Nidoviruses: Maybe a new candidate for the next pandemic

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Nidoviruses form 14 virus families within the order Nidovirales, which the International Committee on Taxonomy of Viruses (ICTV) currently recognizes as comprising 48 genera and 130 species. Members of eight nidovirus families (Arteriviridae, Coronaviridae, Cremegaviridae, Gresnaviridae, Nangoshaviridae, Nanhypoviridae, Olifoviridae, Tobaniviridae) have vertebrate hosts, while those of the remaining six families (Abyssoviridae, Euroniviridae, Medioniviridae, Mesoniviridae, Monidoviridae, Roniviridae) infect invertebrates. Among these, the family Coronaviridae has garnered significant scientific and public interest due to the emergence of severe acute respiratory syndrome coronavirus (SARS-CoV) in 2002, Middle East respiratory syndrome coronavirus (MERS-CoV) in 2012, and SARS-CoV-2 in 2019. Studies on coronaviruses have long driven advancements in virology and related fields.

A notable characteristic of corona- and other nidoviruses is their large single-segment genomes. These genomes range up to the 35.9 kb genome of Aplysia abyssovirus 1 (AAbV) and the largest known RNA virus genome of 41.1 kb from the planarian secretory cell nidovirus (PSCNV). Most nidovirus genomes follow the canonical architecture: 5’ untranslated region (5’UTR), open reading frame (ORF) 1a, ORF1b, 3’-proximal ORFs (3’ORFs), and 3’UTR. Products encoded in ORF1a/b are generated through translation of the genomic RNA, including a -1 ribosomal frameshift in the ORF1a/b overlap region. The 3’ORFs are expressed via subgenomic RNAs, varying in number between nidovirus species.

Comparative genomics has been crucial in advancing our understanding of coronaviruses and other nidoviruses by assigning putative functions to many nidovirus proteins, later confirmed and elaborated through experimental studies. All nidoviruses express a conserved array of five protein domains in ORF1a/ORF1b controlling genome expression and replication. These include the 3C-like main protease (3CLpro or Mpro), the nidovirus RNA-dependent RNA polymerase (RdRp)-Associated Nucleotidyltransferase (NiRAN), the RdRp, a zinc-binding domain (ZBD), and a superfamily 1 helicase (HEL1). NiRAN and ZBD, without known virus homologs outside nidoviruses, serve as genetic markers of nidoviruses. Nidoviruses with genomes exceeding 20 kb also encode an exoribonuclease (ExoN) with proofreading activity, linked to genome expansion by improving the otherwise low fidelity of the RdRp.

Coronaviruses express four structural proteins from their 3’ORFs in the order: spike glycoprotein (S), envelope protein (E), matrix protein (M), and nucleocapsid phosphoprotein (N). This region may also encode non-essential accessory proteins. The C-terminal half of S (S2) is conserved within the family Coronaviridae and includes determinants of lipid association and infectivity, with partial conservation extending to the sister family Tobaniviridae. However, there is little to no sequence similarity between coronaviral structural proteins and those of other nidoviruses.

Evidence for homologous recombination within coronavirus species and between closely related species is accumulating, but similar evidence between different nidovirus families is lacking. Studies may be complicated by limited inter-family conservation. Heterologous recombination plays a pivotal role in generating nidovirus diversity, evident in domain duplication and restricted phyletic distribution of many conserved proteins to some nidovirus lineages. Nidoviruses, like other RNA and DNA viruses, exhibit genetic recombination patterns evident through comparative genomics, particularly quantifying incongruences in phylogenetic trees for different genome regions.

Characteristic nidoviral protein domains (ZBD and NiRAN) and phylogenetic clustering using RdRp and HEL1 allow reliable nidovirus identification through comparative genomics. Recently described nidoviruses have been discovered via bioinformatics analysis of next or third-generation sequencing data from meta-genomic and -transcriptomic studies of diverse specimens. These datasets consist of overlapping sequence fragments of variable lengths (reads) and origins, assembled into contigs, some representing full-length or partial viral genomes. Discrimination of viral from non-viral contigs typically involves sequence-based comparisons with known reference organisms. Unclassified sequences, often termed ‘dark matter,’ may include highly divergent, undescribed viruses.

Here, we introduce a highly parallelized computing workflow centered on a sequence homology search with advanced sensitivity, implementing a targeted assembly approach to reconstruct full-length viral genome sequences. Applying this approach to over 269,000 SRA datasets, we reconstructed numerous vertebrate nidovirus genome sequences. A subset includes prototype members of 18 tentative novel genera of nidoviruses, including novel coronavirus genera and five tentative novel nidovirus subfamilies. The newly discovered viruses include 11 coronaviruses with bisegmented genomes forming a monophyletic lineage within the subfamily Pitovirinae, family Coronaviridae, infecting aquatic hosts. We recovered sequences of both segments from newly generated RNA samples of 69 infected fishes and described a new coronavirus with a giant 36.1 kb genome encoding two genes with significant sequence similarity to the S gene of other corona- and tobaniviruses. The identification of leader and body transcription regulatory sequences (TRSs) in two discovered nidoviruses supports the production of subgenomic RNAs. Comparative genomics and phylogenetic analyses suggest possible structural protein swapping between corona- and tobanivirus ancestors.

The Recovered Viral Genome Sequences

This study builds on advances in sequencing technology, data management, and bioinformatics research, guiding the characterization of nidovirus life cycles and host interactions. Using raw genomic datasets of numerous organisms compiled independently for unrelated projects minimizes biases and serendipity in findings. Observations of very few single nucleotide polymorphisms in assembled sequences indicate a lack of mixed infections by closely related but different viruses in analyzed samples. Identical genome sequences recovered from independently collected samples infected with StyCoV-1 and StyCoV-2 indicate consensus sequences of newly discovered viruses. These sequences align with established protein and nucleotide variation patterns in prior coronavirus research, making them as reliable as any conventionally characterized viral genomes.

Coronaviruses with Bisegmented Genomes

Genome segmentation in positive-sense RNA viruses infecting animals was once considered rare. Discoveries of segmented flaviviruses and novel RNA virus lineages challenged this view. Reports of nidoviruses with putatively bisegmented genomes forming a sister lineage to Orthocoronavirinae lacked molecular evidence for genome segmentation. Here, we describe 12 viruses with bisegmented genomes in 11 genus-like OTUs within the subfamily Pitovirinae, family Coronaviridae, including the incorrectly annotated Pacific salmon nidovirus. Phylogenetic analysis shows genome bisegmentation as a rare evolutionary event confined to a single subfamily in known vertebrate nidoviruses. Association with aquatic hosts is notable but observed in non-segmented nidoviruses of ten other subfamilies.

Genome bisegmentation in coronaviruses might have emerged in fish, followed by an inter-class host jump into amphibians. Bisegmented coronaviruses, unlike unsegmented ones, encode a LAP1C-like coding region upstream of the S ORF, suggesting a link between LAP1C acquisition and genome bisegmentation. Segment reassortment’s role in generating genetic variation in Pitovirinae remains to be explored.

Spike Gene Exchange Between Corona- and Tobaniviruses

Homologous and heterologous recombination are main mechanisms of genetic variation, generating major genetic novelties. Incongruences in phylogenetic trees for non-structural and structural proteins and PED ratios suggest five viruses in Coronaviridae and Tobaniviridae may have exchanged the S gene with distantly related viruses. The identified viruses with recombinant S2, including HTCV with its 36 kb genome encoding two spike genes, present strong evidence for S gene recombination. Further analyses are needed to reveal the full extent of recombinant S proteins among vertebrate nidoviruses, indicating that aquatic hosts or environments might be conducive to cross-species transmissions of corona- and tobaniviruses.

Novel Protein Domains Near S Protein in Corona- and Tobaniviruses

Many novel nidoviruses encode protein domains not previously observed in nidoviruses, some in proximity to the S protein, suggesting functional links. These include new GH18 glycosidases, possibly involved in virion release or cell entry. The LAP1C-like domain, an integral membrane protein, might be related to genome bisegmentation or adaptation of bisegmented viral genomes. The US22-like protein, involved in counteracting antiviral responses, indicates a potential role in coronaviruses.

Future studies should aim to further sample nidovirus genetic diversity and advance computational tools for functional predictions of highly divergent proteins, addressing gaps in our understanding of nidovirus biology.

The SRA as a Source of Complete Viral Genomes

The SRA and similar data repositories are increasingly recognized for their potential in data-driven virus discovery. These resources offer a vast amount of data for comprehensive virus diversity descriptions, essential for accurate viral classification and understanding genetic material exchanges between viral lineages. Our computational pipeline, designed for complete genome sequence reconstruction, was crucial in revealing numerous insights discussed above.

SRA-based virus discovery provides a large-scale platform for validating genetic patterns in newly discovered viruses and linking discovered viruses to specific hosts or physiological conditions. Continued analysis of these datasets will enhance our understanding of nidovirus diversity and evolution.

Quality Standards for SRA-Based Viral Sequence Assemblies

Most SRA experiments are unrelated to virus research, resulting in low viral sequence amounts. To address this, we employed a meta-assembly approach, pooling sequencing experiments with the same virus species, and introduced metrics for ranking viral contig assembly quality. This approach, involving reference viral genome sequences, will improve with future viral sequence discoveries.

Incomplete genome fragments from the SRA, despite associated caveats, provide valuable information on unknown viral diversity. These fragments are essential for approaching a comprehensive virosphere description, akin to the role of expressed sequence tags (ESTs) in gene discovery before the human genome sequence availability.


APPENDIX 1 – Nidoviruses: Taxonomy, Structure, and Hosts

Nidoviruses form a diverse and complex order of viruses known as Nidovirales. This order is recognized by the International Committee on Taxonomy of Viruses (ICTV) and includes 14 virus families, which are further subdivided into 48 genera and 130 species. Nidoviruses are characterized by their large RNA genomes and unique replication strategies. The families within the Nidovirales order include both viruses that infect vertebrates and those that infect invertebrates.

Taxonomy and Classification

The Nidovirales order is categorized into eight suborders based on genome size and phylogenetic relationships. These suborders include:

  1. Cornidovirineae: Includes the family Coronaviridae, which is divided into the subfamilies Orthocoronavirinae (e.g., SARS-CoV, MERS-CoV) and Letovirinae.
  2. Arnidovirineae: Includes families such as Arteriviridae, Cremegaviridae, and Olifoviridae.
  3. Nanidovirinae: Contains the families Nanghoshaviridae and Nanhypoviridae.
  4. Mesnidovirineae: Includes Mesoniviridae and Medioniviridae.
  5. Ronidovirineae: Contains the families Roniviridae and Euroniviridae.
  6. Monidovirineae: Includes Mononiviridae.
  7. Tornidovirineae: Contains Tobaniviridae.
  8. Abnidovirineae: Contains Abyssoviridae.

The Nidovirales order features the largest RNA genomes among known viruses, with lengths ranging from 12.7 kb in Arteriviridae to over 41.1 kb in Planarian secretory cell nidovirus (PSCNV)​.

Structural Characteristics

Nidoviruses possess enveloped, positive-sense single-stranded RNA genomes. They share common genomic organization and replication strategies, which include:

  • Transcription of 3′-nested subgenomic RNAs.
  • Encoding a large polyprotein that is cleaved into functional nonstructural proteins (NSPs).
  • Presence of unique protease activities and replication enzymes such as RNA-dependent RNA polymerase (RdRp) and helicase​​.

Coronaviruses, a prominent family within this order, have been extensively studied for their structural proteins, which include the spike (S) protein, membrane (M) protein, envelope (E) protein, and nucleocapsid (N) protein. The S protein facilitates receptor binding and membrane fusion, while the M protein is involved in viral assembly​​.

Host Range and Pathogenicity

Nidoviruses exhibit a broad host range, infecting various vertebrates and invertebrates. The vertebrate-infecting families include:

  • Arteriviridae: Infects mammals, causing diseases like equine arteritis and porcine reproductive and respiratory syndrome (PRRS).
  • Coronaviridae: Infects mammals, birds, and fish, with notable members like SARS-CoV, MERS-CoV, and the recent SARS-CoV-2 causing significant respiratory diseases in humans.
  • Tobaniviridae: Includes viruses that infect reptiles and other vertebrates.

Invertebrate-infecting families include:

  • Roniviridae: Infects crustaceans.
  • Mesoniviridae: Primarily found in mosquitoes and other insects​.

Recent Findings and Future Directions

Recent studies have expanded the understanding of nidovirus diversity and evolution. Advanced genomic analyses have revealed complex evolutionary relationships and host-switching events. Continued research is expected to uncover new species and provide deeper insights into nidovirus pathogenesis and cross-species transmission​​.

In summary, the Nidovirales order comprises a vast and diverse group of viruses with significant implications for both human and animal health. Understanding their taxonomy, structure, and host interactions is crucial for developing strategies to combat nidovirus-related diseases.


reference link : https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1012163

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