SUMMARY: Genome detective is a web-based, user-friendly software application to quickly and accurately assemble all known virus genomes from next-generation sequencing datasets. This application allows the identification of phylogenetic clusters and genotypes from assembled genomes in FASTA format. Since its release in 2019, we have produced a number of typing tools for emergent viruses that have caused large outbreaks, such as Zika and Yellow Fever Virus in Brazil. Here, we present the Genome Detective Coronavirus Typing Tool that can accurately identify the novel severe acute respiratory syndrome (SARS)-related coronavirus (SARS-CoV-2) sequences isolated in China and around the world. The tool can accept up to 2000 sequences per submission and the analysis of a new whole-genome sequence will take approximately 1 min. The tool has been tested and validated with hundreds of whole genomes from 10 coronavirus species, and correctly classified all of the SARS-related coronavirus (SARSr-CoV) and all of the available public data for SARS-CoV-2. The tool also allows tracking of new viral mutations as the outbreak expands globally, which may help to accelerate the development of novel diagnostics, drugs and vaccines to stop the COVID-19 disease. AVAILABILITY AND IMPLEMENTATION: https://www.genomedetective.com/app/typingtool/cov. CONTACT: [email protected] or [email protected]. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

SUMMARY: Virus sequence data are an essential resource for reconstructing spatiotemporal dynamics of viral spread as well as to inform treatment and prevention strategies. However, the potential benefit of these applications critically depends on accurate and correctly annotated alignments of genetically heterogeneous data. VIRULIGN was built for fast codon-correct alignments of large datasets, with standardized and formalized genome annotation and various alignment export formats. AVAILABILITY AND IMPLEMENTATION: VIRULIGN is freely available at https://github.com/rega-cev/virulign as an open source software project. SUPPLEMENTARY INFORMATION: Supplementary data is available at Bioinformatics online.

SUMMARY: Genome Detective is an easy to use web-based software application that assembles the genomes of viruses quickly and accurately. The application uses a novel alignment method that constructs genomes by reference-based linking of de novo contigs by combining amino-acids and nucleotide scores. The software was optimized using synthetic datasets to represent the great diversity of virus genomes. The application was then validated with next generation sequencing data of hundreds of viruses. User time is minimal and it is limited to the time required to upload the data. AVAILABILITY AND IMPLEMENTATION: Available online: http://www.genomedetective.com/app/typingtool/virus/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

SUMMARY: RegaDB is a free and open source data management and analysis environment for infectious diseases. RegaDB allows clinicians to store, manage and analyse patient data, including viral genetic sequences. Moreover, RegaDB provides researchers with a mechanism to collect data in a uniform format and offers them a canvas to make newly developed bioinformatics tools available to clinicians and virologists through a user friendly interface. AVAILABILITY AND IMPLEMENTATION: Source code, binaries and documentation are available on http://rega.kuleuven.be/cev/regadb. RegaDB is written in the Java programming language, using a web-service-oriented architecture.

BACKGROUND: Failure on Highly Active Anti-Retroviral Treatment is often accompanied with development of antiviral resistance to one or more drugs included in the treatment. In general, the virus is more likely to develop resistance to drugs with a lower genetic barrier. Previously, we developed a method to reverse engineer, from clinical sequence data, a fitness landscape experienced by HIV-1 under nelfinavir (NFV) treatment. By simulation of evolution over this landscape, the individualized genetic barrier to NFV resistance may be estimated for an isolate. RESULTS: We investigated the association of estimated genetic barrier with risk of development of NFV resistance at virological failure, in 201 patients that were predicted fully susceptible to NFV at baseline, and found that a higher estimated genetic barrier was indeed associated with lower odds for development of resistance at failure (OR 0.62 (0.45 - 0.94), per additional mutation needed, p = .02). CONCLUSIONS: Thus, variation in individualized genetic barrier to NFV resistance may impact effective treatment options available after treatment failure. If similar results apply for other drugs, then estimated genetic barrier may be a new clinical tool for choice of treatment regimen, which allows consideration of available treatment options after virological failure.

MOTIVATION: HIV-1 antiviral resistance is a major cause of antiviral treatment failure. The in vivo fitness landscape experienced by the virus in presence of treatment could in principle be used to determine both the susceptibility of the virus to the treatment and the genetic barrier to resistance. We propose a method to estimate this fitness landscape from cross-sectional clinical genetic sequence data of different subtypes, by reverse engineering the required selective pressure for HIV-1 sequences obtained from treatment naive patients, to evolve towards sequences obtained from treated patients. The method was evaluated for recovering 10 random fictive selective pressures in simulation experiments, and for modeling the selective pressure under treatment with the protease inhibitor nelfinavir. RESULTS: The estimated fitness function under nelfinavir treatment considered fitness contributions of 114 mutations at 48 sites. Estimated fitness correlated significantly with the in vitro resistance phenotype in 519 matched genotype-phenotype pairs (R(2) = 0.47 (0.41 - 0.54)) and variation in predicted evolution under nelfinavir selective pressure correlated significantly with observed in vivo evolution during nelfinavir treatment for 39 mutations (with FDR = 0.05). AVAILABILITY: The software is available on request from the authors, and data sets are available from http://jose.med.kuleuven.be/~kdforc0/nfv-fitness-data/.

Human Immunodeficiency Virus-1 (HIV-1) antiviral resistance is a major cause of antiviral therapy failure and compromises future treatment options. As a consequence, resistance testing is the standard of care. Because of the high degree of HIV-1 natural variation and complex interactions, the role of resistance mutations is in many cases insufficiently understood. We applied a probabilistic model, Bayesian networks, to analyze direct influences between protein residues and exposure to treatment in clinical HIV-1 protease sequences from diverse subtypes. We can determine the specific role of many resistance mutations against the protease inhibitor nelfinavir, and determine relationships between resistance mutations and polymorphisms. We can show for example that in addition to the well-known major mutations 90M and 30N for nelfinavir resistance, 88S should not be treated as 88D but instead considered as a major mutation and explain the subtype-dependent prevalence of the 30N resistance pathway.

Abstract Summary: We developed a software tool (SlidingBayes) for recombination analysis based on Bayesian phylogenetic inference. Sliding-Bayes provides a powerful approach for detecting potential recombination, especially between highly divergent sequences and complex HIV-1 recombinants for which simpler methods like neighbor joining (NJ) may be less powerful. SlidingBayes guides Markov Chain Monte Carlo (MCMC) sampling performed by MrBayes in a sliding window across the alignment (Bayesian scanning). The tool can be used for nucleotide and amino acid sequences and combines all the modeling possibilities of MrBayes with the ability to plot the posterior probability support for clustering of various combinations of taxa. Availability: SlidingBayes is available at http://www.kuleuven.ac.be/rega/cev/Software/ Contact: [email protected] Supplementary information: A quick guide and examples for SlidingBayes are available at http://www.kuleuven.ac.be/rega/cev/Software/

MOTIVATION: Genetic analysis of HIV-1 is important not only for vaccine development, but also to guide treatment strategies, track the emergence of new viral variants and ensure that diagnostic assays are contemporary and fully optimized. However, most genotyping methods are laborious and complex, and involve the use of multiple software applications. Here, we describe the development of an automated genotyping system that can be easily applied to HIV-1 and other rapidly evolving viral pathogens. RESULTS: The new REGA subtyping tool, developed using Java programming and PERL scripts, combines phylogenetic analyses with boot-scanning methods for the genetic subtyping of full-length and subgenomic fragments of HIV-1. When used to investigate the subtype of previously published reference datasets that were analysed using manual phylogenetic methods, the automated method correctly identified 97.5-100% of non-recombinant and circulating recombinant forms of HIV-1, including 108 full-length, 108 gag and 221 env sequences downloaded from the Los Alamos database.