Benchmarking bioinformatic virus identification tools using real-world metagenomic data across biomes.

BACKGROUND: As most viruses remain uncultivated, metagenomics is currently the main method for virus discovery. Detecting viruses in metagenomic data is not trivial. In the past few years, many bioinformatic virus identification tools have been developed for this task, making it challenging to choose the right tools, parameters, and cutoffs. As all these tools measure different biological signals, and use different algorithms and training and reference databases, it is imperative to conduct an independent benchmarking to give users objective guidance. RESULTS: We compare the performance of nine state-of-the-art virus identification tools in thirteen modes on eight paired viral and microbial datasets from three distinct biomes, including a new complex dataset from Antarctic coastal waters. The tools have highly variable true positive rates (0-97%) and false positive rates (0-30%). PPR-Meta best distinguishes viral from microbial contigs, followed by DeepVirFinder, VirSorter2, and VIBRANT. Different tools identify different subsets of the benchmarking data and all tools, except for Sourmash, find unique viral contigs. Performance of tools improved with adjusted parameter cutoffs, indicating that adjustment of parameter cutoffs before usage should be considered. CONCLUSIONS: Together, our independent benchmarking facilitates selecting choices of bioinformatic virus identification tools and gives suggestions for parameter adjustments to viromics researchers.

A Single Electrochemical Biosensor Designed to Detect Any Virus.

Viruses are the primary cause of many infectious diseases in both humans and animals. Various testing methods require an amplification step of the viral RNA sample before detection, with quantitative reverse transcription polymerase chain reaction (RT-qPCR) being one of the most widely used along with lesser-known methods like Nucleic Acid Sequence-Based Amplification (NASBA). NASBA offers several advantages, such as isothermal amplification and high selectivity for specific sequences, making it an attractive option for low-income facilities. In this research, we employed a single electrochemical biosensor (E-Biosensor) designed for potentially detecting any virus by modifying the NASBA protocol. In this modified protocol, a reverse primer is designed with an additional 22-nucleotide sequence (tag region) at the 5'-end, which is added to the NASBA process. This tag region becomes part of the final amplicon generated by NASBA. It can hybridize with a single specific E-Biosensor probe set, enabling subsequent virus detection. Using this approach, we successfully detected three different viruses with a single E-Biosensor design, demonstrating the platform's potential for virus detection.

Development of a High-throughput Sequencing Platform for Detection of Viral Encephalitis Pathogens Based on Amplicon Sequencing.

Objective: Viral encephalitis is an infectious disease severely affecting human health. It is caused by a wide variety of viral pathogens, including herpes viruses, flaviviruses, enteroviruses, and other viruses. The laboratory diagnosis of viral encephalitis is a worldwide challenge. Recently, high-throughput sequencing technology has provided new tools for diagnosing central nervous system infections. Thus, In this study, we established a multipathogen detection platform for viral encephalitis based on amplicon sequencing. Methods: We designed nine pairs of specific polymerase chain reaction (PCR) primers for the 12 viruses by reviewing the relevant literature. The detection ability of the primers was verified by software simulation and the detection of known positive samples. Amplicon sequencing was used to validate the samples, and consistency was compared with Sanger sequencing. Results: The results showed that the target sequences of various pathogens were obtained at a coverage depth level greater than 20×, and the sequence lengths were consistent with the sizes of the predicted amplicons. The sequences were verified using the National Center for Biotechnology Information BLAST, and all results were consistent with the results of Sanger sequencing. Conclusion: Amplicon-based high-throughput sequencing technology is feasible as a supplementary method for the pathogenic detection of viral encephalitis. It is also a useful tool for the high-volume screening of clinical samples.

Advances in application of CRISPR-Cas13a system.

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) and CRISPR-associated (Cas) proteins serve as an adaptive immune system that safeguards prokaryotes and some of the viruses that infect prokaryotes from foreign nucleic acids (such as viruses and plasmids). The genomes of the majority of archaea and about half of all bacteria contain various CRISPR-Cas systems. CRISPR-Cas systems depend on CRISPR RNAs (crRNAs). They act as a navigation system to specifically cut and destroy foreign nucleic acids by recognizing invading foreign nucleic acids and binding Cas proteins. In this review, we provide a brief overview of the evolution and classification of the CRISPR-Cas system, focusing on the functions and applications of the CRISPR-Cas13a system. We describe the CRISPR-Cas13a system and discuss its RNA-directed ribonuclease function. Meanwhile, we briefly introduce the mechanism of action of the CRISPR-Cas13a system and summarize the applications of the CRISPR-Cas13a system in pathogen detection, eukaryotes, agriculture, biosensors, and human gene therapy. We are right understanding of CRISPR-Cas13a has been broadened, and the CRISPR-Cas13a system will be useful for developing new RNA targeting tools. Therefore, understanding the basic details of the structure, function, and biological characterization of CRISPR-Cas13a effector proteins is critical for optimizing RNA targeting tools.

Predictors of severity and prolonged hospital stay of viral acute respiratory infections (ARI) among children under five years in Burkina Faso, 2016-2019.

BACKGROUND: Viruses are the leading etiology of acute respiratory infections (ARI) in children. However, there is limited knowledge on drivers of severe acute respiratory infection (SARI) cases involving viruses. We aimed to identify factors associated with severity and prolonged hospitalization of viral SARI among children < 5 years in Burkina Faso. METHODS: Data were collected from four SARI sentinel surveillance sites during October 2016 through April 2019. A SARI case was a child < 5 years with an acute respiratory infection with history of fever or measured fever ≥ 38 °C and cough with onset within the last ten days, requiring hospitalization. Very severe ARI cases required intensive care or had at least one danger sign. Oropharyngeal/nasopharyngeal specimens were collected and analyzed by multiplex real-time reverse-transcription polymerase chain reaction (rRT-PCR) using FTD-33 Kit. For this analysis, we included only SARI cases with rRT-PCR positive test results for at least one respiratory virus. We used simple and multilevel logistic regression models to assess factors associated with very severe viral ARI and viral SARI with prolonged hospitalization. RESULTS: Overall, 1159 viral SARI cases were included in the analysis after excluding exclusively bacterial SARI cases (n = 273)very severe viral ARI cases were common among children living in urban areas (AdjOR = 1.3; 95% CI: 1.1-1.6), those < 3 months old (AdjOR = 1.5; 95% CI: 1.1-2.3), and those coinfected with Klebsiella pneumoniae (AdjOR = 1.9; 95% CI: 1.2-2.2). Malnutrition (AdjOR = 2.2; 95% CI: 1.1-4.2), hospitalization during the rainy season (AdjOR = 1.71; 95% CI: 1.2-2.5), and infection with human CoronavirusOC43 (AdjOR = 3; 95% CI: 1.2-8) were significantly associated with prolonged length of hospital stay (> 7 days). CONCLUSION: Younger age, malnutrition, codetection of Klebsiella pneumoniae, and illness during the rainy season were associated with very severe cases and prolonged hospitalization of SARI involving viruses in children under five years. These findings emphasize the need for preventive actions targeting these factors in young children.

Does the Recovery of Respiratory Viruses Impact Pulmonary Function at Baseline and 1-, 6-, and 12-Month Follow-Up in People Living with HIV and Pneumonia?

The frequency of respiratory viruses in people living with HIV (PLHIV) and their impact on lung function remain unclear. We aimed to determine the frequency of respiratory viruses in bronchoalveolar lavage and induced sputum samples in PLHIV and correlate their presence with lung function. A prospective cohort of adults hospitalized in Medellín between September 2016 and December 2018 included three groups: group 1 = people diagnosed with HIV and a diagnosis of community-acquired pneumonia (CAP), group 2 = HIV, and group 3 = CAP. People were followed up with at months 1, 6, and 12. Clinical, microbiological, and spirometric data were collected. Respiratory viruses were detected by multiplex RT-PCR. Sixty-five patients were included. At least 1 respiratory virus was identified in 51.9%, 45.1%, and 57.1% of groups 1, 2 and 3, respectively. Among these, 89% of respiratory viruses were detected with another pathogen, mainly Mycobacterium tuberculosis (40.7%) and Pneumocystis jirovecii (22.2%). The most frequent respiratory virus was rhinovirus (24/65, 37%). On admission, 30.4% of group 1, 16.6% of group 2, and 50% of group 3 had airflow limitation, with alteration in forced expiratory volume at first second in both groups with pneumonia compared to HIV. Respiratory viruses are frequent in people diagnosed with HIV, generally coexisting with other pathogens. Pulmonary function on admission was affected in patients with pneumonia, improving significantly in the 1st, 6th, and 12th months after CAP onset.

Evolution of Virology: Science History through Milestones and Technological Advancements.

The history of virology, which is marked by transformative breakthroughs, spans microbiology, biochemistry, genetics, and molecular biology. From the development of Jenner's smallpox vaccine in 1796 to 20th-century innovations such as ultrafiltration and electron microscopy, the field of virology has undergone significant development. In 1898, Beijerinck laid the conceptual foundation for virology, marking a pivotal moment in the evolution of the discipline. Advancements in influenza A virus research in 1933 by Richard Shope furthered our understanding of respiratory pathogens. Additionally, in 1935, Stanley's determination of viruses as solid particles provided substantial progress in the field of virology. Key milestones include elucidation of reverse transcriptase by Baltimore and Temin in 1970, late 20th-century revelations linking viruses and cancer, and the discovery of HIV by Sinoussi, Montagnier, and Gallo in 1983, which has since shaped AIDS research. In the 21st century, breakthroughs such as gene technology, mRNA vaccines, and phage display tools were achieved in virology, demonstrating its potential for integration with molecular biology. The achievements of COVID-19 vaccines highlight the adaptability of virology to global health.

Novel Viral Vectors for Gene Therapy.

Viral vectors are gene transfer tools assembled from the backbones of naturally occurring viruses [...].

Duplex Droplet Digital PCR Assay for Quantification of Hepatitis E Virus in Food.

Hepatitis E virus (HEV) represents an emerging risk in industrialized countries where the consumption of contaminated food plays a pivotal role. Quantitative real-time RT-PCR (RT-qPCR) is one of the most suitable methods for the detection and quantification of viruses in food. Nevertheless, quantification using RT-qPCR has limitations. Droplet digital PCR (ddPCR) provides the precise quantification of nucleic acids without the need for a standard curve and a reduction in the effect on virus quantification due to the presence of inhibitors. The objectives of the present work were (i) to develop a method for the absolute quantification of HEV in swine tissues based on ddPCR technology and provide internal process control for recovery assessment and (ii) to evaluate the performance of the method by analyzing a selection of naturally contaminated wild boar muscle samples previously tested using RT-qPCR. The method was optimized using a set of in vitro synthesized HEV RNA and quantified dsDNA. The limit of detection of the developed ddPCR assay was 0.34 genome copies/µL. The analysis of the wild boar samples confirmed the validity of the ddPCR assay. The duplex ddPCR method showed no reduction in efficiency compared to individual assays. The method developed in the present study could represent a sensitive assay for the detection and absolute quantification of HEV RNA in food samples with the advantage of presenting the co-amplification of internal process control.

Identification and Characterisation of a Myxoma Virus Detected in the Italian Hare (Lepus corsicanus).

Myxoma virus (MYXV) is a Leporipoxvirus (genus) belonging to the family Poxviridae; it is characterised by a genome of approximately 161 kb dsDNA encoding for several proteins that play an essential role in both host spectrum determination and immunomodulation. The healthy reservoir of the virus is Sylvilagus spp. At the same time, in wild and domestic European rabbits (Oryctolagus cuniculus), MYXV is the etiologic agent of myxomatosis, a disease with an extremely high mortality rate. In 2014, an interspecies jump of MYXV was reported in Lepus europaeus in the UK. In 2018, myxomatosis induced by a new recombinant strain called MYXV-To was identified during a large outbreak in Iberian hares (Lepus granatensis) in Spain. Here, we describe the case of myxomatosis in another hare species: an adult male Italian hare (Lepus corsicanus) found dead in 2018 in Sicily with lesions suggestive of myxomatosis and treponema infection. Laboratory tests, e.g., end-point PCR and negative staining electron microscopy, confirmed the presence of both pathogens. MYXV was then isolated from tissue samples in permissive cells and sequenced using NGS technology. Main genomic differences concerning known MYXV strains are discussed.

A Balancing Act: The Viral-Host Battle over RNA Binding Proteins.

A defining feature of a productive viral infection is the co-opting of host cell resources for viral replication. Despite the host repertoire of molecular functions and biological counter measures, viruses still subvert host defenses to take control of cellular factors such as RNA binding proteins (RBPs). RBPs are involved in virtually all steps of mRNA life, forming ribonucleoprotein complexes (mRNPs) in a highly ordered and regulated process to control RNA fate and stability in the cell. As such, the hallmark of the viral takeover of a cell is the reshaping of RNA fate to modulate host gene expression and evade immune responses by altering RBP interactions. Here, we provide an extensive review of work in this area, particularly on the duality of the formation of RNP complexes that can be either pro- or antiviral. Overall, in this review, we highlight the various ways viruses co-opt RBPs to regulate RNA stability and modulate the outcome of infection by gathering novel insights gained from research studies in this field.

Host-like RNA Elements Regulate Virus Translation.

Viruses are obligate, intracellular parasites that co-opt host cell machineries for propagation. Critical among these machineries are those that translate RNA into protein and their mechanisms of control. Most regulatory mechanisms effectuate their activity by targeting sequence or structural features at the RNA termini, i.e., at the 5' or 3' ends, including the untranslated regions (UTRs). Translation of most eukaryotic mRNAs is initiated by 5' cap-dependent scanning. In contrast, many viruses initiate translation at internal RNA regions at internal ribosome entry sites (IRESs). Eukaryotic mRNAs often contain upstream open reading frames (uORFs) that permit condition-dependent control of downstream major ORFs. To offset genome compression and increase coding capacity, some viruses take advantage of out-of-frame overlapping uORFs (oORFs). Lacking the essential machinery of protein synthesis, for example, ribosomes and other translation factors, all viruses utilize the host apparatus to generate virus protein. In addition, some viruses exhibit RNA elements that bind host regulatory factors that are not essential components of the translation machinery. SARS-CoV-2 is a paradigm example of a virus taking advantage of multiple features of eukaryotic host translation control: the virus mimics the established human GAIT regulatory element and co-opts four host aminoacyl tRNA synthetases to form a stimulatory binding complex. Utilizing discontinuous transcription, the elements are present and identical in all SARS-CoV-2 subgenomic RNAs (and the genomic RNA). Thus, the virus exhibits a post-transcriptional regulon that improves upon analogous eukaryotic regulons, in which a family of functionally related mRNA targets contain elements that are structurally similar but lacking sequence identity. This "thrifty" virus strategy can be exploited against the virus since targeting the element can suppress the expression of all subgenomic RNAs as well as the genomic RNA. Other 3' end viral elements include 3'-cap-independent translation elements (3'-CITEs) and 3'-tRNA-like structures. Elucidation of virus translation control elements, their binding proteins, and their mechanisms can lead to novel therapeutic approaches to reduce virus replication and pathogenicity.

Viruses exploit growth factor mechanisms to achieve augmented pathogenicity and promote tumorigenesis.

Cellular homeostasis is regulated by growth factors (GFs) which orchestrate various cellular processes including proliferation, survival, differentiation, motility, inflammation and angiogenesis. Dysregulation of GFs in microbial infections and malignancies have been reported previously. Viral pathogens exemplify the exploitation of host cell GFs and their signalling pathways contributing to viral entry, virulence, and evasion of anti-viral immune responses. Viruses can also perturb cellular metabolism and the cell cycle by manipulation of GF signaling. In some cases, this disturbance may promote oncogenesis. Viral pathogens can encode viral GF homologues and induce the endogenous biosynthesis of GFs and their corresponding receptors or manipulate their activity to infect the host cells. Close investigation of how viral strategies exploit and regulate GFs, a will shed light on how to improve anti-viral therapy and cancer treatment. In this review, we discuss and provide insights on how various viral pathogens exploit different GFs to promote viral survival and oncogenic transformation, and how this knowledge can be leveraged toward the design of more efficient therapeutics or novel drug delivery systems in the treatment of both viral infections and malignancies.

Taxonomic difference in marine bloom-forming phytoplanktonic species affects the dynamics of both bloom-responding prokaryotes and prokaryotic viruses.

The production of dissolved organic matter during phytoplankton blooms and consumption by heterotrophic prokaryotes promote marine carbon biogeochemical cycling. Although prokaryotic viruses presumably affect this process, their dynamics during blooms are not fully understood. Here, we investigated the effects of taxonomic difference in bloom-forming phytoplankton on prokaryotes and their viruses. We analyzed the dynamics of coastal prokaryotic communities and viruses under the addition of dissolved intracellular fractions from taxonomically distinct phytoplankton, the diatom Chaetoceros sp. (CIF) and the raphidophycean alga Heterosigma akashiwo (HIF), using microcosm experiments. Ribosomal RNA gene amplicon and viral metagenomic analyses revealed that particular prokaryotes and prokaryotic viruses specifically increased in either CIF or HIF, indicating that taxonomic difference in bloom-forming phytoplankton promotes distinct dynamics of not only the prokaryotic community but also prokaryotic viruses. Furthermore, combining our microcosm experiments with publicly available environmental data mining, we identified both known and novel possible host-virus pairs. In particular, the growth of prokaryotes associating with phytoplanktonic organic matter, such as Bacteroidetes (Polaribacter and NS9 marine group), Vibrio spp., and Rhodobacteriales (Nereida and Planktomarina), was accompanied by an increase in viruses predicted to infect Bacteroidetes, Vibrio, and Rhodobacteriales, respectively. Collectively, our findings suggest that changes in bloom-forming species can be followed by an increase in a specific group of prokaryotes and their viruses and that elucidating these tripartite relationships among specific phytoplankton, prokaryotes, and prokaryotic viruses improves our understanding of coastal biogeochemical cycling in blooms.IMPORTANCEThe primary production during marine phytoplankton bloom and the consumption of the produced organic matter by heterotrophic prokaryotes significantly contribute to coastal biogeochemical cycles. While the activities of those heterotrophic prokaryotes are presumably affected by viral infection, the dynamics of their viruses during blooms are not fully understood. In this study, we experimentally demonstrated that intracellular fractions of taxonomically distinct bloom-forming phytoplankton species, the diatom Chaetoceros sp. and the raphidophycean alga Heterosigma akashiwo, promoted the growth of taxonomically different prokaryotes and prokaryotic viruses. Based on their dynamics and predicted hosts of those viruses, we succeeded in detecting already-known and novel possible host-virus pairs associating with either phytoplankton species. Altogether, we propose that the succession of bloom-forming phytoplankton would change the composition of the abundant prokaryotes, resulting in an increase in their viruses. These changes in viral composition, depending on bloom-forming species, would alter the dynamics and metabolism of prokaryotes, affecting biogeochemical cycling in blooms.

Challenges to mapping and defining m6A function in viral RNA.

Viral RNA molecules contain multiple layers of regulatory information. This includes features beyond the primary sequence, such as RNA structures and RNA modifications, including N6-methyladenosine (m6A). Many recent studies have identified the presence and location of m6A in viral RNA and have found diverse regulatory roles for this modification during viral infection. However, to date, viral m6A mapping strategies have limitations that prevent a complete understanding of the function of m6A on individual viral RNA molecules. While m6A sites have been profiled on bulk RNA from many viruses, the resulting m6A maps of viral RNAs described to date present a composite picture of m6A across viral RNA molecules in the infected cell. Thus, for most viruses, it is unknown if unique viral m6A profiles exist throughout infection, nor if they regulate specific viral life cycle stages. Here, we describe several challenges to defining the function of m6A in viral RNA molecules and provide a framework for future studies to help in the understanding of how m6A regulates viral infection.

The Expanding Diversity of Viruses from Extreme Environments.

Viruses are nonliving biological entities whose host range encompasses all known forms of life. They are deceptively simple in description (a protein shell surrounding genetic material with an occasional lipid envelope) and yet can infect all known forms of life. Recently, due to technological advancements, viruses from more extreme environments can be studied through both culture-dependent and independent means. Viruses with thermophilic, halophilic, psychrophilic, and barophilic properties are highlighted in this paper with an emphasis on the properties that allow them to exist in said environments. Unfortunately, much of this field is extremely novel and thus, not much is yet known about these viruses or the microbes they infect when compared to non-extremophilic host-virus systems. With this review, we hope to shed some light on these relatively new studies and highlight their intrinsic value.

High niche specificity and host genetic diversity of groundwater viruses.

Viruses are key members of microbial communities that exert control over host abundance and metabolism, thereby influencing ecosystem processes and biogeochemical cycles. Aquifers are known to host taxonomically diverse microbial life, yet little is known about viruses infecting groundwater microbial communities. Here, we analysed 16 metagenomes from a broad range of groundwater physicochemistries. We recovered 1571 viral genomes that clustered into 468 high-quality viral operational taxonomic units. At least 15% were observed to be transcriptionally active, although lysis was likely constrained by the resource-limited groundwater environment. Most were unclassified (95%), and the remaining 5% were Caudoviricetes. Comparisons with viruses inhabiting other aquifers revealed no shared species, indicating substantial unexplored viral diversity. In silico predictions linked 22.4% of the viruses to microbial host populations, including to ultra-small prokaryotes, such as Patescibacteria and Nanoarchaeota. Many predicted hosts were associated with the biogeochemical cycling of carbon, nitrogen, and sulfur. Metabolic predictions revealed the presence of 205 putative auxiliary metabolic genes, involved in diverse processes associated with the utilization of the host's intracellular resources for biosynthesis and transformation reactions, including those involved in nucleotide sugar, glycan, cofactor, and vitamin metabolism. Viruses, prokaryotes overall, and predicted prokaryotic hosts exhibited narrow spatial distributions, and relative abundance correlations with the same groundwater parameters (e.g. dissolved oxygen, nitrate, and iron), consistent with host control over viral distributions. Results provide insights into underexplored groundwater viruses, and indicate the large extent to which viruses may manipulate microbial communities and biogeochemistry in the terrestrial subsurface.

Isolation of viruses, including mollivirus, with the potential to infect Acanthamoeba from a Japanese warm temperate zone.

Acanthamoeba castellanii is infected with diverse nucleocytoplasmic large DNA viruses. Here, we report the co-isolation of 12 viral strains from marine sediments in Uranouchi Inlet, Kochi, Japan. Based on the morphological features revealed by electron microscopy, these isolates were classified into four viral groups including Megamimiviridae, Molliviridae, Pandoraviridae, and Pithoviridae. Genomic analyses indicated that these isolates showed high similarities to the known viral genomes with which they are taxonomically clustered, and their phylogenetic relationships were also supported by core gene similarities. It is noteworthy that Molliviridae was isolated from the marine sediments in the Japanese warm temperate zone because other strains have only been found in the subarctic region. Furthermore, this strain has 19 and 4 strain-specific genes found in Mollivirus sibericum and Mollivirus kamchatka, respectively. This study extends our knowledge about the habitat and genomic diversity of Molliviridae.

High yield sterile filtration process for highly concentrated lentiviral vectors.

The development and manufacture of biopharmaceuticals are subject to strict regulations that specify the required minimum quality of the products. A key measure to meet these quality requirements is the integration of a sterile filtration step into the commercial manufacturing process. Whereas common procedures for most biologics exist, this is challenging for lentiviral vector (LVV) production for ex vivo gene therapy. LVVs nominal size is more than half the pore size (0.2 µm) of filters used for sterile filtration. Hence, highly concentrated virus solutions are prone to filter clogging if aggregation of viruses occurs or impurities attach to the viruses. Several filters were screened aiming to identify those which allow filtering highly concentrated stocks of LVVs of up to 1E + 9 transducing units mL-1 , which corresponds to 4.5E + 12 particles mL-1 . In addition, the effect of endonuclease treatment upstream of the purification process on filter performance was studied. In summary, three suitable filters were identified in a small-scale study (<15 mL) with virus yields >80% and the process was successfully scaled-up to a final scale of 100 mL LVV stock solution.

Mottle: Accurate pairwise substitution distance at high divergence through the exploitation of short-read mappers and gradient descent.

Current tools for estimating the substitution distance between two related sequences struggle to remain accurate at a high divergence. Difficulties at distant homologies, such as false seeding and over-alignment, create a high barrier for the development of a stable estimator. This is especially true for viral genomes, which carry a high rate of mutation, small size, and sparse taxonomy. Developing an accurate substitution distance measure would help to elucidate the relationship between highly divergent sequences, interrogate their evolutionary history, and better facilitate the discovery of new viral genomes. To tackle these problems, we propose an approach that uses short-read mappers to create whole-genome maps, and gradient descent to isolate the homologous fraction and calculate the final distance value. We implement this approach as Mottle. With the use of simulated and biological sequences, Mottle was able to remain stable to 0.66-0.96 substitutions per base pair and identify viral outgroup genomes with 95% accuracy at the family-order level. Our results indicate that Mottle performs as well as existing programs in identifying taxonomic relationships, with more accurate numerical estimation of genomic distance over greater divergences. By contrast, one limitation is a reduced numerical accuracy at low divergences, and on genomes where insertions and deletions are uncommon, when compared to alternative approaches. We propose that Mottle may therefore be of particular interest in the study of viruses, viral relationships, and notably for viral discovery platforms, helping in benchmarking of homology search tools and defining the limits of taxonomic classification methods. The code for Mottle is available at https://github.com/tphoward/Mottle_Repo.