The International Virus Bioinformatics Meeting 2023.

The 2023 International Virus Bioinformatics Meeting was held in Valencia, Spain, from 24-26 May 2023, attracting approximately 180 participants worldwide. The primary objective of the conference was to establish a dynamic scientific environment conducive to discussion, collaboration, and the generation of novel research ideas. As the first in-person event following the SARS-CoV-2 pandemic, the meeting facilitated highly interactive exchanges among attendees. It served as a pivotal gathering for gaining insights into the current status of virus bioinformatics research and engaging with leading researchers and emerging scientists. The event comprised eight invited talks, 19 contributed talks, and 74 poster presentations across eleven sessions spanning three days. Topics covered included machine learning, bacteriophages, virus discovery, virus classification, virus visualization, viral infection, viromics, molecular epidemiology, phylodynamic analysis, RNA viruses, viral sequence analysis, viral surveillance, and metagenomics. This report provides rewritten abstracts of the presentations, a summary of the key research findings, and highlights shared during the meeting.

Genomic analysis of 61 Chlamydia psittaci strains reveals extensive divergence associated with host preference.

BACKGROUND: Chlamydia (C.) psittaci, the causative agent of avian chlamydiosis and human psittacosis, is a genetically heterogeneous species. Its broad host range includes parrots and many other birds, but occasionally also humans (via zoonotic transmission), ruminants, horses, swine and rodents. To assess whether there are genetic markers associated with host tropism we comparatively analyzed whole-genome sequences of 61 C. psittaci strains, 47 of which carrying a 7.6-kbp plasmid. RESULTS: Following clean-up, reassembly and polishing of poorly assembled genomes from public databases, phylogenetic analyses using C. psittaci whole-genome sequence alignment revealed four major clades within this species. Clade 1 represents the most recent lineage comprising 40/61 strains and contains 9/10 of the psittacine strains, including type strain 6BC, and 10/13 of human isolates. Strains from different non-psittacine hosts clustered in Clades 2- 4. We found that clade membership correlates with typing schemes based on SNP types, ompA genotypes, multilocus sequence types as well as plasticity zone (PZ) structure and host preference. Genome analysis also revealed that i) sequence variation in the major outer membrane porin MOMP can result in 3D structural changes of immunogenic domains, ii) past host change of Clade 3 and 4 strains could be associated with loss of MAC/perforin in the PZ, rather than the large cytotoxin, iii) the distinct phylogeny of atypical strains (Clades 3 and 4) is also reflected in their repertoire of inclusion proteins (Inc family) and polymorphic membrane proteins (Pmps). CONCLUSIONS: Our study identified a number of genomic features that can be correlated with the phylogeny and host preference of C. psittaci strains. Our data show that intra-species genomic divergence is associated with past host change and includes deletions in the plasticity zone, structural variations in immunogenic domains and distinct repertoires of virulence factors.

Sequential disruption of SPLASH-identified vRNA-vRNA interactions challenges their role in influenza A virus genome packaging.

A fundamental step in the influenza A virus (IAV) replication cycle is the coordinated packaging of eight distinct genomic RNA segments (i.e. vRNAs) into a viral particle. Although this process is thought to be controlled by specific vRNA-vRNA interactions between the genome segments, few functional interactions have been validated. Recently, a large number of potentially functional vRNA-vRNA interactions have been detected in purified virions using the RNA interactome capture method SPLASH. However, their functional significance in coordinated genome packaging remains largely unclear. Here, we show by systematic mutational analysis that mutant A/SC35M (H7N7) viruses lacking several prominent SPLASH-identified vRNA-vRNA interactions involving the HA segment package the eight genome segments as efficiently as the wild-type virus. We therefore propose that the vRNA-vRNA interactions identified by SPLASH in IAV particles are not necessarily critical for the genome packaging process, leaving the underlying molecular mechanism elusive.

Genome Structure, Life Cycle, and Taxonomy of Coronaviruses and the Evolution of SARS-CoV-2.

Coronaviruses have a broad host range and exhibit high zoonotic potential. In this chapter, we describe their genomic organization in terms of encoded proteins and provide an introduction to the peculiar discontinuous transcription mechanism. Further, we present evolutionary conserved genomic RNA secondary structure features, which are involved in the complex replication mechanism. With a focus on computational methods, we review the emergence of SARS-CoV-2 starting with the 2019 strains. In that context, we also discuss the debated hypothesis of whether SARS-CoV-2 was created in a laboratory. We focus on the molecular evolution and the epidemiological dynamics of this recently emerged pathogen and we explain how variants of concern are detected and characterised. COVID-19, the disease caused by SARS-CoV-2, can spread through different transmission routes and also depends on a number of risk factors. We describe how current computational models of viral epidemiology, or more specifically, phylodynamics, have facilitated and will continue to enable a better understanding of the epidemic dynamics of SARS-CoV-2.

A genome-wide CRISPR activation screen reveals Hexokinase 1 as a critical factor in promoting resistance to multi-kinase inhibitors in hepatocellular carcinoma cells.

Hepatocellular carcinoma (HCC) is often diagnosed at an advanced stage and is, therefore, treated with systemic drugs, such as tyrosine-kinase inhibitors (TKIs). These drugs, however, offer only modest survival benefits due to the rapid development of drug resistance. To identify genes implicated in TKI resistance, a cluster of regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 activation screen was performed in hepatoma cells treated with regorafenib, a TKI used as second-line therapy for advanced HCC. The screen results show that Hexokinase 1 (HK1), catalyzing the first step in glucose metabolism, is a top candidate for conferring TKI resistance. Compatible with this, HK1 was upregulated in regorafenib-resistant cells. Using several experimental approaches, both in vitro and in vivo, we show that TKI resistance correlates with HK1 expression. Furthermore, an HK inhibitor resensitized resistant cells to TKI treatment. Together, our data indicate that HK1 may function as a critical factor modulating TKI resistance in hepatoma cells and, therefore, may serve as a biomarker for treatment success.

Comparative Study of Ten Thogotovirus Isolates and Their Distinct In Vivo Characteristics.

Thogotoviruses are tick-borne arboviruses that comprise a unique genus within the Orthomyxoviridae family. Infections with thogotoviruses primarily cause disease in livestock with occasional reports of human infections suggesting a zoonotic potential. In the past, multiple genetically distinct thogotoviruses were isolated mostly from collected ticks. However, many aspects regarding their phylogenetic relationships, morphological characteristics, and virulence in mammals remain unclear. For the present comparative study, we used a collection of 10 different thogotovirus isolates from different geographic areas. Next-generation sequencing and subsequent phylogenetic analyses revealed a distinct separation of these viruses into two major clades, the Thogoto-like and Dhori-like viruses. Electron microscopy demonstrated a heterogeneous morphology with spherical and filamentous particles being present in virus preparations. To study their pathogenicity, we analyzed the viruses in a small animal model system. In intraperitoneally infected C57BL/6 mice, all isolates showed a tropism for liver, lung, and spleen. Importantly, we did not observe horizontal transmission to uninfected, highly susceptible contact mice. The isolates enormously differed in their capacity to induce disease, ranging from subclinical to fatal outcomes. In vivo multistep passaging experiments of two low-pathogenic isolates showed no increased virulence and sequence analyses of the passaged viruses indicated a high stability of the viral genomes after 10 mouse passages. In summary, our analysis demonstrates the broad genetic and phenotypic variability within the thogotovirus genus. Moreover, thogotoviruses are well adapted to mammals but their horizontal transmission seems to depend on ticks as their vectors. IMPORTANCE Since their discovery over 60 years ago, 15 genetically distinct members of the thogotovirus genus have been isolated. These arboviruses belong to the Orthomyxovirus family and share many features with influenza viruses. However, numerous of these isolates have not been characterized in depth. In the present study, we comparatively analyzed a collection of 10 different thogotovirus isolates to answer basic questions about their phylogenetic relationships, morphology, and pathogenicity in mice. Our results highlight shared and unique characteristics of this diverse genus. Taken together, these observations provide a framework for the phylogenic classification and phenotypic characterization of newly identified thogotovirus isolates that could potentially cause severe human infections as exemplified by the recently reported, fatal Bourbon virus cases in the United States.

Computational strategies to combat COVID-19: useful tools to accelerate SARS-CoV-2 and coronavirus research.

SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) is a novel virus of the family Coronaviridae. The virus causes the infectious disease COVID-19. The biology of coronaviruses has been studied for many years. However, bioinformatics tools designed explicitly for SARS-CoV-2 have only recently been developed as a rapid reaction to the need for fast detection, understanding and treatment of COVID-19. To control the ongoing COVID-19 pandemic, it is of utmost importance to get insight into the evolution and pathogenesis of the virus. In this review, we cover bioinformatics workflows and tools for the routine detection of SARS-CoV-2 infection, the reliable analysis of sequencing data, the tracking of the COVID-19 pandemic and evaluation of containment measures, the study of coronavirus evolution, the discovery of potential drug targets and development of therapeutic strategies. For each tool, we briefly describe its use case and how it advances research specifically for SARS-CoV-2. All tools are free to use and available online, either through web applications or public code repositories. Contact:evbc@unj-jena.de.

Rfam 14: expanded coverage of metagenomic, viral and microRNA families.

Rfam is a database of RNA families where each of the 3444 families is represented by a multiple sequence alignment of known RNA sequences and a covariance model that can be used to search for additional members of the family. Recent developments have involved expert collaborations to improve the quality and coverage of Rfam data, focusing on microRNAs, viral and bacterial RNAs. We have completed the first phase of synchronising microRNA families in Rfam and miRBase, creating 356 new Rfam families and updating 40. We established a procedure for comprehensive annotation of viral RNA families starting with Flavivirus and Coronaviridae RNAs. We have also increased the coverage of bacterial and metagenome-based RNA families from the ZWD database. These developments have enabled a significant growth of the database, with the addition of 759 new families in Rfam 14. To facilitate further community contribution to Rfam, expert users are now able to build and submit new families using the newly developed Rfam Cloud family curation system. New Rfam website features include a new sequence similarity search powered by RNAcentral, as well as search and visualisation of families with pseudoknots. Rfam is freely available at https://rfam.org.

The International Virus Bioinformatics Meeting 2020.

The International Virus Bioinformatics Meeting 2020 was originally planned to take place in Bern, Switzerland, in March 2020. However, the COVID-19 pandemic put a spoke in the wheel of almost all conferences to be held in 2020. After moving the conference to 8-9 October 2020, we got hit by the second wave and finally decided at short notice to go fully online. On the other hand, the pandemic has made us even more aware of the importance of accelerating research in viral bioinformatics. Advances in bioinformatics have led to improved approaches to investigate viral infections and outbreaks. The International Virus Bioinformatics Meeting 2020 has attracted approximately 120 experts in virology and bioinformatics from all over the world to join the two-day virtual meeting. Despite concerns being raised that virtual meetings lack possibilities for face-to-face discussion, the participants from this small community created a highly interactive scientific environment, engaging in lively and inspiring discussions and suggesting new research directions and questions. The meeting featured five invited and twelve contributed talks, on the four main topics: (1) proteome and RNAome of RNA viruses, (2) viral metagenomics and ecology, (3) virus evolution and classification and (4) viral infections and immunology. Further, the meeting featured 20 oral poster presentations, all of which focused on specific areas of virus bioinformatics. This report summarizes the main research findings and highlights presented at the meeting.

Comparative Genome Analysis of 33 Chlamydia Strains Reveals Characteristic Features of Chlamydia Psittaci and Closely Related Species.

To identify genome-based features characteristic of the avian and human pathogen Chlamydia(C.) psittaci and related chlamydiae, we analyzed whole-genome sequences of 33 strains belonging to 12 species. Using a novel genome analysis tool termed Roary ILP Bacterial Annotation Pipeline (RIBAP), this panel of strains was shown to share a large core genome comprising 784 genes and representing approximately 80% of individual genomes. Analyzing the most variable genomic sites, we identified a set of features of C. psittaci that in its entirety is characteristic of this species: (i) a relatively short plasticity zone of less than 30,000 nt without a tryptophan operon (also in C. abortus, C. avium, C. gallinacea, C. pneumoniae), (ii) a characteristic set of of Inc proteins comprising IncA, B, C, V, X, Y (with homologs in C. abortus, C. caviae and C. felis as closest relatives), (iii) a 502-aa SinC protein, the largest among Chlamydia spp., and (iv) an elevated number of Pmp proteins of subtype G (14 in C. psittaci, 14 in Cand. C. ibidis). In combination with future functional studies, the common and distinctive criteria revealed in this study provide important clues for understanding the complexity of host-specific behavior of individual Chlamydia spp.

HIV-1 Lethality and Loss of Env Protein Expression Induced by Single Synonymous Substitutions in the Virus Genome Intronic-Splicing Silencer.

Synonymous genome recoding has been widely used to study different aspects of virus biology. Codon usage affects the temporal regulation of viral gene expression. In this study, we performed synonymous codon mutagenesis to investigate whether codon usage affected HIV-1 Env protein expression and virus viability. We replaced the codons AGG, GAG, CCU, ACU, CUC, and GGG of the HIV-1 env gene with the synonymous codons CGU, GAA, CCG, ACG, UUA, and GGA, respectively. We found that recoding the Env protein gp120 coding region (excluding the Rev response element [RRE]) did not significantly affect virus replication capacity, even though we introduced 15 new CpG dinucleotides. In contrast, changing a single codon (AGG to CGU) located in the gp41 coding region (HXB2 env position 2125 to 2127), which was included in the intronic splicing silencer (ISS), completely abolished virus replication and Env expression. Computational analyses of this mutant revealed a severe disruption in the ISS RNA secondary structure. A variant that restored ISS secondary RNA structure also reestablished Env production and virus viability. Interestingly, this codon variant prevented both virus replication and Env translation in a eukaryotic expression system. These findings suggested that disrupting mRNA splicing was not the only means of inhibiting translation. Our findings indicated that synonymous gp120 recoding was not always deleterious to HIV-1 replication. Importantly¸ we found that disrupting an external ISS loop strongly affected HIV-1 replication and Env translation.IMPORTANCE Synonymous substitutions can influence virus phenotype, replication capacity, and virulence. In this study, we explored how synonymous codon mutations impacted HIV-1 Env protein expression and virus replication capacity. We changed a single codon, AGG to CGU, which was located in the gp41 coding region (env nucleotide residues 2125 to 2127) and was included in the HIV-1 intronic splicing silencer. This change completely abolished virus replication and Env expression. We also found that changing codon usage in the gp120 region by including an increased number of CpG dinucleotides did not significantly affect Env expression or virus viability. Our findings showed that synonymous recoding was useful for altering viral phenotype and exploring virus biology.

A comprehensive annotation and differential expression analysis of short and long non-coding RNAs in 16 bat genomes.

Although bats are increasingly becoming the focus of scientific studies due to their unique properties, these exceptional animals are still among the least studied mammals. Assembly quality and completeness of bat genomes vary a lot and especially non-coding RNA (ncRNA) annotations are incomplete or simply missing. Accordingly, standard bioinformatics pipelines for gene expression analysis often ignore ncRNAs such as microRNAs or long antisense RNAs. The main cause of this problem is the use of incomplete genome annotations. We present a complete screening for ncRNAs within 16 bat genomes. NcRNAs affect a remarkable variety of vital biological functions, including gene expression regulation, RNA processing, RNA interference and, as recently described, regulatory processes in viral infections. Within all investigated bat assemblies, we annotated 667 ncRNA families including 162 snoRNAs and 193 miRNAs as well as rRNAs, tRNAs, several snRNAs and lncRNAs, and other structural ncRNA elements. We validated our ncRNA candidates by six RNA-Seq data sets and show significant expression patterns that have never been described before in a bat species on such a large scale. Our annotations will be usable as a resource (rna.uni-jena.de/supplements/bats) for deeper studying of bat evolution, ncRNAs repertoire, gene expression and regulation, ecology and important host-virus interactions.

Direct RNA nanopore sequencing of full-length coronavirus genomes provides novel insights into structural variants and enables modification analysis.

Sequence analyses of RNA virus genomes remain challenging owing to the exceptional genetic plasticity of these viruses. Because of high mutation and recombination rates, genome replication by viral RNA-dependent RNA polymerases leads to populations of closely related viruses, so-called "quasispecies." Standard (short-read) sequencing technologies are ill-suited to reconstruct large numbers of full-length haplotypes of (1) RNA virus genomes and (2) subgenome-length (sg) RNAs composed of noncontiguous genome regions. Here, we used a full-length, direct RNA sequencing (DRS) approach based on nanopores to characterize viral RNAs produced in cells infected with a human coronavirus. By using DRS, we were able to map the longest (∼26-kb) contiguous read to the viral reference genome. By combining Illumina and Oxford Nanopore sequencing, we reconstructed a highly accurate consensus sequence of the human coronavirus (HCoV)-229E genome (27.3 kb). Furthermore, by using long reads that did not require an assembly step, we were able to identify, in infected cells, diverse and novel HCoV-229E sg RNAs that remain to be characterized. Also, the DRS approach, which circumvents reverse transcription and amplification of RNA, allowed us to detect methylation sites in viral RNAs. Our work paves the way for haplotype-based analyses of viral quasispecies by showing the feasibility of intra-sample haplotype separation. Even though several technical challenges remain to be addressed to exploit the potential of the nanopore technology fully, our work illustrates that DRS may significantly advance genomic studies of complex virus populations, including predictions on long-range interactions in individual full-length viral RNA haplotypes.

A CRISPR Activation Screen Identifies Genes That Protect against Zika Virus Infection.

Zika virus (ZIKV) is an arthropod-borne emerging pathogen causing febrile illness. ZIKV is associated Guillain-Barré syndrome and other neurological complications. Infection during pregnancy is associated with pregnancy complications and developmental and neurological abnormalities collectively defined as congenital Zika syndrome. There is still no vaccine or specific treatment for ZIKV infection. To identify host factors that can rescue cells from ZIKV infection, we used a genome-scale CRISPR activation screen. Our highly ranking hits included a short list of interferon-stimulated genes (ISGs) previously reported to have antiviral activity. Validation of the screen results highlighted interferon lambda 2 (IFN-λ2) and interferon alpha-inducible protein 6 (IFI6) as genes providing high levels of protection from ZIKV. Activation of these genes had an effect on an early stage in viral infection. In addition, infected cells expressing single guide RNAs (sgRNAs) for both of these genes displayed lower levels of cell death than did the controls. Furthermore, the identified genes were significantly induced in ZIKV-infected placenta explants. Thus, these results highlight a set of ISGs directly relevant for rescuing cells from ZIKV infection or its associated cell death and substantiate CRISPR activation screens as a tool to identify host factors impeding pathogen infection.IMPORTANCE Zika virus (ZIKV) is an emerging vector-borne pathogen causing a febrile disease. ZIKV infection might also trigger Guillain-Barré syndrome, neuropathy, and myelitis. Vertical transmission of ZIKV can cause fetus demise, stillbirth, or severe congenital abnormalities and neurological complications. There is no vaccine or specific antiviral treatment against ZIKV. We used a genome-wide CRISPR activation screen, where genes are activated from their native promoters to identify host cell factors that protect cells from ZIKV infection or associated cell death. The results provide a better understanding of key host factors that protect cells from ZIKV infection and might assist in identifying novel antiviral targets.

Structure and Hierarchy of Influenza Virus Models Revealed by Reaction Network Analysis.

Influenza A virus is recognized today as one of the most challenging viruses that threatens both human and animal health worldwide. Understanding the control mechanisms of influenza infection and dynamics is crucial and could result in effective future treatment strategies. Many kinetic models based on differential equations have been developed in recent decades to capture viral dynamics within a host. These models differ in their complexity in terms of number of species elements and number of reactions. Here, we present a new approach to understanding the overall structure of twelve influenza A virus infection models and their relationship to each other. To this end, we apply chemical organization theory to obtain a hierarchical decomposition of the models into chemical organizations. The decomposition is based on the model structure (reaction rules) but is independent of kinetic details such as rate constants. We found different types of model structures ranging from two to eight organizations. Furthermore, the model's organizations imply a partial order among models entailing a hierarchy of model, revealing a high model diversity with respect to their long-term behavior. Our methods and results can be helpful in model development and model integration, also beyond the influenza area.

The Third Annual Meeting of the European Virus Bioinformatics Center.

The Third Annual Meeting of the European Virus Bioinformatics Center (EVBC) took place in Glasgow, United Kingdom, 28-29 March 2019. Virus bioinformatics has become central to virology research, and advances in bioinformatics have led to improved approaches to investigate viral infections and outbreaks, being successfully used to detect, control, and treat infections of humans and animals. This active field of research has attracted approximately 110 experts in virology and bioinformatics/computational biology from Europe and other parts of the world to attend the two-day meeting in Glasgow to increase scientific exchange between laboratory- and computer-based researchers. The meeting was held at the McIntyre Building of the University of Glasgow; a perfect location, as it was originally built to be a place for "rubbing your brains with those of other people", as Rector Stanley Baldwin described it. The goal of the meeting was to provide a meaningful and interactive scientific environment to promote discussion and collaboration and to inspire and suggest new research directions and questions. The meeting featured eight invited and twelve contributed talks, on the four main topics: (1) systems virology, (2) virus-host interactions and the virome, (3) virus classification and evolution and (4) epidemiology, surveillance and evolution. Further, the meeting featured 34 oral poster presentations, all of which focused on specific areas of virus bioinformatics. This report summarizes the main research findings and highlights presented at the meeting.

Structural and functional conservation of cis-acting RNA elements in coronavirus 5'-terminal genome regions.

Structure predictions suggest a partial conservation of RNA structure elements in coronavirus terminal genome regions. Here, we determined the structures of stem-loops (SL) 1 and 2 of two alphacoronaviruses, human coronavirus (HCoV) 229E and NL63, by RNA structure probing and studied the functional relevance of these putative cis-acting elements. HCoV-229E SL1 and SL2 mutants generated by reverse genetics were used to study the effects on viral replication of single-nucleotide substitutions predicted to destabilize the SL1 and SL2 structures. The data provide conclusive evidence for the critical role of SL1 and SL2 in HCoV-229E replication and, in some cases, revealed parallels with previously characterized betacoronavirus SL1 and SL2 elements. Also, we were able to rescue viable HCoV-229E mutants carrying replacements of SL2 with equivalent betacoronavirus structural elements. The data obtained in this study reveal a remarkable degree of structural and functional conservation of 5'-terminal RNA structural elements across coronavirus genus boundaries.