Deep mining of the Sequence Read Archive reveals major genetic innovations in coronaviruses and other nidoviruses of aquatic vertebrates.

Virus discovery by genomics and metagenomics empowered studies of viromes, facilitated characterization of pathogen epidemiology, and redefined our understanding of the natural genetic diversity of viruses with profound functional and structural implications. Here we employed a data-driven virus discovery approach that directly queries unprocessed sequencing data in a highly parallelized way and involves a targeted viral genome assembly strategy in a wide range of sequence similarity. By screening more than 269,000 datasets of numerous authors from the Sequence Read Archive and using two metrics that quantitatively assess assembly quality, we discovered 40 nidoviruses from six virus families whose members infect vertebrate hosts. They form 13 and 32 putative viral subfamilies and genera, respectively, and include 11 coronaviruses with bisegmented genomes from fishes and amphibians, a giant 36.1 kilobase coronavirus genome with a duplicated spike glycoprotein (S) gene, 11 tobaniviruses and 17 additional corona-, arteri-, cremega-, nanhypo- and nangoshaviruses. Genome segmentation emerged in a single evolutionary event in the monophyletic lineage encompassing the subfamily Pitovirinae. We recovered the bisegmented genome sequences of two coronaviruses from RNA samples of 69 infected fishes and validated the presence of poly(A) tails at both segments using 3'RACE PCR and subsequent Sanger sequencing. We report a genetic linkage between accessory and structural proteins whose phylogenetic relationships and evolutionary distances are incongruent with the phylogeny of replicase proteins. We rationalize these observations in a model of inter-family S recombination involving at least five ancestral corona- and tobaniviruses of aquatic hosts. In support of this model, we describe an individual fish co-infected with members from the families Coronaviridae and Tobaniviridae. Our results expand the scale of the known extraordinary evolutionary plasticity in nidoviral genome architecture and call for revisiting fundamentals of genome expression, virus particle biology, host range and ecology of vertebrate nidoviruses.

What's what in a pandemic? Virus, disease, and societal disaster must be differentiated.

Viruses, the diseases they can trigger, and the possible associated societal disaster represent different entities. To engage with the complexities of viral pandemics, we need to recognize each entity by using a distinctive name.

Genome-wide diversity of Zika virus: Exploring spatio-temporal dynamics to guide a new nomenclature proposal.

The Zika virus (ZIKV) disease caused a public health emergency of international concern that started in February 2016. The overall number of ZIKV-related cases increased until November 2016, after which it declined sharply. While the evaluation of the potential risk and impact of future arbovirus epidemics remains challenging, intensified surveillance efforts along with a scale-up of ZIKV whole-genome sequencing provide an opportunity to understand the patterns of genetic diversity, evolution, and spread of ZIKV. However, a classification system that reflects the true extent of ZIKV genetic variation is lacking. Our objective was to characterize ZIKV genetic diversity and phylodynamics, identify genomic footprints of differentiation patterns, and propose a dynamic classification system that reflects its divergence levels. We analysed a curated dataset of 762 publicly available sequences spanning the full-length coding region of ZIKV from across its geographical span and collected between 1947 and 2021. The definition of genetic groups was based on comprehensive evolutionary dynamics analyses, which included recombination and phylogenetic analyses, within- and between-group pairwise genetic distances comparison, detection of selective pressure, and clustering analyses. Evidence for potential recombination events was detected in a few sequences. However, we argue that these events are likely due to sequencing errors as proposed in previous studies. There was evidence of strong purifying selection, widespread across the genome, as also detected for other arboviruses. A total of 50 sites showed evidence of positive selection, and for a few of these sites, there was amino acid (AA) differentiation between genetic clusters. Two main genetic clusters were defined, ZA and ZB, which correspond to the already characterized 'African' and 'Asian' genotypes, respectively. Within ZB, two subgroups, ZB.1 and ZB.2, represent the Asiatic and the American (and Oceania) lineages, respectively. ZB.1 is further subdivided into ZB.1.0 (a basal Malaysia sequence sampled in the 1960s and a recent Indian sequence), ZB.1.1 (South-Eastern Asia, Southern Asia, and Micronesia sequences), and ZB.1.2 (very similar sequences from the outbreak in Singapore). ZB.2 is subdivided into ZB.2.0 (basal American sequences and the sequences from French Polynesia, the putative origin of South America introduction), ZB.2.1 (Central America), and ZB.2.2 (Caribbean and North America). This classification system does not use geographical references and is flexible to accommodate potential future lineages. It will be a helpful tool for studies that involve analyses of ZIKV genomic variation and its association with pathogenicity and serve as a starting point for the public health surveillance and response to on-going and future epidemics and to outbreaks that lead to the emergence of new variants.

N7-Methylation of the Coronavirus RNA Cap Is Required for Maximal Virulence by Preventing Innate Immune Recognition.

The ongoing coronavirus (CoV) disease 2019 (COVID-19) pandemic caused by infection with severe acute respiratory syndrome CoV 2 (SARS-CoV-2) is associated with substantial morbidity and mortality. Understanding the immunological and pathological processes of coronavirus diseases is crucial for the rational design of effective vaccines and therapies for COVID-19. Previous studies showed that 2'-O-methylation of the viral RNA cap structure is required to prevent the recognition of viral RNAs by intracellular innate sensors. Here, we demonstrate that the guanine N7-methylation of the 5' cap mediated by coronavirus nonstructural protein 14 (nsp14) contributes to viral evasion of the type I interferon (IFN-I)-mediated immune response and pathogenesis in mice. A Y414A substitution in nsp14 of the coronavirus mouse hepatitis virus (MHV) significantly decreased N7-methyltransferase activity and reduced guanine N7-methylation of the 5' cap in vitro. Infection of myeloid cells with recombinant MHV harboring the nsp14-Y414A mutation (rMHVnsp14-Y414A) resulted in upregulated expression of IFN-I and ISG15 mainly via MDA5 signaling and in reduced viral replication compared to that of wild-type rMHV. rMHVnsp14-Y414A replicated to lower titers in livers and brains and exhibited an attenuated phenotype in mice. This attenuated phenotype was IFN-I dependent because the virulence of the rMHVnsp14-Y414A mutant was restored in Ifnar-/- mice. We further found that the comparable mutation (Y420A) in SARS-CoV-2 nsp14 (rSARS-CoV-2nsp14-Y420A) also significantly decreased N7-methyltransferase activity in vitro, and the mutant virus was attenuated in K18-human ACE2 transgenic mice. Moreover, infection with rSARS-CoV-2nsp14-Y420A conferred complete protection against subsequent and otherwise lethal SARS-CoV-2 infection in mice, indicating the vaccine potential of this mutant. IMPORTANCE Coronaviruses (CoVs), including SARS-CoV-2, the cause of COVID-19, use several strategies to evade the host innate immune responses. While the cap structure of RNA, including CoV RNA, is important for translation, previous studies indicate that the cap also contributes to viral evasion from the host immune response. In this study, we demonstrate that the N7-methylated cap structure of CoV RNA is pivotal for virus immunoevasion. Using recombinant MHV and SARS-CoV-2 encoding an inactive N7-methyltransferase, we demonstrate that these mutant viruses are highly attenuated in vivo and that attenuation is apparent at very early times after infection. Virulence is restored in mice lacking interferon signaling. Further, we show that infection with virus defective in N7-methylation protects mice from lethal SARS-CoV-2, suggesting that the N7-methylase might be a useful target in drug and vaccine development.

Translating genomic exploration of the family Polyomaviridae into confident human polyomavirus detection.

The Polyomaviridae is a family of ubiquitous dsDNA viruses that establish persistent infection early in life. Screening for human polyomaviruses (HPyVs), which comprise 14 diverse species, relies upon species-specific qPCRs whose validity may be challenged by accelerating genomic exploration of the virosphere. Using this reasoning, we tested 64 published HPyV qPCR assays in silico against the 1781 PyV genome sequences that were divided in targets and nontargets, based on anticipated species specificity of each qPCR. We identified several cases of problematic qPCR performance that were confirmed in vitro and corrected through using degenerate oligos. Furthermore, our study ranked 8 out of 52 tested BKPyV qPCRs as remaining of consistently high quality in the wake of recent PyV discoveries and showed how sensitivity of most other qPCRs could be rescued by annealing temperature adjustment. This study establishes an efficient framework for ensuring confidence in available HPyV qPCRs in the genomic era.

Bioinformatics of virus taxonomy: foundations and tools for developing sequence-based hierarchical classification.

The genome sequence is the only characteristic readily obtainable for all known viruses, underlying the growing role of comparative genomics in organizing knowledge about viruses in a systematic evolution-aware way, known as virus taxonomy. Overseen by the International Committee on Taxonomy of Viruses (ICTV), development of virus taxonomy involves taxa demarcation at 15 ranks of a hierarchical classification, often in host-specific manner. Outside the ICTV remit, researchers assess fitting numerous unclassified viruses into the established taxa. They employ different metrics of virus clustering, basing on conserved domain(s), separation of viruses in rooted phylogenetic trees and pair-wise distance space. Computational approaches differ further in respect to methodology, number of ranks considered, sensitivity to uneven virus sampling, and visualization of results. Advancing and using computational tools will be critical for improving taxa demarcation across the virosphere and resolving rank origins in research that may also inform experimental virology.

ICTV Virus Taxonomy Profile: Arteriviridae 2021.

The family Arteriviridae comprises enveloped RNA viruses with a linear, positive-sense genome of approximately 12.7 to 15.7 kb. The spherical, pleomorphic virions have a median diameter of 50-74 nm and include eight to eleven viral proteins. Arteriviruses infect non-human mammals in a vector-independent manner. Infections are often persistent and can either be asymptomatic or produce overt disease. Some arteriviruses are important veterinary pathogens while others infect particular species of wild rodents or African non-human primates. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Arteriviridae, which is available at ictv.global/report/arteriviridae.

Conflicting and ambiguous names of overlapping ORFs in the SARS-CoV-2 genome: A homology-based resolution.

At least six small alternative-frame open reading frames (ORFs) overlapping well-characterized SARS-CoV-2 genes have been hypothesized to encode accessory proteins. Researchers have used different names for the same ORF or the same name for different ORFs, resulting in erroneous homological and functional inferences. We propose standard names for these ORFs and their shorter isoforms, developed in consultation with the Coronaviridae Study Group of the International Committee on Taxonomy of Viruses. We recommend calling the 39 codon Spike-overlapping ORF ORF2b; the 41, 57, and 22 codon ORF3a-overlapping ORFs ORF3c, ORF3d, and ORF3b; the 33 codon ORF3d isoform ORF3d-2; and the 97 and 73 codon Nucleocapsid-overlapping ORFs ORF9b and ORF9c. Finally, we document conflicting usage of the name ORF3b in 32 studies, and consequent erroneous inferences, stressing the importance of reserving identical names for homologs. We recommend that authors referring to these ORFs provide lengths and coordinates to minimize ambiguity caused by prior usage of alternative names.

Recognizing species as a new focus of virus research.

Species taxa are the units of taxonomy most suited to measure virus diversity, and they account for more than 70% of all virus taxa. Yet, as evidenced by the content of GenBank entries and illustrated by the recent literature on SARS-CoV-2, they are the most neglected taxa of virus research. To correct this disparity, we propose to make species taxa a first choice for communicating virus taxonomy in publications concerning viruses. We see it as a key step toward promoting research on diverse viruses, including pathogens, at this fundamental level of biology.

A nidovirus perspective on SARS-CoV-2.

Two pandemics of respiratory distress diseases associated with zoonotic introductions of the species Severe acute respiratory syndrome-related coronavirus in the human population during 21st century raised unprecedented interest in coronavirus research and assigned it unseen urgency. The two viruses responsible for the outbreaks, SARS-CoV and SARS-CoV-2, respectively, are in the spotlight, and SARS-CoV-2 is the focus of the current fast-paced research. Its foundation was laid down by studies of many corona- and related viruses that collectively form the vast order Nidovirales. Comparative genomics of nidoviruses played a key role in this advancement over more than 30 years. It facilitated the transfer of knowledge from characterized to newly identified viruses, including SARS-CoV and SARS-CoV-2, as well as contributed to the dissection of the nidovirus proteome and identification of patterns of variations between different taxonomic groups, from species to families. This review revisits selected cases of protein conservation and variation that define nidoviruses, illustrates the remarkable plasticity of the proteome during nidovirus adaptation, and asks questions at the interface of the proteome and processes that are vital for nidovirus reproduction and could inform the ongoing research of SARS-CoV-2.

Coronavirus replication-transcription complex: Vital and selective NMPylation of a conserved site in nsp9 by the NiRAN-RdRp subunit.

RNA-dependent RNA polymerases (RdRps) of the Nidovirales (Coronaviridae, Arteriviridae, and 12 other families) are linked to an amino-terminal (N-terminal) domain, called NiRAN, in a nonstructural protein (nsp) that is released from polyprotein 1ab by the viral main protease (Mpro). Previously, self-GMPylation/UMPylation activities were reported for an arterivirus NiRAN-RdRp nsp and suggested to generate a transient state primed for transferring nucleoside monophosphate (NMP) to (currently unknown) viral and/or cellular biopolymers. Here, we show that the coronavirus (human coronavirus [HCoV]-229E and severe acute respiratory syndrome coronavirus 2) nsp12 (NiRAN-RdRp) has Mn2+-dependent NMPylation activity that catalyzes the transfer of a single NMP to the cognate nsp9 by forming a phosphoramidate bond with the primary amine at the nsp9 N terminus (N3825) following Mpro-mediated proteolytic release of nsp9 from N-terminally flanking nsps. Uridine triphosphate was the preferred nucleotide in this reaction, but also adenosine triphosphate, guanosine triphosphate, and cytidine triphosphate were suitable cosubstrates. Mutational studies using recombinant coronavirus nsp9 and nsp12 proteins and genetically engineered HCoV-229E mutants identified residues essential for NiRAN-mediated nsp9 NMPylation and virus replication in cell culture. The data corroborate predictions on NiRAN active-site residues and establish an essential role for the nsp9 N3826 residue in both nsp9 NMPylation in vitro and virus replication. This residue is part of a conserved N-terminal NNE tripeptide sequence and shown to be the only invariant residue in nsp9 and its homologs in viruses of the family Coronaviridae The study provides a solid basis for functional studies of other nidovirus NMPylation activities and suggests a possible target for antiviral drug development.

LAMPA, LArge Multidomain Protein Annotator, and its application to RNA virus polyproteins.

MOTIVATION: To facilitate accurate estimation of statistical significance of sequence similarity in profile-profile searches, queries should ideally correspond to protein domains. For multidomain proteins, using domains as queries depends on delineation of domain borders, which may be unknown. Thus, proteins are commonly used as queries that complicate establishing homology for similarities close to cutoff levels of statistical significance. RESULTS: In this article, we describe an iterative approach, called LAMPA, LArge Multidomain Protein Annotator, that resolves the above conundrum by gradual expansion of hit coverage of multidomain proteins through re-evaluating statistical significance of hit similarity using ever smaller queries defined at each iteration. LAMPA employs TMHMM and HHsearch for recognition of transmembrane regions and homology, respectively. We used Pfam database for annotating 2985 multidomain proteins (polyproteins) composed of >1000 amino acid residues, which dominate proteomes of RNA viruses. Under strict cutoffs, LAMPA outperformed HHsearch-mediated runs using intact polyproteins as queries by three measures: number of and coverage by identified homologous regions, and number of hit Pfam profiles. Compared to HHsearch, LAMPA identified 507 extra homologous regions in 14.4% of polyproteins. This Pfam-based annotation of RNA virus polyproteins by LAMPA was also superior to RefSeq expert annotation by two measures, region number and annotated length, for 69.3% of RNA virus polyprotein entries. We rationalized the obtained results based on dependencies of HHsearch hit statistical significance for local alignment similarity score from lengths and diversities of query-target pairs in computational experiments. AVAILABILITY AND IMPLEMENTATION: LAMPA 1.0.0 R package is placed at github (https://github.com/Gorbalenya-Lab/LAMPA). SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Structural basis for catalysis and substrate specificity of a 3C-like cysteine protease from a mosquito mesonivirus.

Cavally virus (CavV) is a mosquito-borne plus-strand RNA virus in the family Mesoniviridae (order Nidovirales). We present X-ray structures for the CavV 3C-like protease (3CLpro), as a free enzyme and in complex with a peptide aldehyde inhibitor mimicking the P4-to-P1 residues of a natural substrate. The 3CLpro structure (refined to 1.94 Å) shows that the protein forms dimers. The monomers are comprised of N-terminal domains I and II, which adopt a chymotrypsin-like fold, and a C-terminal α-helical domain III. The catalytic Cys-His dyad is assisted by a complex network of interactions involving a water molecule that mediates polar contacts between the catalytic His and a conserved Asp located in the domain II-III junction and is suitably positioned to stabilize the developing positive charge of the catalytic His in the transition state during catalysis. The study also reveals the structural basis for the distinct P2 Asn-specific substrate-binding pocket of mesonivirus 3CLpros.

Additional changes to taxonomy ratified in a special vote by the International Committee on Taxonomy of Viruses (October 2018).

This article reports the changes to virus taxonomy approved and ratified by the International Committee on Taxonomy of Viruses (ICTV) in October 2018. Of note, the ICTV has approved, by an absolute majority, the creation of additional taxonomical ranks above those recognized previously. A total of 15 ranks (realm, subrealm, kingdom, subkingdom, phylum, subphylum, class, subclass, order, suborder, family, subfamily, genus, subgenus, and species) are now available to encompass the entire spectrum of virus diversity. Classification at ranks above genus is not obligatory but can be used by the authors of new taxonomic proposals when scientific justification is provided.

A planarian nidovirus expands the limits of RNA genome size.

RNA viruses are the only known RNA-protein (RNP) entities capable of autonomous replication (albeit within a permissive host environment). A 33.5 kilobase (kb) nidovirus has been considered close to the upper size limit for such entities; conversely, the minimal cellular DNA genome is in the 100-300 kb range. This large difference presents a daunting gap for the transition from primordial RNP to contemporary DNA-RNP-based life. Whether or not RNA viruses represent transitional steps towards DNA-based life, studies of larger RNA viruses advance our understanding of the size constraints on RNP entities and the role of genome size in virus adaptation. For example, emergence of the largest previously known RNA genomes (20-34 kb in positive-stranded nidoviruses, including coronaviruses) is associated with the acquisition of a proofreading exoribonuclease (ExoN) encoded in the open reading frame 1b (ORF1b) in a monophyletic subset of nidoviruses. However, apparent constraints on the size of ORF1b, which encodes this and other key replicative enzymes, have been hypothesized to limit further expansion of these viral RNA genomes. Here, we characterize a novel nidovirus (planarian secretory cell nidovirus; PSCNV) whose disproportionately large ORF1b-like region including unannotated domains, and overall 41.1-kb genome, substantially extend the presumed limits on RNA genome size. This genome encodes a predicted 13,556-aa polyprotein in an unconventional single ORF, yet retains canonical nidoviral genome organization and expression, as well as key replicative domains. These domains may include functionally relevant substitutions rarely or never before observed in highly conserved sites of RdRp, NiRAN, ExoN and 3CLpro. Our evolutionary analysis suggests that PSCNV diverged early from multi-ORF nidoviruses, and acquired additional genes, including those typical of large DNA viruses or hosts, e.g. Ankyrin and Fibronectin type II, which might modulate virus-host interactions. PSCNV's greatly expanded genome, proteomic complexity, and unique features-impressive in themselves-attest to the likelihood of still-larger RNA genomes awaiting discovery.

Increasing the number of available ranks in virus taxonomy from five to ten and adopting the Baltimore classes as taxa at the basal rank.

This opinion article makes a case for increasing the number of ranks used in virus taxonomy from the current five to ten (as are used to classify cellular life forms) and placing the Baltimore classes in the proposed basal rank of domain. These suggestions aim at initiating the process of accommodation of Baltimore classes in virus taxonomy and extension of the virus taxonomy scale to encompass also the most distant relationships.