Entourage: all-in-one sequence analysis software for genome assembly, virus detection, virus discovery, and intrasample variation profiling.

BACKGROUND: Pan-virus detection, and virome investigation in general, can be challenging, mainly due to the lack of universally conserved genetic elements in viruses. Metagenomic next-generation sequencing can offer a promising solution to this problem by providing an unbiased overview of the microbial community, enabling detection of any viruses without prior target selection. However, a major challenge in utilising metagenomic next-generation sequencing for virome investigation is that data analysis can be highly complex, involving numerous data processing steps. RESULTS: Here, we present Entourage to address this challenge. Entourage enables short-read sequence assembly, viral sequence search with or without reference virus targets using contig-based approaches, and intrasample sequence variation quantification. Several workflows are implemented in Entourage to facilitate end-to-end virus sequence detection analysis through a single command line, from read cleaning, sequence assembly, to virus sequence searching. The results generated are comprehensive, allowing for thorough quality control, reliability assessment, and interpretation. We illustrate Entourage's utility as a streamlined workflow for virus detection by employing it to comprehensively search for target virus sequences and beyond in raw sequence read data generated from HeLa cell culture samples spiked with viruses. Furthermore, we showcase its flexibility and performance on a real-world dataset by analysing a preassembled Tara Oceans dataset. Overall, our results show that Entourage performs well even with low virus sequencing depth in single digits, and it can be used to discover novel viruses effectively. Additionally, by using sequence data generated from a patient with chronic SARS-CoV-2 infection, we demonstrate Entourage's capability to quantify virus intrasample genetic variations, and generate publication-quality figures illustrating the results. CONCLUSIONS: Entourage is an all-in-one, versatile, and streamlined bioinformatics software for virome investigation, developed with a focus on ease of use. Entourage is available at https://codeberg.org/CENMIG/Entourage under the MIT license.

Genomic Interactions Between Mycobacterium tuberculosis and Humans.

Mycobacterium tuberculosis is considered by many to be the deadliest microbe, with the estimated annual cases numbering more than 10 million. The bacteria, including Mycobacterium africanum, are classified into nine major lineages and hundreds of sublineages, each with different geographical distributions and levels of virulence. The phylogeographic patterns can be a result of recent and early human migrations as well as coevolution between the bacteria and various human populations, which may explain why many studies on human genetic factors contributing to tuberculosis have not been replicable in different areas. Moreover, several studies have revealed the significance of interactions between human genetic variations and bacterial genotypes in determining the development of tuberculosis, suggesting coadaptation. The increased availability of whole-genome sequence data from both humans and bacteria has enabled a better understanding of these interactions, which can inform the development of vaccines and other control measures.

SARS-CoV-2 variant with the spike protein mutation F306L in the southern border provinces of Thailand.

The southernmost part of Thailand is a unique and culturally diverse region that has been greatly affected by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) outbreak during the coronavirus disease-2019 pandemic. To gain insights into this situation, we analyzed 1942 whole-genome sequences of SARS-CoV-2 obtained from the five southernmost provinces of Thailand between April 2021 and March 2022, together with those publicly available in the Global Initiative on Sharing All Influenza Data database. Our analysis revealed evidence for transboundary transmissions of the virus in and out of the five southernmost provinces during the study period, from both domestic and international sources. The most prevalent viral variant in our sequence dataset was the Delta B.1.617.2.85 variant, also known as the Delta AY.85 variant, with many samples carrying a non-synonymous mutation F306L in their spike protein. Protein-protein docking and binding interface analyses suggested that the mutation may enhance the binding between the spike protein and host cell receptor protein angiotensin-converting enzyme 2, and we found that the mutation was significantly associated with an increased fatality rate. This mutation has also been observed in other SARS-CoV-2 variants, suggesting that it is of particular interest and should be monitored.

Spatiotemporal evolution of SARS-CoV-2 in the Bangkok metropolitan region, Thailand, 2020-2022: implications for future outbreak preparedness.

Thailand experienced five waves of coronavirus disease 2019 (COVID-19) between 2020 and 2022, with the Bangkok Metropolitan Region (BMR) being at the centre of all outbreaks. The molecular evolution of the causative agent of the disease, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has previously been characterized in Thailand, but a detailed spatiotemporal analysis is still lacking. In this study, we comprehensively reviewed the development and timelines of the five COVID-19 outbreaks in Thailand and the public health responses, and also conducted a phylogenetic analysis of 27 913 SARS-CoV-2 genomes from Thailand, together with 7330 global references, to investigate the virus's spatiotemporal evolution during 2020 and 2022, with a particular focus on the BMR. Limited cross-border transmission was observed during the first four waves in 2020 and 2021, but was common in 2022, aligning well with the timeline of change in the international travel restrictions. Within the country, viruses were mostly restricted to the BMR during the first two waves in 2020, but subsequent waves in 2021 and 2022 saw extensive nationwide transmission of the virus, consistent with the timeline of relaxation of disease control measures employed within the country. Our results also suggest frequent epidemiological connections between Thailand and neighbouring countries during 2020 and 2021 despite relatively stringent international travel controls. The overall sequencing rate of the viruses circulating in the BMR was ~0.525 %, meeting the recommended benchmark, and our analysis supports that this is sufficient for tracking of the trend of the virus burden and genetic diversity. Our findings reveal insights into the local transmission dynamics of SARS-CoV-2 in Thailand, and provide a valuable reference for planning responses to future outbreaks.

Analysis of whiB7 in Mycobacterium tuberculosis reveals novel AT-hook deletion mutations.

Mutations in whiB7 have been associated with both hypersusceptibility and resistance to various antibiotics in Mycobacterium tuberculosis (Mtb). Unlocking the secrets of antibiotic resistance in the bacterium, we examined mutations in the coding sequences of whiB7 of over 40,000 diverse Mtb isolates. Our results unveil the dominant c.191delG (Gly64delG) mutation, present in all members of the lineage L1.2.2 and its impact on WhiB7's conserved GVWGG-motif, causing conformational changes and deletion of the C-terminal AT-hook. Excitingly, we discovered six unique mutations associated with partial or total deletion of the AT-hook, specific to certain sublineages. Our findings suggest the selective pressures driving these mutations, underlining the potential of genomics to advance our understanding of Mtb's antibiotic resistance. As tuberculosis remains a global health threat, our study offers valuable insights into the diverse nature and functional consequences of whiB7 mutations, paving the way for the development of novel therapeutic interventions.

Four principles to establish a universal virus taxonomy.

A universal taxonomy of viruses is essential for a comprehensive view of the virus world and for communicating the complicated evolutionary relationships among viruses. However, there are major differences in the conceptualisation and approaches to virus classification and nomenclature among virologists, clinicians, agronomists, and other interested parties. Here, we provide recommendations to guide the construction of a coherent and comprehensive virus taxonomy, based on expert scientific consensus. Firstly, assignments of viruses should be congruent with the best attainable reconstruction of their evolutionary histories, i.e., taxa should be monophyletic. This fundamental principle for classification of viruses is currently included in the International Committee on Taxonomy of Viruses (ICTV) code only for the rank of species. Secondly, phenotypic and ecological properties of viruses may inform, but not override, evolutionary relatedness in the placement of ranks. Thirdly, alternative classifications that consider phenotypic attributes, such as being vector-borne (e.g., "arboviruses"), infecting a certain type of host (e.g., "mycoviruses," "bacteriophages") or displaying specific pathogenicity (e.g., "human immunodeficiency viruses"), may serve important clinical and regulatory purposes but often create polyphyletic categories that do not reflect evolutionary relationships. Nevertheless, such classifications ought to be maintained if they serve the needs of specific communities or play a practical clinical or regulatory role. However, they should not be considered or called taxonomies. Finally, while an evolution-based framework enables viruses discovered by metagenomics to be incorporated into the ICTV taxonomy, there are essential requirements for quality control of the sequence data used for these assignments. Combined, these four principles will enable future development and expansion of virus taxonomy as the true evolutionary diversity of viruses becomes apparent.

Systematic Exploration of SARS-CoV-2 Adaptation to Vero E6, Vero E6/TMPRSS2, and Calu-3 Cells.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to spread globally, and scientists around the world are currently studying the virus intensively in order to fight against the on-going pandemic of the virus. To do so, SARS-CoV-2 is typically grown in the lab to generate viral stocks for various kinds of experimental investigations. However, accumulating evidence suggests that such viruses often undergo cell culture adaptation. Here, we systematically explored cell culture adaptation of two SARS-CoV-2 variants, namely the B.1.36.16 variant and the AY.30 variant, a sub lineage of the B.1.617.2 (Delta) variant, propagated in three different cell lines, including Vero E6, Vero E6/TMPRSS2, and Calu-3 cells. Our analyses detected numerous potential cell culture adaptation changes scattering across the entire virus genome, many of which could be found in naturally circulating isolates. Notable ones included mutations around the spike glycoprotein's multibasic cleavage site, and the Omicron-defining H655Y mutation on the spike glycoprotein, as well as mutations in the nucleocapsid protein's linker region, all of which were found to be Vero E6-specific. Our analyses also identified deletion mutations on the non-structural protein 1 and membrane glycoprotein as potential Calu-3-specific adaptation changes. S848C mutation on the non-structural protein 3, located to the protein's papain-like protease domain, was also identified as a potential adaptation change, found in viruses propagated in all three cell lines. Our results highlight SARS-CoV-2 high adaptability, emphasize the need to deep-sequence cultured viral samples when used in intricate and sensitive biological experiments, and illustrate the power of experimental evolutionary study in shedding lights on the virus evolutionary landscape.

Target Enrichment Metagenomics Reveals Human Pegivirus-1 in Pediatric Hematopoietic Stem Cell Transplantation Recipients.

Human pegivirus-1 (HPgV-1) is a lymphotropic human virus, typically considered nonpathogenic, but its infection can sometimes cause persistent viremia both in immunocompetent and immunosuppressed individuals. In a viral discovery research program in hematopoietic stem cell transplant (HSCT) pediatric patients, HPgV-1 was detected in 3 out of 14 patients (21.4%) using a target enrichment next-generation sequencing method, and the presence of the viruses was confirmed by agent-specific qRT-PCR assays. For the first time in this patient cohort, complete genomes of HPgV-1 were acquired and characterized. Phylogenetic analyses indicated that two patients had HPgV-1 genotype 2 and one had HPgV-1 genotype 3. Intra-host genomic variations were described and discussed. Our results highlight the necessity to screen HSCT patients and blood and stem cell donors to reduce the potential risk of HPgV-1 transmission.

Genomic epidemiology of Streptococcus agalactiae ST283 in Southeast Asia.

Streptococcus agalactiae, also known as Lancefield Group B Streptococcus (GBS), is typically regarded as a neonatal pathogen; however, several studies have shown that the bacteria are capable of causing invasive diseases in non-pregnant adults as well. The majority of documented cases were from Southeast Asian countries, and the most common genotype found was ST283, which is also known to be able to infect fish. This study sequenced 12 GBS ST283 samples collected from adult patients in Thailand. Together with publicly available sequences, we performed temporo-spatial analysis and estimated population dynamics of the bacteria. Putative drug resistance genes were also identified and characterized, and the drug resistance phenotypes were validated experimentally. The results, together with historical records, draw a detailed picture of the past transmission history of GBS ST283 in Southeast Asia.

SARS-CoV-2 genetic variations associated with COVID-19 pathogenicity.

In this study, we performed genome-wide association analyses on SARS-CoV-2 genomes to identify genetic mutations associated with pre-symptomatic/asymptomatic COVID-19 cases. Various potential covariates and confounding factors of COVID-19 severity, including patient age, gender and country, as well as virus phylogenetic relatedness were adjusted for. In total, 3021 full-length genomes of SARS-CoV-2 generated from original clinical samples and whose patient status could be determined conclusively as either 'pre-symptomatic/asymptomatic' or 'symptomatic' were retrieved from the GISAID database. We found that the mutation 11 083G>T, located in the coding region of non-structural protein 6, is significantly associated with asymptomatic COVID-19. Patient age is positively correlated with symptomatic infection, while gender is not significantly correlated with the development of the disease. We also found that the effects of the mutation, patient age and gender do not vary significantly among countries, although each country appears to have varying baseline chances of COVID-19 symptom development.

Diversity, taxonomy, and evolution of archaeal viruses of the class Caudoviricetes.

The archaeal tailed viruses (arTV), evolutionarily related to tailed double-stranded DNA (dsDNA) bacteriophages of the class Caudoviricetes, represent the most common isolates infecting halophilic archaea. Only a handful of these viruses have been genomically characterized, limiting our appreciation of their ecological impacts and evolution. Here, we present 37 new genomes of haloarchaeal tailed virus isolates, more than doubling the current number of sequenced arTVs. Analysis of all 63 available complete genomes of arTVs, which we propose to classify into 14 new families and 3 orders, suggests ancient divergence of archaeal and bacterial tailed viruses and points to an extensive sharing of genes involved in DNA metabolism and counterdefense mechanisms, illuminating common strategies of virus-host interactions with tailed bacteriophages. Coupling of the comparative genomics with the host range analysis on a broad panel of haloarchaeal species uncovered 4 distinct groups of viral tail fiber adhesins controlling the host range expansion. The survey of metagenomes using viral hallmark genes suggests that the global architecture of the arTV community is shaped through recurrent transfers between different biomes, including hypersaline, marine, and anoxic environments.

Transcriptional response to the host cell environment of a multidrug-resistant Mycobacterium tuberculosis clonal outbreak Beijing strain reveals its pathogenic features.

Tuberculosis is a global public health problem with emergence of multidrug-resistant infections. Previous epidemiological studies of tuberculosis in Thailand have identified a clonal outbreak multidrug-resistant strain of Mycobacterium tuberculosis in the Kanchanaburi province, designated "MKR superspreader", and this particular strain later was found to also spread to other regions. In this study, we elucidated its biology through RNA-Seq analyses and identified a set of genes involved in cholesterol degradation to be up-regulated in the MKR during the macrophage cell infection, but not in the H37Rv reference strain. We also found that the bacterium up-regulated genes associated with the ESX-1 secretion system during its intracellular growth phase, while the H37Rv did not. All results were confirmed by qRT-PCR. Moreover, we showed that compounds previously shown to inhibit the mycobacterial ESX-1 secretion system and cholesterol utilisation, and FDA-approved drugs known to interfere with the host cholesterol transportation were able to decrease the intracellular survival of the MKR when compared to the untreated control, while not that of the H37Rv. Altogether, our findings suggested that such pathways are important for the MKR's intracellular growth, and potentially could be targets for the discovery of new drugs against this emerging multidrug-resistant strain of M. tuberculosis.

Tilapia lake virus (TiLV): Genomic epidemiology and its early origin.

Tilapia lake virus (TiLV) is an emerging virus that is rapidly spreading across the world. Over the past 6 years (2014-2020), TiLV outbreaks had been reported in at least 16 countries, spanning three continents, including Asia, Africa, and America. Despite its enormous economic impact, its origin, evolution and epidemiology are still largely poorly characterized. Here, we report eight TiLV whole-genome sequences from Thailand sampled between 2014 and 2019. Together with publicly available sequences from various regions of the world, we estimated the origin of TiLV to be between 2003 and 2009, 5-10 years before the first report of the virus in Israel in 2014. Our analyses consistently showed that TiLV started to spread in 2000s, and reached its peak in 2014-2016, matching well with the timing of its first report. From 2016 onwards, the global TiLV population declined steadily. This could be a result of herd immunity building up in the fish population, and/or a reflection of a better awareness of the virus coupled with a better and more cautious protocol of Tilapia importation. Despite the fact that we included all publicly available sequences, our analyses revealed long unsampled histories of TiLVs in many countries, especially towards its basal diversification. This result highlights the lack and the need for systematic surveillance of TiLV in fish.

Early origin and global colonisation of foot-and-mouth disease virus.

In this study, we compiled 84-year worth (1934-2017) of genomic and epidemiological data of foot-and-mouth disease virus (FMDV), and performed comprehensive analyses to determine its early origin and transmission route. We found that recombination is a key feature of FMDV, and that the genomic regions coding for structural and non-structural proteins have markedly different evolutionary histories, and evolve at different rates. Despite all of these differences, analyses of both structural and non-structural protein coding regions consistently suggested that the most recent common ancestor of FMDV could be dated back to the Middle Age, ~ 200 to 300 years earlier than previously thought. The ancestors of the Euro-Asiatic and SAT strains could be dated back to the mid-seventeenth century, and to the mid-fifteenth to mid-sixteenth century, respectively. Our results implicated Mediterranean counties as an early geographical origin of FMDV before spreading to Europe and subsequently to Asia and South America.

Erratum: Avian and serpentine endogenous foamy viruses, and new insights into the macroevolutionary history of foamy viruses.

[This corrects the article DOI: 10.1093/ve/vez057.][This corrects the article DOI: 10.1093/ve/vez057.].

Avian and serpentine endogenous foamy viruses, and new insights into the macroevolutionary history of foamy viruses.

This study reports and characterises two novel distinct lineages of foamy viruses (FVs) in the forms of endogenous retroviruses (ERVs). Several closely related elements were found in the genome of oriental stork (Ciconia boyciana) and other was found in the genome of spine-bellied sea snake (Hydrophis hardwickii), designated ERV-Spuma.N-Cbo (where 'N' runs from one to thirteen) and ERV-Spuma.1-Hha, respectively. This discovery of avian and serpentine endogenous FVs adds snakes, and perhaps more crucially, birds to the list of currently known hosts of FVs, in addition to mammals, reptiles, amphibians, and fish. This indicates that FVs are, or at least were, capable of infecting all major lineages of vertebrates. Moreover, together with other FVs, phylogenetic analyses showed that both of them are most closely related to mammalian FVs. Further examination revealed that reptilian FVs form a deep paraphyletic group that is basal to mammalian and avian FVs, suggesting that there were multiple ancient FV cross-class transmissions among their hosts. Evolutionary timescales of various FV lineages were estimated in this study, in particular, the timescales of reptilian FVs and that of the clade of mammalian, avian, and serpentine FVs. This was accomplished by using the recently established time-dependent rate phenomenon models, inferred using mainly the knowledge of the co-speciation history between FVs and mammals. It was found that the estimated timescales matched very well with those of reptiles. Combined with the observed phylogenetic patterns, these results suggested that FVs likely co-speciated with ancient reptilian animals, but later jumped to a protomammal and/or a bird, which ultimately gave rise to mammalian and avian FVs. These results contribute to our understanding of FV emergence, specifically the emergence of mammalian and avian FVs, and provide new insights into how FVs co-evolved with their non-mammalian vertebrate hosts in the distant past.

Analysis of Spounaviruses as a Case Study for the Overdue Reclassification of Tailed Phages.

Tailed bacteriophages are the most abundant and diverse viruses in the world, with genome sizes ranging from 10 kbp to over 500 kbp. Yet, due to historical reasons, all this diversity is confined to a single virus order-Caudovirales, composed of just four families: Myoviridae, Siphoviridae, Podoviridae, and the newly created Ackermannviridae family. In recent years, this morphology-based classification scheme has started to crumble under the constant flood of phage sequences, revealing that tailed phages are even more genetically diverse than once thought. This prompted us, the Bacterial and Archaeal Viruses Subcommittee of the International Committee on Taxonomy of Viruses (ICTV), to consider overall reorganization of phage taxonomy. In this study, we used a wide range of complementary methods-including comparative genomics, core genome analysis, and marker gene phylogenetics-to show that the group of Bacillus phage SPO1-related viruses previously classified into the Spounavirinae subfamily, is clearly distinct from other members of the family Myoviridae and its diversity deserves the rank of an autonomous family. Thus, we removed this group from the Myoviridae family and created the family Herelleviridae-a new taxon of the same rank. In the process of the taxon evaluation, we explored the feasibility of different demarcation criteria and critically evaluated the usefulness of our methods for phage classification. The convergence of results, drawing a consistent and comprehensive picture of a new family with associated subfamilies, regardless of method, demonstrates that the tools applied here are particularly useful in phage taxonomy. We are convinced that creation of this novel family is a crucial milestone toward much-needed reclassification in the Caudovirales order.

Modular nature of simian foamy virus genomes and their evolutionary history.

Among all known retroviruses, foamy viruses (FVs) have the most stable virus-host co-speciation history, co-diverging in concert with their vertebrate hosts for hundreds of millions of years. However, detailed molecular analyses indicate that different parts of their genome might have different evolutionary histories. While their polymerase gene displays a robust and straightforward virus-host co-speciation pattern, the evolutionary history of their envelope (env) gene, is much more complicated. Here, we report eleven new FV env sequences in two mandrill populations in Central Africa, geographically separated by the Ogooué River into the North and the South populations. Phylogenetic reconstruction of the polymerase gene shows that the two virus populations are distinct, and each contains two variants of env genes co-existing with one another. The distinction between the two env variants can be mapped to the surface domain, flanked by two recombination hotspots, as previously reported for chimpanzee and gorilla FVs. Our analyses suggest that the two env variants originated during the diversification of Old World monkeys and apes, ∼30 million years ago. We also show that this env gene region forms two phylogenetically distinct clades, each displaying a host co-divergence and geographical separation pattern, while the rest of the genome of the two strains is phylogenetically indistinguishable in each of the host-specific groups. We propose possible evolutionary mechanisms to explain the modular nature of the FV genome.