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It is commonly held that there is a fundamental relationship between genome size and error rate, manifest as a notional "error threshold" that sets an upper limit on genome sizes. The genome sizes of RNA viruses, which have intrinsically high mutation rates due to a lack of mechanisms for error correction, must therefore be small to avoid accumulating an excessive number of deleterious mutations that will ultimately lead to population extinction. The proposed exceptions to this evolutionary rule are RNA viruses from the order Nidovirales (such as coronaviruses) that encode error-correcting exonucleases, enabling them to reach genome lengths greater than 40 kb. The recent discovery of large-genome flavi-like viruses (Flaviviridae), which comprise genomes up to 27 kb in length yet seemingly do not encode exonuclease domains, has led to the proposal that a proofreading mechanism is required to facilitate the expansion of nonsegmented RNA virus genomes above 30 kb. Herein, we describe a ~40 kb flavi-like virus identified in a Haliclona sponge metatranscriptome that does not encode a known exonuclease. Structural analysis revealed that this virus may have instead captured cellular domains associated with nucleic acid metabolism that have not been previously found in RNA viruses. Phylogenetic inference placed this virus as a divergent pesti-like lineage, such that we have provisionally termed it "Maximus pesti-like virus." This virus represents an instance of a flavi-like virus achieving a genome size comparable to that of the Nidovirales and demonstrates that RNA viruses have evolved multiple solutions to overcome the error threshold. [ABSTRACT FROM AUTHOR]

During infection, positive-stranded RNA causes a rearrangement of the host cell membrane, resulting in specialized membrane structure formation aiding viral genome replication. Double-membrane vesicles (DMVs), typical structures produced by virus-induced membrane rearrangements, are platforms for viral replication. Nidoviruses, one of the most complex positive-strand RNA viruses, have the ability to infect not only mammals and a few birds but also invertebrates. Nidoviruses possess a distinctive replication mechanism, wherein their nonstructural proteins (nsps) play a crucial role in DMV biogenesis. With the participation of host factors related to autophagy and lipid synthesis pathways, several viral nsps hijack the membrane rearrangement process of host endoplasmic reticulum (ER), Golgi apparatus, and other organelles to induce DMV formation. An understanding of the mechanisms of DMV formation and its structure and function in the infectious cycle of nidovirus may be essential for the development of new and effective antiviral strategies in the future. [ABSTRACT FROM AUTHOR]

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. Author summary: Research in virology is primarily motivated by human pathogens, such as SARS-CoV-2 in the case of the family Coronaviridae in the order Nidovirales. Studies of these and few model viruses describe virus-host interactions on the molecular level and are essential for developing virus control measures, but they must accommodate a vast range of viral natural diversity to allow generalizations. Here, we redefine our understanding of the genetic and genomic diversity in corona- and other nidoviruses of poorly sampled hosts. We mine more than 269,000 publicly accessible raw sequencing datasets for the presence of viral sequences using high-performance computing and discover 40 nidoviruses including 13 coronaviruses from a wide range of vertebrates. Some of the novel viruses from aquatic hosts have extraordinary features such as segmented genomes and recombinant genes coding for structural proteins. Our study suggests that gene exchange between diverse nidovirus species from different virus families might be more frequent than previously thought and can result in abrupt genomic innovations that in turn might facilitate host jumps even across vertebrate class borders. The growing list of newly discovered (corona)viruses enables an evolutionary perspective across virus divergency scales in different hosts on the wet lab-acquired knowledge about few viruses. [ABSTRACT FROM AUTHOR]

Serpentoviruses are a subfamily of positive sense RNA viruses in the order Nidovirales, family Tobaniviridae, associated with respiratory disease in multiple clades of reptiles. While the broadest viral diversity is reported from captive pythons, other reptiles, including colubrid snakes, turtles, and lizards of captive and free-ranging origin are also known hosts. To better define serpentoviral diversity, eleven novel serpentovirus genomes were sequenced with an Illumina MiSeq and, when necessary, completed with other Sanger sequencing methods. The novel serpentoviral genomes, along with 57 other previously published serpentovirus genomes, were analyzed alongside four outgroup genomes. Genomic analyses included identifying unique genome templates for each serpentovirus clade, as well as analysis of coded protein composition, potential protein function, protein glycosylation sites, differences in phylogenetic history between open-reading frames, and recombination. Serpentoviral genomes contained diverse protein compositions. In addition to the fundamental structural spike, matrix, and nucleoprotein proteins required for virion formation, serpentovirus genomes also included 20 previously uncharacterized proteins. The uncharacterized proteins were homologous to a number of previously characterized proteins, including enzymes, transcription factors, scaffolding, viral resistance, and apoptosis-related proteins. Evidence for recombination was detected in multiple instances in genomes from both captive and free-ranging snakes. These results show serpentovirus as a diverse clade of viruses with genomes that code for a wide diversity of proteins potentially enhanced by recombination events. [ABSTRACT FROM AUTHOR]

Several viruses are now known to code for deubiquitinating proteases in their genomes. Ubiquitination is an essential post-translational modification of cellular substrates involved in many processes in the cell, including in innate immune signalling. This post-translational modification is regulated by the ubiquitin conjugation machinery, as well as various host deubiquitinating enzymes. The conjugation of ubiquitin chains to several innate immune related factors is often needed to induce downstream signalling, shaping the antiviral response. Viral deubiquitinating proteins, besides often having a primary function in the viral replication cycle by cleaving the viral polyprotein, are also able to cleave ubiquitin chains from such host substrates, in that way exerting a function in innate immune evasion. The presence of viral deubiquitinating enzymes has been firmly established for numerous animal-infecting viruses, such as some well-researched and clinically important nidoviruses, and their presence has now been confirmed in several plant viruses as well. Viral proteases in general have long been highlighted as promising drug targets, with a current focus on small molecule inhibitors. In this review, we will discuss the range of viral deubiquitinating proteases known to date, summarise the various avenues explored to inhibit such proteases and discuss novel strategies and models intended to inhibit and study these specific viral enzymes.

During infection, positive-stranded RNA causes a rearrangement of the host cell membrane, resulting in specialized membrane structure formation aiding viral genome replication. Double-membrane vesicles (DMVs), typical structures produced by virus-induced membrane rearrangements, are platforms for viral replication. Nidoviruses, one of the most complex positive-strand RNA viruses, have the ability to infect not only mammals and a few birds but also invertebrates. Nidoviruses possess a distinctive replication mechanism, wherein their nonstructural proteins (nsps) play a crucial role in DMV biogenesis. With the participation of host factors related to autophagy and lipid synthesis pathways, several viral nsps hijack the membrane rearrangement process of host endoplasmic reticulum (ER), Golgi apparatus, and other organelles to induce DMV formation. An understanding of the mechanisms of DMV formation and its structure and function in the infectious cycle of nidovirus may be essential for the development of new and effective antiviral strategies in the future.

Phosphodiesterases (PDEs) encoded by viruses are putatively acquired by horizontal transfer of cellular PDE ancestor genes. Viral PDEs inhibit the OAS-RNase L antiviral pathway, a key effector component of the innate immune response. Although the function of these proteins is well-characterized, the origins of these gene acquisitions are less clear. Phylogenetic analysis revealed at least five independent PDE acquisition events by ancestral viruses. We found evidence that PDE-encoding genes were horizontally transferred between coronaviruses belonging to different genera. Three clades of viruses within Nidovirales: merbecoviruses (MERS-CoV), embecoviruses (HCoV-OC43), and toroviruses encode independently acquired PDEs, and a clade of rodent alphacoronaviruses acquired an embecovirus PDE via recent horizontal transfer. Among rotaviruses, the PDE of rotavirus A was acquired independently from rotavirus B and G PDEs, which share a common ancestor. Conserved motif analysis suggests a link between all viral PDEs and a similar ancestor among the mammalian AKAP7 proteins despite low levels of sequence conservation. Additionally, we used ancestral sequence reconstruction and structural modeling to reveal that sequence and structural divergence are not well-correlated among these proteins. Specifically, merbecovirus PDEs are as structurally divergent from the ancestral protein and the solved structure of human AKAP7 PDE as they are from each other. In contrast, comparisons of rotavirus B and G PDEs reveal virtually unchanged structures despite evidence for loss of function in one, suggesting impactful changes that lie outside conserved catalytic sites. These findings highlight the complex and volatile evolutionary history of viral PDEs and provide a framework to facilitate future studies. [ABSTRACT FROM AUTHOR]

SARS-CoV-2 nsp13 is a multifunctional helicase from helicase superfamily 1B. It unwinds the viral RNA genome for replication and is thought to play a role in 5' mRNA capping to produce mature mRNA using its triphosphatase activity. The sequence and structure are highly conserved in nidovirales and the protein is essential to the viral infection cycle, acting as a standalone enzyme and in conjunction with other SARS-CoV-2 proteins, making SARS-CoV-2 helicase a promising target for structure-based drug design. By inhibiting helicase activity, phosphatase activity, or its interaction with the RNA-dependent RNA polymerase we could interrupt viral replication. A total of 72 structures of SARS-CoV-2 nsp13 have been published in the protein databank (PDB) to date, 56 monomers and 16 as part of a complex. The structure of nsp13 is made up of five conserved folds, from N- to C-terminus, a zinc-binding domain, stalk domain, beta barrel domain 1B, RecA-like subdomain 1A, and RecA-like subdomain 1B. This review summarizes the current structural and functional knowledge surrounding SARS-CoV-2 nsp13 and related helicases, as well as the structure-based drug design efforts to date, and other complementary knowledge to provide downstream users of SARS-CoV-2 structures with a solid foundation to better inform their work. [ABSTRACT FROM AUTHOR]

The pandemic coronavirus disease 2019 outbreak's causative agent was identified as severe acute respiratory syndrome coronavirus 2. It is a positive-sense single-stranded RNA virus with a ~30 kb size genome that belongs to the Nidovirales. Molecular analysis revealed that severe acute respiratory syndrome coronavirus 2 is a variant of severe acute respiratory syndrome coronavirus and Middle East respiratory syndrome coronavirus with some sequence similarity. The confirmed cases and death toll are high in severe acute respiratory syndrome coronavirus 2 compared to severe acute respiratory syndrome coronavirus and the estimated R0 is >1. The data on pathological findings on severe acute respiratory syndrome coronavirus 2 are scarce and present treatment management is based on symptoms that are similar to severe acute respiratory syndrome coronavirus. In this review, we have discussed the transmission, viral replication, and cytokine storm and highlighted the recent pathological findings of coronavirus disease 2019. The reported severe acute respiratory syndrome coronavirus 2 pathological findings were similar to that of severe acute respiratory syndrome coronavirus. Though these findings help notify the clinical course of the disease, it warrants further in vivo and ex vivo studies with larger samples obtained from the coronavirus disease 2019 patients. [ABSTRACT FROM AUTHOR]

• Viral proteases with deubiquitinating activity suggest an antiviral immune evasive effect. • Antiviral strategies against viral proteases of nidoviruses have been explored extensively. • Ubiquitin variants show promising inhibition of nidovirus PLpro. Several viruses are now known to code for deubiquitinating proteases in their genomes. Ubiquitination is an essential post-translational modification of cellular substrates involved in many processes in the cell, including in innate immune signalling. This post-translational modification is regulated by the ubiquitin conjugation machinery, as well as various host deubiquitinating enzymes. The conjugation of ubiquitin chains to several innate immune related factors is often needed to induce downstream signalling, shaping the antiviral response. Viral deubiquitinating proteins, besides often having a primary function in the viral replication cycle by cleaving the viral polyprotein, are also able to cleave ubiquitin chains from such host substrates, in that way exerting a function in innate immune evasion. The presence of viral deubiquitinating enzymes has been firmly established for numerous animal-infecting viruses, such as some well-researched and clinically important nidoviruses, and their presence has now been confirmed in several plant viruses as well. Viral proteases in general have long been highlighted as promising drug targets, with a current focus on small molecule inhibitors. In this review, we will discuss the range of viral deubiquitinating proteases known to date, summarise the various avenues explored to inhibit such proteases and discuss novel strategies and models intended to inhibit and study these specific viral enzymes. [ABSTRACT FROM AUTHOR]

Nidoviruses comprise the most complex grouping among other viruses with respect to their multiplicity, phylogeny, systematics, species identification, genetic relationships within a taxon, progressive variability. Like other single-stranded RNA viruses, nidoviruses have a relatively high ability to mutate and recombine, which allows them to quickly adapt to new hosts and new ecological niches. Although most of the known representatives of nidoviruses are associated with terrestrial hosts, more and more data has recently appeared on nidoviruses recovered from aquatic organisms. This review is the analysis of current data on the representatives of the order Nidovirales associated with aquatic animals. They are all included in the eight families based on the current classification of viruses. The most studied among them are members of the families Coronaviridae, Tobaniviridae and Roniviridae. Representatives of the other families of aquatic animal nidoviruses were identified using metagenomic deep sequencing (metagenomics), but their effect on the host organism has not yet been adequately studied. Data on the distribution of nidoviruses among aquatic animals in different global aquatic systems are presented, clinical signs of the disease are described, a brief description of nidoviruses and their genomes is given. Nidoviruses of aquatic animals as the earliest members of the animal kingdom are supposed to have played a possible role in the evolution of terrestrial animal nidoviruses. Therefore, aquatic animal nidoviruses could play a significant role in the formation of new natural reservoirs unknown to science, as well as in their interspecies transfer between marine, freshwater and terrestrial hosts.

During infection, positive-stranded RNA causes a rearrangement of the host cell membrane, resulting in specialized membrane structure formation aiding viral genome replication. Double-membrane vesicles (DMVs), typical structures produced by virus-induced membrane rearrangements, are platforms for viral replication. Nidoviruses, one of the most complex positive-strand RNA viruses, have the ability to infect not only mammals and a few birds but also invertebrates. Nidoviruses possess a distinctive replication mechanism, wherein their nonstructural proteins (nsps) play a crucial role in DMV biogenesis. With the participation of host factors related to autophagy and lipid synthesis pathways, several viral nsps hijack the membrane rearrangement process of host endoplasmic reticulum (ER), Golgi apparatus, and other organelles to induce DMV formation. An understanding of the mechanisms of DMV formation and its structure and function in the infectious cycle of nidovirus may be essential for the development of new and effective antiviral strategies in the future., Competing Interests: Authors HO and DP were employed by the company Chongqing Jitang Biotechnology Research Institute Co., Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Wang, Chen, Qi, Li, Zhang, Zhou, Wu, Zhang, Qi, Ouyang, Xie and Pang.)

Introduction: Existing strategies to control porcine reproductive and respiratory syndrome (PRRS) are not completely effective and require alternative approaches. Although intrauterine growth restricted (IUGR) fetuses are more resilient to transplacental PRRS virus-2 (PRRSV2) infection compared to normal fetuses, the exact mechanisms are unknown. The objective of this research was to assess abundance and localization of a subset of tight junction (TJ) proteins in the maternal-fetal interface and any alterations that may affect the movement of nutrients or PRRSV2 across the epitheliochorial placenta.Methods: Paraffin-embedded samples of placenta from non-infected control (CTRL) and PRRSV2 infected fetuses (IUGR, non(N)-IUGR, meconium-stained (MEC) (n = 6 per group) were randomly selected from a large challenge trial and immunostained for claudins (CLDN) 1, 3, 4, 7 and tight junction protein 1 (TJP1). Immunostaining intensity was semi-subjectively scored by region.Results: Intensity of CLDN1 was lower in placenta of IUGR, MEC, and N-IUGR fetuses compared to CTRL, mainly in fetal epithelium and maternal endothelial cells (MECL). CLDN4 intensity was lower in MECL of IUGR compared to CTRL and MEC fetuses. TJP1 intensity was lower in maternal and fetal epithelia of placenta within IUGR, MEC, and N-IUGR fetuses versus CTRL.Discussion: Differences were mainly observed between PRRSV2 infected and non-infected groups indicating TJ integrity was affected by PRRSV2 infection. These results provide insights into the potential mechanisms of transplacental transmission of PRRSV2; however, since only CLDN4 differed amongst the infected groups, PRRSV2 induced changes in TJ integrity do not appear to explain variation in fetal outcomes after infection. [ABSTRACT FROM AUTHOR]

Viral positive-sense RNA genomes evolve rapidly due to the high mutation rates during replication and RNA recombination, which allowing the viruses to acquire and modify genes for their adaptation. The size of RNA genome is limited by several factors, including low fidelity of RNA polymerases and packaging constraints. However, the 12-kb size limit is exceeded in the two groups of eukaryotic (+)RNA viruses – animal nidoviruses and plant closteroviruses. These virus groups have several traits in common. Their genomes contain 5′-proximal genes that are expressed via ribosomal frameshifting and encode one or two papain-like protease domains, membrane-binding domain(s), methyltransferase, RNA helicase, and RNA polymerase. In addition, some nidoviruses (i.e., coronaviruses) contain replication-associated domains, such as proofreading exonuclease, putative primase, nucleotidyltransferase, and endonuclease. In both nidoviruses and closteroviruses, the 3′-terminal part of the genome contains genes for structural and accessory proteins expressed via a nested set of coterminal subgenomic RNAs. Coronaviruses and closteroviruses have evolved to form flexuous helically symmetrical nucleocapsids as a mean to resolve packaging constraints. Since phylogenetic reconstructions of the RNA polymerase domains indicate only a marginal relationship between the nidoviruses and closteroviruses, their similar properties likely have evolved convergently, along with the increase in the genome size. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Nidovirales , which accommodates viruses with the largest RNA genomes, includes the notorious coronaviruses; however, the evolutionary route for nidoviruses is not well understood. We have characterized a positive-sense (+) single-stranded (ss) RNA mycovirus, R hizoctonia s olani h ypo v irus 2 (RsHV2), from the phytopathogenic fungus Rhizoctonia solani. RsHV2 has the largest RNA genome size of 22,219 nucleotides, excluding the poly(A) tail, in all known mycoviruses, and contains two open reading frames (ORF1 and ORF2). ORF1 encodes a protein of 2,009 amino acid (aa) that includes a conserved helicase domain belonging to helicase superfamily I (SFI). In contrast, ORF2 encodes a polyprotein of 4459 aa containing the hallmark genes of hypoviruses. The latter includes a helicase belonging to SFII. Following phylogenetic analysis, the ORF1-encoded helicase (Hel1) unexpectedly clustered in an independent evolutionary branch together with nidovirus helicases, including coronaviruses, and bacteria helicases. Thus, Hel1 presence indicates the occurrence of horizontal gene transfer between viruses and bacteria. These findings also suggest that RsHV2 is most likely a recombinant arising between hypoviruses and nidoviruses. [ABSTRACT FROM AUTHOR]

The coronavirus, Severe Acute Respiratory Syndrome (SARS)-CoV-2, responsible for the ongoing coronavirus disease 2019 (COVID-19) pandemic, has emphasized the need for a better understanding of the evolution of virus-host interactions. ORF3a in both SARS-CoV-1 and SARS-CoV-2 are ion channels (viroporins) implicated in virion assembly and membrane budding. Using sensitive profile-based homology detection methods, we unify the SARS-CoV ORF3a family with several families of viral proteins, including ORF5 from MERS-CoVs, proteins from beta-CoVs (ORF3c), alpha-CoVs (ORF3b), most importantly, the Matrix (M) proteins from CoVs, and more distant homologs from other nidoviruses. We present computational evidence that these viral families might utilize specific conserved polar residues to constitute an aqueous pore within the membrane-spanning region. We reconstruct an evolutionary history of these families and objectively establish the common origin of the M proteins of CoVs and Toroviruses. We also show that the divergent ORF3 clade (ORF3a/ORF3b/ORF3c/ORF5 families) represents a duplication stemming from the M protein in alpha- and beta-CoVs. By phyletic profiling of major structural components of primary nidoviruses, we present a hypothesis for their role in virion assembly of CoVs, ToroVs, and Arteriviruses. The unification of diverse M/ORF3 ion channel families in a wide range of nidoviruses, especially the typical M protein in CoVs, reveal a conserved, previously under-appreciated role of ion channels in virion assembly and membrane budding. We show that M and ORF3 are under different evolutionary pressures; in contrast to the slow evolution of M as core structural component, the ORF3 clade is under selection for diversification, which suggests it might act at the interface with host molecules and/or immune attack. [ABSTRACT FROM AUTHOR]