cluster by the bacterial host family, phage morphotype, and genome size. We expect that the findings reported in this seminal study will facilitate future analyses that will improve our knowledge of the phages that infect these hosts.

Characterization of the Attachment of Three New Coliphages onto the Ferrichrome Transporter FhuA.

Receptor-binding proteins (RBPs) allow phages to dock onto their host and initiate infection through the recognition of proteinaceous or saccharidic receptors located on the cell surface. FhuA is the ferrichrome hydroxamate transporter in Escherichia coli and serves as a receptor for the well-characterized phages T1, T5, and phi80. To further characterize how other FhuA-dependent phages attach to FhuA, we isolated and published the genomes of three new FhuA-dependent coliphages: JLBYU37, JLBYU41, and JLBYU60. We identified the egions of FhuA involved in phage attachment by testing the effect of mutant fhuA alleles containing single-loop deletions of extracellular loops (L3, L4, L5, L8, L10, and L11) on phage infectivity. Deletion of loop 8 resulted in complete resistance to SO1-like phages JLBYU37 and JLBYU60 and the previously isolated vB_EcoD_Teewinot phage, but no single-loop deletions significantly altered the infection of T1-like JLBYU41. Additionally, lipopolysaccharide (LPS) truncation coupled with the L5 mutant significantly impaired the infectivity of JLBYU37 and JLBYU60. Moreover, significant reductions in the infectivity of JLBYU41 were observed upon LPS truncation in the L8 mutant strain. Analysis of the evolutionary relationships among FhuA-dependent phage RBPs highlights the conservation of L8 dependence in JLBYU37, JLBYU60, Teewinot, T5, and phi80, but also showcases how positive selective pressure and/or homologous recombination also selected for L4 dependence in T1 and even the lack of complete loop dependence in JLBYU41. IMPORTANCE Phage attachment is the first step of phage infection and plays a role in governing host specificity. Characterizing the interactions taking place between phage tail fibers and bacterial receptors that better equip bacteria to survive within the human body may provide insights to aid the development of phage therapeutics.

Identifying the core genome of the nucleus-forming bacteriophage family and characterization of Erwinia phage RAY.

We recently discovered that some bacteriophages establish a nucleus-like replication compartment (phage nucleus), but the core genes that define nucleus-based phage replication and their phylogenetic distribution were still to be determined. Here, we show that phages encoding the major phage nucleus protein chimallin share 72 conserved genes encoded within seven gene blocks. Of these, 21 core genes are unique to nucleus-forming phage, and all but one of these genes encode proteins of unknown function. We propose that these phages comprise a novel viral family we term Chimalliviridae. Fluorescence microscopy and cryoelectron tomography studies of Erwinia phage vB_EamM_RAY confirm that many of the key steps of nucleus-based replication are conserved among diverse chimalliviruses and reveal variations on this replication mechanism. This work expands our understanding of phage nucleus and PhuZ spindle diversity and function, providing a roadmap for identifying key mechanisms underlying nucleus-based phage replication.

Identifying the core genome of the nucleus-forming bacteriophage family and characterization of Erwinia phage RAY.

We recently discovered that some bacteriophages establish a nucleus-like replication compartment (phage nucleus), but the core genes that define nucleus-based phage replication and their phylogenetic distribution were unknown. By studying phages that encode the major phage nucleus protein chimallin, including previously sequenced yet uncharacterized phages, we discovered that chimallin-encoding phages share a set of 72 highly conserved genes encoded within seven distinct gene blocks. Of these, 21 core genes are unique to this group, and all but one of these unique genes encode proteins of unknown function. We propose that phages with this core genome comprise a novel viral family we term Chimalliviridae. Fluorescence microscopy and cryo-electron tomography studies of Erwinia phage vB_EamM_RAY confirm that many of the key steps of nucleus-based replication encoded in the core genome are conserved among diverse chimalliviruses, and reveal that non-core components can confer intriguing variations on this replication mechanism. For instance, unlike previously studied nucleus-forming phages, RAY doesn't degrade the host genome, and its PhuZ homolog appears to form a five-stranded filament with a lumen. This work expands our understanding of phage nucleus and PhuZ spindle diversity and function, providing a roadmap for identifying key mechanisms underlying nucleus-based phage replication.

The small genome, virulent, non-contractile tailed bacteriophages that infect Enterobacteriales hosts.

Tailed bacteriophages are abundant and extremely diverse. Understanding this diversity is a challenge, and here we examine a small slice of that diversity in some detail. We contrast and compare the small genome, virulent, non-contractile tailed phages that infect the bacterial order Enterobacteriales. These phages, with genomes in the 35-60 kbp range, have very similar virions that are often difficult to distinguish by negative stain electron microscopy. There are currently 651 genome sequences of such phages in the public database. We show that these can be robustly parsed into fifteen well-defined clusters that have very different nucleotide sequences. We examine the similarities and differences among these clusters, as well as genetic exchange among clusters and the relationships between host species and phage clusters.

Genome Sequences of 14 Siphophages That Infect Serratia marcescens.

We announce the complete genome sequences of 14 Serratia bacteriophages isolated from wastewater treatment plants. These phages define two previously undescribed types which we call the Carrot-like phage cluster (phages Carrot, BigDog, LittleDog, Niamh, Opt-148, Opt-169, PhooPhighters, Rovert, Serratianator, Stoker, Swain, and Ulliraptor) and Tlacuache-like phage cluster (Tlacuache and Opt-155).

Complete Genome Sequences of Five Bacteriophages That Infect Enterobacteriales Hosts.

Full genome sequences of five bacteriophages that were isolated from raw sewage samples and infect Enterobacteriales hosts are presented. Brookers is a P22-like Proteus phage, OddieOddie is a 9g-like Escherichia coli phage, Diencephelon is a Kp3-like Klebsiella phage, and Rgz1 and Lilpapawes are classic T4-like and T7-like virulent Proteus phages, respectively.

Complete Genome Sequences of Six Chi-Like Bacteriophages That Infect Proteus and Klebsiella.

Proteus mirabilis and Klebsiella aerogenes are Gram-negative opportunistic pathogens that are responsible for nosocomial and health care-associated infections, including urinary tract infections. Here, the full genome sequences of six Chi-like Proteus (DanisaurMW, DoubleBarrel, Inception, Jing313, and NotEvenPhaged) or Klebsiella (Phraden) bacteriophages are announced, contributing to the understanding of Chi-like phages.

Complete Genome Sequences of Five SO-1-Like Siphoviridae Bacteriophages That Infect Enterobacteriales.

The Enterobacteriales order is composed of Gram-negative bacteria that range from harmless symbionts to well-studied pathogens. We announce complete genome sequences of five related SO-1-like Enterobacteriales bacteriophages (also known as the Dhillonvirus genus) isolated from wastewater that infect Escherichia coli (Opt-212, Over9000, Pubbukkers, and Teewinot) or Shigella boydii (StarDew).

Presence and stability of SARS-CoV-2 on environmental currency and money cards in Utah reveals a lack of live virus.

The highly contagious nature of SARS-CoV-2 has led to several studies on the transmission of the virus. A little studied potential fomite of great concern in the community is currency, which has been shown to harbor microbial pathogens in several studies. Since the onset of the COVID-19 pandemic, many businesses in the United States have limited the use of banknotes in favor of credit cards. However, SARS-CoV-2 has shown greater stability on plastic in several studies. Herein, the stability of SARS-CoV-2 at room temperature on banknotes, money cards and coins was investigated. In vitro studies with live virus suggested SARS-CoV-2 was highly unstable on banknotes, showing an initial rapid reduction in viable virus and no viral detection by 24 hours. In contrast, SARS-CoV-2 displayed increased stability on money cards with live virus detected after 48 hours. Environmental swabbing of currency and money cards on and near the campus of Brigham Young University supported these results, with no detection of SARS-CoV-2 RNA on banknotes, and a low level on money cards. However, no viable virus was detected on either. These preliminary results suggest that the use of money cards over banknotes in order to slow the spread of this virus may be ill-advised. These findings should be investigated further through larger environmental studies involving more locations.

Gut Microbiota Regulates the Interaction between Diet and Genetics to Influence Glucose Tolerance.

Background: Metabolic phenotypes are the result of an intricate interplay between multiple factors, including diet, genotype, and the gut microbiome. Per-Arnt-Sim (PAS) kinase is a nutrient-sensing serine/threonine kinase, whose absence (PASK-/-) protects against triglyceride accumulation, insulin resistance, and weight gain on a high-fat diet; conditions that are associated with dysbiosis of the gut microbiome. Methods: Herein, we report the metabolic effects of the interplay of diet (high fat high sugar, HFHS), genotype (PASK-/-), and microbiome (16S sequencing). Results: Microbiome analysis identified a diet-induced, genotype-independent forked shift, with two discrete clusters of HFHS mice having increased beta and decreased alpha diversity. A "lower" cluster contained elevated levels of Firmicutes, Bacteroidetes, Actinobacteria, Proteobacteria and Defferibacteres, and was associated with increased weight gain, glucose intolerance, triglyceride accumulation, and decreased claudin-1 expression. Genotypic effects were observed within the clusters, lower cluster PASK-/- mice displayed increased weight gain and decreased triglyceride accumulation, whereas upper PASK-/- were resistant to decreased claudin-1. Conclusions: These results confirm previous reports that PAS kinase deficiency can protect mice against the deleterious effects of diet, and they suggest that microbiome imbalances can override protection. In addition, these results support a healthy diet for beneficial microbiome maintenance and suggest microbial culprits associated with metabolic disease.

The Use of Bacteriophages and Immunological Monitoring for the Treatment of a Case of Chronic Septicemic Cutaneous Ulcerative Disease in a Loggerhead Sea Turtle Caretta caretta.

In this case study, phage therapy was applied to treat a multidrug-resistant case of septicemic cutaneous ulcerative disease (SCUD) caused by Citrobacter freundii in a loggerhead sea turtle Caretta caretta. Phages were applied topically, intravenously, into the carapace, and into the exhibit water using various phage cocktails specific to the causative agent over an 8-month period. This was performed in conjunction with antimicrobial therapy. The animal was monitored through weekly cultures, photographs, and complete blood cell counts, as well as immune assays (phagocytosis, plasma lysozyme and superoxide dismutase activity, and plasma electrophoresis profiles). The animal, in comparison to an untreated, unaffected control, had elevated antibody titers to the administered phages, which persisted for at least 35 weeks. Although cultures were clear of C. freundii after phage treatment, the infection did return over time and immune assays confirmed deficiencies when compared to a healthy loggerhead sea turtle. Immune parameters with statistically significant changes over the study period included the following: decreased phagocytosis, increased alpha- and gamma-globulin protein components, and an increased albumin : globulin ratio. When C. freundii appeared again, the multidrug-resistant status had reverted back to normal susceptibility patterns. Although not completely known whether it was another subspecies of bacteria, the therapy did resolve the multidrug-resistant challenge. Phage therapy in combination with antimicrobial agents may be an effective treatment for sea turtles with normally functioning immune systems or less-severe infections. Additional research is needed to better understand and quantify sea turtle immunology.

Atypical chemokine receptor ACKR2-V41A has decreased CCL2 binding, scavenging, and activation, supporting sustained inflammation and increased Alzheimer's disease risk.

A recent genome-wide association study (GWAS) of 59 cerebrospinal fluid (CSF) proteins with a connection to Alzheimer's disease (AD) demonstrated an association between increased levels of chemokine ligand 2 (CCL2) with an atypical chemokine receptor chemokine-binding protein 2 variant V41A (ACKR2-V41A; rs2228467). High levels of CCL2 are associated with increased risk of AD development as well as other inflammatory diseases. In this study we characterized the biological function of the ACKR2-V41A receptor compared to the wild type allele by measuring its ligand binding affinity, CCL2 scavenging efficiency, and cell activation sensitivity. We transfected Chinese hamster ovary cells with plasmids carrying wild type ACKR2 (ACKR2-WT) or the mutant ACKR2-V41A receptor. Binding affinity assays showed that ACKR2-V41A has a lower binding affinity for CCL2 and CCL4 than ACKR2-WT. CCL2 scavenging results aligned with binding affinity assays, with ACKR2-V41A cells scavenging CCL2 with a lower efficiency than ACKR2-WT. Cell activation assays also showed that ACKR2-V41A cells had significantly lower receptor upregulation (ß-Arrestin-dependent signaling pathway) upon stimulation compared to ACKR2-WT cells. These findings provide molecular and biological mechanistic insights into the GWAS association of ACKR2-V41A with increased levels of CCL2 in CSF and possibly other chemokine ligands. Increased CCL2 levels are associated with accelerated cognitive decline and increased risk of AD. Understanding how this atypical chemokine receptor allele increases serum markers of inflammation could lead to novel therapeutic solutions for AD.

Genome Sequences of 12 Phages That Infect Klebsiella pneumoniae.

Klebsiella pneumoniae is a pathogen responsible for significant proportions of nosocomial and health care-associated infections and is known to acquire multiple antibiotic resistance genes. Here, we announce the full genome sequences of 12 K. pneumoniae bacteriophages from samples collected in wastewater treatment facilities across the western United States.

Genome Sequences of Two Bacillus Phages Isolated from Indonesia.

Here, the genome sequences of two soil bacteriophages isolated from a red chili plantation in Indonesia are presented. The genome of vB_BspS_SplendidRed (42,859 bp) is highly similar to Bacillus phage Ray17 from the United States, while vB_BspM_MarvelLand (156,945 bp) is highly similar to Bacillus phage BC01 from South Korea.

A Novel, Highly Related Jumbo Family of Bacteriophages That Were Isolated Against Erwinia.

Erwinia amylovora is a plant pathogen from the Erwiniaceae family and a causative agent of the devastating agricultural disease fire blight. Here we characterize eight lytic bacteriophages of E. amylovora that we isolated from the Wasatch front (Utah, United States) that are highly similar to vB_EamM_Ea35-70 which was isolated in Ontario, Canada. With the genome size ranging from 271 to 275 kb, this is a novel jumbo family of bacteriophages. These jumbo bacteriophages were further characterized through genomic and proteomic comparison, mass spectrometry, host range and burst size. Their proteomes are highly unstudied, with over 200 putative proteins with no known homologs. The production of 27 of these putative proteins was confirmed by mass spectrometry analysis. These bacteriophages appear to be most similar to bacteriophages that infect Pseudomonas and Ralstonia rather than Enterobacteriales bacteria by protein similarity, however, we were only able to detect infection of Erwinia and the closely related strains of Pantoea.