Discovery of the First Insect Nidovirus, a Missing Evolutionary Link in the Emergence of the Largest RNA Virus Genomes

Abstract

Nidoviruses with large genomes (26.3–31.7 kb; ‘large nidoviruses’), including Coronaviridae and Roniviridae, are the most complex positive-sense single-stranded RNA (ssRNA+) viruses. Based on genome size, they are far separated from all other ssRNA+ viruses (below 19.6 kb), including the distantly related Arteriviridae (12.7–15.7 kb; ‘small nidoviruses’). Exceptionally for ssRNA+ viruses, large nidoviruses encode a 3′-5′exoribonuclease (ExoN) that was implicated in controlling RNA replication fidelity. Its acquisition may have given rise to the ancestor of large nidoviruses, a hypothesis for which we here provide evolutionary support using comparative genomics involving the newly discovered first insect-borne nidovirus. This Nam Dinh virus (NDiV), named after a Vietnamese province, was isolated from mosquitoes and is yet to be linked to any pathology. The genome of this enveloped 60–80 nm virus is 20,192 nt and has a nidovirus-like polycistronic organization including two large, partially overlapping open reading frames (ORF) 1a and 1b followed by several smaller 3′-proximal ORFs. Peptide sequencing assigned three virion proteins to ORFs 2a, 2b, and 3, which are expressed from two 3′-coterminal subgenomic RNAs. The NDiV ORF1a/ORF1b frameshifting signal and various replicative proteins were tentatively mapped to canonical positions in the nidovirus genome. They include six nidovirus-wide conserved replicase domains, as well as the ExoN and 2′-O-methyltransferase that are specific to large nidoviruses. NDiV ORF1b also encodes a putative N7-methyltransferase, identified in a subset of large nidoviruses, but not the uridylate-specific endonuclease that – in deviation from the current paradigm - is present exclusively in the currently known vertebrate nidoviruses. Rooted phylogenetic inference by Bayesian and Maximum Likelihood methods indicates that NDiV clusters with roniviruses and that its branch diverged from large nidoviruses early after they split from small nidoviruses. Together these characteristics identify NDiV as the prototype of a new nidovirus family and a missing link in the transition from small to large nidoviruses.

Discovery of a novel nidovirus in cattle with respiratory disease

“…The expansion of the nidovirus genome has been correlated with an evolutionary procurement of enzymes controlling RNA replication fidelity. The acquisition of 39 to 59 exoribonuclease (ExoN), a homologue of a DNA proofreading enzyme, has been proposed to be of particular importance in the transition from 'small' genomes (arteriviruses) to 'intermediate' and 'large' (genomes of ,20 kb) (Gorbalenya et al, 2006;Lauber et al, 2013;Nga et al, 2011). Consistent with this hypothesis, we identified an ExoN homologue within the 'intermediate'-sized genome of BoNV.…”

“…In addition to phylogenetic classification, genome size is a distinctive feature of these viral families. Arteriviruses (genome size of 12.7-15.7 kb) have been referred to as 'small nidoviruses', mesoniviruses (,20 kb) as 'intermediate ', and both roniviruses (,26 kb) and coronaviruses (26-33 kb) as 'large ' (deGroot et al, 2011a;Gorbalenya et al, 2006;Nga et al, 2011). On the basis of genetic and phenotypic differences, the family Coronaviridae is further divided into the subfamilies Coronavirinae and Torovirinae (deGroot et al, 2011b).…”

“…If, as proposed by Lauber et al (2013), NendoU was present in an ancestral lineage that culminated in the four known families of nidoviruses, it would suggest that this domain was lost at some point in the BoNV evolutionary history. Such domain loss would not be unprecedented as NendoU has been lost in both mesoniviruses and roniviruses (Lauber et al, 2013;Nga et al, 2011). In contrast, the O-MT domain is present in all nidoviruses with 'intermediate' and 'large' genomes.…”

A Unique Nodavirus with Novel Features: Mosinovirus Expresses Two Subgenomic RNAs, a Capsid Gene of Unknown Origin, and a Suppressor of the Antiviral RNA Interference Pathway

“…The characterization of novel viruses provides important new insights into genome organization and replication strategies. For example, a new nidovirus family named Mesoniviridae has recently been identified in mosquitoes (1,2), and it uses a unique mechanism to generate subgenomic RNAs and express the spike glycoprotein (3). Another example is the discovery of clades of putatively insect-specific bunyaviruses with less complex genomes than vertebrate-infecting bunyaviruses, suggesting that they encode only the basic genes necessary for virus maintenance in insects (4,5).…”

The Coronavirus Endoribonuclease Nsp15 Interacts with Retinoblastoma Tumor Suppressor Protein

“…Coronaviruses are also of interest for their ϳ30-kb positive-strand genomes and novel mechanisms to express and process this large RNA (12,22,25). All known members of nidovirus family that includes coronaviruses encode an endoribonuclease, with the exception of Nam Dinh virus (33). A recombinant endoribonuclease of the SARS-CoV (Nsp15) cleaves RNAs immediately 3= of uridylates.…”