Guidance for creating individual and batch latinized binomial virus species names.

The International Committee on Taxonomy of Viruses recently adopted, and is gradually implementing, a binomial naming format for virus species. Although full Latinization of these names remains optional, a standardized nomenclature based on Latinized binomials has the advantage of comparability with all other biological taxonomies. As a language without living native speakers, Latin is more culturally neutral than many contemporary languages, and words built from Latin roots are already widely used in the language of science across the world. Conversion of established species names to Latinized binomials or creation of Latinized binomials de novo may seem daunting, but the rules for name creation are straightforward and can be implemented in a formulaic manner. Here, we describe approaches, strategies and steps for creating Latinized binomials for virus species without prior knowledge of Latin. We also discuss a novel approach to the automated generation of large batches of novel genus and species names. Importantly, conversion to a binomial format does not affect virus names, many of which are created from local languages.

The lactococcal phages Tuc2009 and TP901-1 incorporate two alternate forms of their tail fiber into their virions for infection specialization.

Lactococcal phages Tuc2009 and TP901-1 possess a conserved tail fiber called a tail-associated lysin (referred to as Tal(2009) for Tuc2009, and Tal(901-1) for TP901-1), suspended from their tail tips that projects a peptidoglycan hydrolase domain toward a potential host bacterium. Tal(2009) and Tal(901-1) can undergo proteolytic processing mid-protein at the glycine-rich sequence GG(S/N)SGGG, removing their C-terminal structural lysin. In this study, we show that the peptidoglycan hydrolase of these Tal proteins is an M23 peptidase that exhibits D-Ala-D-Asp endopeptidase activity and that this activity is required for efficient infection of stationary phase cells. Interestingly, the observed proteolytic processing of Tal(2009) and Tal(901-1) facilitates increased host adsorption efficiencies of the resulting phages. This represents, to the best of our knowledge, the first example of tail fiber proteolytic processing that results in a heterogeneous population of two phage types. Phages that possess a full-length tail fiber, or a truncated derivative, are better adapted to efficiently infect cells with an extensively cross-linked cell wall or infect with increased host-adsorption efficiencies, respectively.

Isolation and characterization of a novel lytic Parabacteroides distasonis bacteriophage φPDS1 from the human gut.

The human gut microbiome plays a significant role in health and disease. The viral component (virome) is predominantly composed of bacteriophages (phages) and has received significantly less attention in comparison to the bacteriome. This knowledge gap is largely due to challenges associated with the isolation and characterization of novel gut phages, and bioinformatic hurdles such as the lack of a universal phage marker gene and the absence of sufficient numbers of homologs in viral databases. Here, we describe the isolation from human feces of a novel lytic phage with siphovirus morphology, φPDS1, infecting Parabacteroides distasonis APCS2/PD, and classified within a newly proposed Sagittacolavirus genus. In silico and biological characterization of this phage is presented in this study. Key to the isolation of φPDS1 was the antibiotic-driven selective enrichment of the bacterial host in a fecal fermenter. Despite producing plaques and lacking genes associated with lysogeny, φPDS1 demonstrates the ability to coexist in liquid culture for multiple days without affecting the abundance of its host. Multiple studies have shown that changes in Parabacteroides distasonis abundance can be linked to various disease states, rendering this novel phage-host pair and their interactions of particular interest.

Incorporating plasmid biology and metagenomics into a holistic model of the human gut microbiome.

The human gut microbiome is often described as the collection of bacteria, archaea, fungi, protists, and viruses associated with an individual, with no acknowledgement of the plasmid constituents. However, like viruses, plasmids are autonomous intracellular replicating entities that can influence the genotype and phenotype of their host and mediate trans-kingdom interactions. Plasmids are frequently noted as vehicles for horizontal gene transfer and for spreading antibiotic resistance, yet their multifaceted contribution to mutualistic and antagonistic interactions within the human microbiome and impact on human health is overlooked. In this review, we highlight the importance of plasmids and their biological properties as overlooked components of microbiomes. Subsequent human microbiome studies should include dedicated analyses of plasmids, particularly as a holistic understanding of human-microbial interactions is required before effective and safe interventions can be implemented to improve human well-being.

An IBD-associated pathobiont synergises with NSAID to promote colitis which is blocked by NLRP3 inflammasome and Caspase-8 inhibitors.

Conflicting evidence exists on the association between consumption of non-steroidal anti-inflammatory drugs (NSAIDs) and symptomatic worsening of inflammatory bowel disease (IBD). We hypothesized that the heterogeneous prevalence of pathobionts [e.g., adherent-invasive Escherichia coli (AIEC)], might explain this inconsistent NSAIDs/IBD correlation. Using IL10-/- mice, we found that NSAID aggravated colitis in AIEC-colonized animals. This was accompanied by activation of the NLRP3 inflammasome, Caspase-8, apoptosis, and pyroptosis, features not seen in mice exposed to AIEC or NSAID alone, revealing an AIEC/NSAID synergistic effect. Inhibition of NLRP3 or Caspase-8 activity ameliorated colitis, with reduction in NLRP3 inflammasome activation, cell death markers, activated T-cells and macrophages, improved histology, and increased abundance of Clostridium cluster XIVa species. Our findings provide new insights into how NSAIDs and an opportunistic gut-pathobiont can synergize to worsen IBD symptoms. Targeting the NLRP3 inflammasome or Caspase-8 could be a potential therapeutic strategy in IBD patients with gut inflammation, which is worsened by NSAIDs.

Interpersonal variability of the human gut virome confounds disease signal detection in IBD.

Viruses are increasingly recognised as important components of the human microbiome, fulfilling numerous ecological roles including bacterial predation, immune stimulation, genetic diversification, horizontal gene transfer, microbial interactions, and augmentation of metabolic functions. However, our current view of the human gut virome is tainted by previous sequencing requirements that necessitated the amplification of starting nucleic acids. In this study, we performed an original longitudinal analysis of 40 healthy control, 19 Crohn's disease, and 20 ulcerative colitis viromes over three time points without an amplification bias, which revealed and highlighted the interpersonal individuality of the human gut virome. In contrast to a 16 S rRNA gene analysis of matched samples, we show that α- and ß-diversity metrics of unamplified viromes are not as efficient at discerning controls from patients with inflammatory bowel disease. Additionally, we explored the intrinsic properties of unamplified gut viromes and show there is considerable interpersonal variability in viral taxa, infrequent longitudinal persistence of intrapersonal viruses, and vast fluctuations in the abundance of temporal viruses. Together, these properties of unamplified faecal viromes confound the ability to discern disease associations but significantly advance toward an unbiased and accurate representation of the human gut virome.

RNA-Seq of untreated wastewater to assess COVID-19 and emerging and endemic viruses for public health surveillance.

Background: The COVID-19 pandemic showcased the power of genomic sequencing to tackle the emergence and spread of infectious diseases. However, metagenomic sequencing of total microbial RNAs in wastewater has the potential to assess multiple infectious diseases simultaneously and has yet to be explored. Methods: A retrospective RNA-Seq epidemiological survey of 140 untreated composite wastewater samples was performed across urban (n = 112) and rural (n = 28) areas of Nagpur, Central India. Composite wastewater samples were prepared by pooling 422 individual grab samples collected prospectively from sewer lines of urban municipality zones and open drains of rural areas from 3rd February to 3rd April 2021, during the second COVID-19 wave in India. Samples were pre-processed and total RNA was extracted prior to genomic sequencing. Findings: This is the first study that has utilised culture and/or probe-independent unbiased RNA-Seq to examine Indian wastewater samples. Our findings reveal the detection of zoonotic viruses including chikungunya, Jingmen tick and rabies viruses, which have not previously been reported in wastewater. SARS-CoV-2 was detectable in 83 locations (59%), with stark abundance variations observed between sampling sites. Hepatitis C virus was the most frequently detected infectious virus, identified in 113 locations and co-occurring 77 times with SARS-CoV-2; and both were more abundantly detected in rural areas than urban zones. Concurrent identification of segmented virus genomic fragments of influenza A virus, norovirus, and rotavirus was observed. Geographical differences were also observed for astrovirus, saffold virus, husavirus, and aichi virus that were more prevalent in urban samples, while the zoonotic viruses chikungunya and rabies, were more abundant in rural environments. Interpretation: RNA-Seq can effectively detect multiple infectious diseases simultaneously, facilitating geographical and epidemiological surveys of endemic viruses that could help direct healthcare interventions against emergent and pre-existent infectious diseases as well as cost-effectively and qualitatively characterising the health status of the population over time. Funding: UK Research and Innovation (UKRI) Global Challenges Research Fund (GCRF) grant number H54810, as supported by Research England.

Viral biogeography of the mammalian gut and parenchymal organs.

The mammalian virome has been linked to health and disease but our understanding of how it is structured along the longitudinal axis of the mammalian gastrointestinal tract (GIT) and other organs is limited. Here, we report a metagenomic analysis of the prokaryotic and eukaryotic virome occupying luminal and mucosa-associated habitats along the GIT, as well as parenchymal organs (liver, lung and spleen), in two representative mammalian species, the domestic pig and rhesus macaque (six animals per species). Luminal samples from the large intestine of both mammals harboured the highest loads and diversity of bacteriophages (class Caudoviricetes, family Microviridae and others). Mucosal samples contained much lower viral loads but a higher proportion of eukaryotic viruses (families Astroviridae, Caliciviridae, Parvoviridae). Parenchymal organs contained bacteriophages of gut origin, in addition to some eukaryotic viruses. Overall, GIT virome composition was specific to anatomical region and host species. Upper GIT and mucosa-specific viruses were greatly under-represented in distal colon samples (a proxy for faeces). Nonetheless, certain viral and phage species were ubiquitous in all samples from the oral cavity to the distal colon. The dataset and its accompanying methodology may provide an important resource for future work investigating the biogeography of the mammalian gut virome.

Progress and prospects of the healthy human gut virome.

Not all viruses associated with humans cause disease. Non-pathogenic human-infecting viruses are predicted as important for immune system induction and preparation. Phages that infect bacteria are the most abundant predators of the human microbial ecosystem, promoting and maintaining bacterial diversity. Metagenomic analyses of the human gut virome and microbiome are unravelling the intricate predator-prey dynamics of phage-bacteria co-existence, co-evolution, and their interplay with the human host. While most adults harbour a distinctly individualistic and persistent community of virulent phages, new-borns are dominated by temperate phages heavily influenced by environmental exposures. The future development of microbiome-based interventions, therapeutics, and manipulation, will require a greater understanding of the human microbiome and the virome.

Biases in Viral Metagenomics-Based Detection, Cataloguing and Quantification of Bacteriophage Genomes in Human Faeces, a Review.

The human gut is colonised by a vast array of microbes that include bacteria, viruses, fungi, and archaea. While interest in these microbial entities has largely focused on the bacterial constituents, recently the viral component has attracted more attention. Metagenomic advances, compared to classical isolation procedures, have greatly enhanced our understanding of the composition, diversity, and function of viruses in the human microbiome (virome). We highlight that viral extraction methodologies are crucial in terms of identifying and characterising communities of viruses infecting eukaryotes and bacteria. Different viral extraction protocols, including those used in some of the most significant human virome publications to date, have introduced biases affecting their a overall conclusions. It is important that protocol variations should be clearly highlighted across studies, with the ultimate goal of identifying and acknowledging biases associated with different protocols and, perhaps, the generation of an unbiased and standardised method for examining this portion of the human microbiome.

Isolation and characterisation of ΦcrAss002, a crAss-like phage from the human gut that infects Bacteroides xylanisolvens.

BACKGROUND: The gut phageome comprises a complex phage community of thousands of individual strains, with a few highly abundant bacteriophages. CrAss-like phages, which infect bacteria of the order Bacteroidales, are the most abundant bacteriophage family in the human gut and make an important contribution to an individual's core virome. Based on metagenomic data, crAss-like phages form a family, with four sub-families and ten candidate genera. To date, only three representatives isolated in pure culture have been reported: ΦcrAss001 and two closely related phages DAC15 and DAC17; all are members of the less abundant candidate genus VI. The persistence at high levels of both crAss-like phage and their Bacteroidales hosts in the human gut has not been explained mechanistically, and this phage-host relationship can only be properly studied with isolated phage-host pairs from as many genera as possible. RESULTS: Faeces from a healthy donor with high levels of crAss-like phage was used to initiate a faecal fermentation in a chemostat, with selected antibiotics chosen to inhibit rapidly growing bacteria and selectively enrich for Gram-negative Bacteroidales. This had the objective of promoting the simultaneous expansion of crAss-like phages on their native hosts. The levels of seven different crAss-like phages expanded during the fermentation, indicating that their hosts were also present in the fermenter. The enriched supernatant was then tested against individual Bacteroidales strains isolated from the same faecal sample. This resulted in the isolation of a previously uncharacterised crAss-like phage of candidate genus IV of the proposed Alphacrassvirinae sub-family, ΦcrAss002, that infects the gut commensal Bacteroides xylanisolvens. ΦcrAss002 does not form plaques or spots on lawns of sensitive cells, nor does it lyse liquid cultures, even at high titres. In keeping with the co-abundance of phage and host in the human gut, ΦcrAss002 and Bacteroides xylanisolvens can also co-exist at high levels when co-cultured in laboratory media. CONCLUSIONS: We report the isolation and characterisation of ΦcrAss002, the first representative of the proposed Alphacrassvirinae sub-family of crAss-like phages. ΦcrAss002 cannot form plaques or spots on bacterial lawns but can co-exist with its host, Bacteroides xylanisolvens, at very high levels in liquid culture without impacting on bacterial numbers. Video abstract.

Alpha-synuclein alters the faecal viromes of rats in a gut-initiated model of Parkinson's disease.

Parkinson's disease (PD) is a chronic neurological disorder associated with the misfolding of alpha-synuclein (α-syn) into aggregates within nerve cells that contribute to their neurodegeneration. Recent evidence suggests α-syn aggregation may begin in the gut and travel to the brain along the vagus nerve, with microbes potentially a trigger initiating α-syn misfolding. However, the effects α-syn alterations on the gut virome have not been investigated. In this study, we show longitudinal faecal virome changes in rats administered either monomeric or preformed fibrils (PFF) of α-syn directly into their enteric nervous system. Differential changes in rat viromes were observed when comparing monomeric and PFF α-syn, with alterations compounded by the addition of LPS. Changes in rat faecal viromes were observed after one month and did not resolve within the study's five-month observational period. These results suggest that virome alterations may be reactive to host α-syn changes that are associated with PD development.

Leviviricetes: expanding and restructuring the taxonomy of bacteria-infecting single-stranded RNA viruses.

The vast majority of described prokaryotic viruses have double-stranded or single-stranded DNA or double-stranded RNA genomes. Until 2020, a mere four prokaryotic single-stranded, positive-sense RNA viruses have been classified in two genera (Riboviria; Lenarviricota; Allassoviricetes; Leviviridae). Several recent metagenomic and metatranscriptomic studies revealed a vastly greater diversity of these viruses in prokaryotic soil communities than ever anticipated. Phylogenetic analysis of these newly discovered viruses prompted the reorganization of class Allassoviricetes, now renamed Leviviricetes, to include two orders, Norzivirales and Timlovirales, and a total of six families, 428 genera and 882 species. Here we outline the new taxonomy of Leviviricetes, approved and ratified in 2021 by the International Committee on Taxonomy of Viruses, and describe open-access hidden Markov models to accommodate the anticipated identification and future classification of hundreds, if not thousands, of additional class members into this new taxonomic framework.

Stability of the human gut virome and effect of gluten-free diet.

The human gut microbiome consists of bacteria, archaea, eukaryotes, and viruses. The gut viruses are relatively underexplored. Here, we longitudinally analyzed the gut virome composition in 11 healthy adults: its stability, variation, and the effect of a gluten-free diet. Using viral enrichment and a de novo assembly-based approach, we demonstrate the quantitative dynamics of the gut virome, including dsDNA, ssDNA, dsRNA, and ssRNA viruses. We observe highly divergent individual viral communities, carrying on an average 2,143 viral genomes, 13.1% of which were present at all 3 time points. In contrast to previous reports, the Siphoviridae family dominates over Microviridae in studied individual viromes. We also show individual viromes to be stable at the family level but to vary substantially at the genera and species levels. Finally, we demonstrate that lower initial diversity of the human gut virome leads to a more pronounced effect of the dietary intervention on its composition.

Isolation of a Novel Jumbo Bacteriophage Effective Against Klebsiella aerogenes.

Increasing levels of bacterial resistance to many common and last resort antibiotics has increased interest in finding new treatments. The low rate of approval of new antibiotics has led to the search for new and alternative antimicrobial compounds. Bacteriophages (phages) are bacterial viruses found in almost every environment. Phage therapy was historically investigated to control bacterial infections and is still in use in Georgia and as a treatment of last resort. Phage therapy is increasingly recognized as an alternative antimicrobial treatment for antibiotic resistant pathogens. A novel lytic Klebsiella aerogenes phage N1M2 was isolated from maize silage. Klebsiella aerogenes, a member of the ESKAPE bacterial pathogens, is an important target for new antimicrobial therapies. Klebsiella aerogenes can form biofilms on medical devices which aids its environmental persistence and for this reason we tested the effect of phage N1M2 against biofilms. Phage N1M2 successfully removed a pre-formed Klebsiella aerogenes biofilm. Biofilm assays were also carried out with Staphylococcus aureus and Phage K. Phage K successfully removed a preformed Staphylococcus aureus biofilm. Phage N1M2 and Phage K in combination were significantly better at removing a mixed community biofilm of Klebsiella aerogenes and Staphylococcus aureus than either phage alone.

Diverse Bacteriocins Produced by Strains From the Human Milk Microbiota.

Microbial colonization of the infant gut is a convoluted process dependent on numerous contributing factors, including age, mode of delivery and diet among others that has lifelong implication for human health. Breast milk also contains a microbiome which acts as a source of colonizing bacteria for the infant. Here, we demonstrate that human milk harbors a wide diversity of bacteriocin-producing strains with the potential to compete among the developing gut microbiota of the infant. We screened 37 human milk samples and found isolates with antimicrobial activity and distinct cross-immunity profiles. From these isolates, we detected 73 putative gene clusters for bacteriocins of all known sub-classes, including 16 novel prepeptides. More specifically, we detected two novel lantibiotics, four sactibiotics and three class IIa bacteriocins with an unusual modification of the pediocin box that is composed of YDNGI instead of the highly conserved motif YGNGV. Moreover, we identified a novel class IIb bacteriocin, four novel class IIc and two class IId bacteriocins. In conclusion, human milk contains a variety of bacteriocin-producing strains which may provide them a competitive advantage in the colonization of the infant gut and suggests that the milk microbiota is a source of antimicrobial potential.

Metagenomics reveals impact of geography and acute diarrheal disease on the Central Indian human gut microbiome.

BACKGROUND: The Central Indian gut microbiome remains grossly understudied. Herein, we sought to investigate the burden of antimicrobial resistance and diarrheal diseases, particularly Clostridioides difficile, in rural-agricultural and urban populations in Central India, where there is widespread unregulated antibiotic use. We utilized shotgun metagenomics to comprehensively characterize the bacterial and viral fractions of the gut microbiome and their encoded functions in 105 participants. RESULTS: We observed distinct rural-urban differences in bacterial and viral populations, with geography exhibiting a greater influence than diarrheal status. Clostridioides difficile disease was more commonly observed in urban subjects, and their microbiomes were enriched in metabolic pathways relating to the metabolism of industrial compounds and genes encoding resistance to 3rd generation cephalosporins and carbapenems. By linking phages present in the microbiome to their bacterial hosts through CRISPR spacers, phage variation could be directly related to shifts in bacterial populations, with the auxiliary metabolic potential of rural-associated phages enriched for carbon and amino acid energy metabolism. CONCLUSIONS: We report distinct differences in antimicrobial resistance gene profiles, enrichment of metabolic pathways and phage composition between rural and urban populations, as well as a higher burden of Clostridioides difficile disease in the urban population. Our results reveal that geography is the key driver of variation in urban and rural Indian microbiomes, with acute diarrheal disease, including C. difficile disease exerting a lesser impact. Future studies will be required to understand the potential role of dietary, cultural, and genetic factors in contributing to microbiome differences between rural and urban populations.

RNA Phage Biology in a Metagenomic Era.

The number of novel bacteriophage sequences has expanded significantly as a result of many metagenomic studies of phage populations in diverse environments. Most of these novel sequences bear little or no homology to existing databases (referred to as the "viral dark matter"). Also, these sequences are primarily derived from DNA-encoded bacteriophages (phages) with few RNA phages included. Despite the rapid advancements in high-throughput sequencing, few studies enrich for RNA viruses, i.e., target viral rather than cellular fraction and/or RNA rather than DNA via a reverse transcriptase step, in an attempt to capture the RNA viruses present in a microbial communities. It is timely to compile existing and relevant information about RNA phages to provide an insight into many of their important biological features, which should aid in sequence-based discovery and in their subsequent annotation. Without comprehensive studies, the biological significance of RNA phages has been largely ignored. Future bacteriophage studies should be adapted to ensure they are properly represented in phageomic studies.

Complete Genome Sequence of Escherichia coli Phage APC_JM3.2 Isolated from a Chicken Cecum.

Avian pathogenic Escherichia coli (APEC) bacteria are a significant challenge to the poultry industry. Bacteriophages (phages) have the potential to control APEC strains, increasing animal welfare and economic productivity. Here, we report the isolation of an E. coli-infecting phage, APC_JM3.2, isolated from the cecum of a broiler chicken in Ireland.

Biology and Taxonomy of crAss-like Bacteriophages, the Most Abundant Virus in the Human Gut.

CrAssphages represent the most abundant virus in the human gut microbiota, but the lack of available genome sequences for comparison has kept them enigmatic. Recently, sequence-based classification of distantly related crAss-like phages from multiple environments was reported, leading to a proposed familial-level taxonomic group. Here, we assembled the metagenomic sequencing reads from 702 human fecal virome/phageome samples and analyzed 99 complete circular crAss-like phage genomes and 150 contigs ≥70 kb. In silico comparative genomics and taxonomic analysis enabled a classification scheme of crAss-like phages from human fecal microbiomes into four candidate subfamilies composed of ten candidate genera. Laboratory analysis was performed on fecal samples from an individual harboring seven distinct crAss-like phages. We achieved crAss-like phage propagation in ex vivo human fecal fermentations and visualized short-tailed podoviruses by electron microscopy. Mass spectrometry of a crAss-like phage capsid protein could be linked to metagenomic sequencing data, confirming crAss-like phage structural annotations.