Taxonomic Structure of the Order
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Order |
Nidovirales |
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Family |
Coronaviridae |
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Genus |
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Genus |
Torovirus |
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Family |
Arteriviridae |
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Genus |
Arterivirus |
The order comprises two families of viruses. The characteristics which define the Nidovirales are:
1. |
Linear, non-segmented, positive-sense, ssRNA genomes. |
2. |
The genome organization from 5- to 3-end contains a replicase gene followed by the structural protein genes. Other non-structural genes are variable in number and may be localized at various positions downstream of the replicase gene. |
3. |
A 3 co-terminal nested set of four or more subgenomic mRNAs. |
4. |
The genomic RNA functions as the mRNA for the translation of the replicase. |
5. |
In general, only the 5 unique regions of the mRNAs, i.e., those absent from the next smaller RNA, are translated. |
6. |
Possession of a virion envelope. |
7. |
An integral membrane protein which spans the membrane at least three times. |
8. |
3 polyadenylation of the genomic RNA. |
9. |
Gene 1 encodes the viral replicase from two large ORFs 1a and 1b with ribosomal frameshifting operating to translate the second ORF. |
10. |
The ORF1b encodes the putative polymerase and helicase domains, the latter being downstream of the polymerase domain. The canonical GDD polymerase motif is SDD. |
The Nidovirales are enveloped viruses with an architecture that shows various similarities and differences, depending on whether the external apperance or the nucleocapsid of the virions is studied (Fig. 1). The two genera of the family Coronaviridae (Coronavirus and Torovirus) show large projections protruding from the envelope (peplomers) formed, at least in coronaviruses, by trimers of the spike protein. These oligomerized structures provide them with the characteristic “crown” observed by electron microscopy that inspired the name of the family. In contrast, the members of the family Arteriviridae have shorter spike proteins protruding from the envelope which do not form easily visible peplomers. Coronaviruses have a core shell that protects an internal nucleocapsid with helical symmetry. The torovirus nucleocapsid shows an unusual morphology resembling a toroid, which inspired their name. In the family Arteriviridae a core shell probably icosahedral, has been identified that contains the genome.
Vitrified coronavirus particles have a diameter of 145 nm (including the extended peplomers) and an internal core shell of 65 nm, which is possibly icosahedral. Within the core shell coronaviruses have a helical nucleocapsid. Toroviruses have a virion size similar to coronaviruses, while arterivirus virions are significantly smaller with a complete particle of 50-70 nm in diameter, a nucleocapsid of 25-35 nm in diameter and lacking surface projections. Cup-like structures with a diameter of 10-15 nm have been observed in arteriviruses.
Physicochemical and Physical Properties
The coronavirus virion Mr is 400 106, the buoyant density in sucrose is 1.15 to 1.20 g/cm3, the density in CsCl is 1.23 to 1.24 g/cm3, and viron S20W is 300 to 500S. Toroviruses have buoyant densities of 1.14-1.18 g/cm3 in sucrose. Arterivirus virion buoyant density is about 1.13-1.17 g/cm3 in sucrose and 1.17 to 1.20 g/cm3 in CsCl. Virion S20W is 200 to 300S. Nidovirales virions are sensitive to heat, lipid solvents, non-ionic detergents, formaldehyde, oxidizing agents and UV irradiation.
The genome of the members of the Order Nidovirales is an infectious, linear, positive-sense, polyadenylated and, at least for arteri- and coronaviruses, 5 capped ssRNA molecule. The size of the Nidovirales genomes are 27.6 to 31 kb (Coronavirus), 20 to 25 kb (Torovirus), and 13 to 16 kb (Arterivirus). The coronavirus genome is the largest known non-fragmented viral RNA genome. The complete sequence of the genome of several coronaviruses (Murine hepatitis virus, MHV; Transmissible gastro-enteritis virus, TGEV; Infectious bronchitis virus, IBV; and Human coronavirus 229E, HCoV-229E) and arteriviruses (Equine arteritis virus, EAV; Lactate dehydrogenase-elevating virus, LDV; Porcine reproductive and respiratory syndrome virus, PRRSV) have been determined and are available from international databases.
The nidovirus proteins are summarized in Table 1.
The viral envelope of coronaviruses contains three to four proteins. The spike proteins (S) of coronaviruses and toroviruses have a highly exposed globular domain and a stem portion containing heptad repeats, indicative of a coiled-coil structure. The membrane (M) proteins of coronaviruses and toroviruses are different in sequence but alike in size, structure and function. The M proteins have a similar triple- or quadruple-spanning membrane topology. In addition, coronaviruses have a small structural protein (E) within the envelope (around 20 per virion). Toroviruses seem to lack a homologue for the E protein. Some coronaviruses (MHV; Human coronavirus OC43, OC43; Bovine coronavirus, BCoV) and toroviruses contain another membrane protein with hemagglutinin-esterase activity (HE).
The structural proteins of arteriviruses are apparently unrelated to those of the members of the family Coronaviridae. There are three envelope proteins common to all arteriviruses: (1) a 16 to 20 103 nonglycosylated membrane protein (M) which transverses the membrane three times and thus structurally resembles the M protein of corona- and toroviruses; (2) a heterogeneously N-glycosylated triple-spanning protein (designated large glycoprotein, GL for EAV) of variable size, and (3) a class I glycoprotein of 25-30 103 (designated small glycoprotein, GS for EAV) which is a minor virion component. The GL and M proteins associate into disulphide-linked heterodimers and probably form the cup-like structures observed on the virion surface.
Nidovirales have lipid envelopes. The S protein (MHV, BCoV) and the E protein (MHV) of coronaviruses are acylated (palmitic acid).
Coronavirus S and HE proteins contain N-linked glycans, the S protein being heavily glycosylated (about 20-35 glycans). The M protein of coronaviruses contains a small number of either N- or O-linked glycans, depending on the species. These side chains are located near the amino-terminus, but the M protein of TGEV also has a potential glycosylation site in the carboxy-terminus. At present there is no evidence to suggest that the E protein is glycosylated or phosphorylated.
Torovirus S protein has 18 potential N-glycosylation sites. Also their HE protein (Bovine torovirus, BoTV) is N-glycosylated and binds 9-O-acetylated receptors, but their M protein is not glycosylated. In the arteriviruses the GS and GL contain N-linked glycans. GL (EAV) results from heterogeneous N-acetyllactosamine addition. The M protein is not glycosylated.
Genome Organization and Replication
Despite the differences in genetic complexity and gene composition, the genome organization of corona-, toro, and arteriviruses are remarkably similar (Fig. 2). Two thirds of each genome encode two large ORFs, designated ORF1a and ORF1b. The more downstream ORF1b, is only expressed after a-1 frameshift introduced by a slippery sequence and a pseudoknot structure. The polypeptides encoded by these ORFs are proteolytically cleaved by virus-encoded proteinases to yield the mature proteins involved in viral RNA synthesis.
Downstream of ORF 1b there are four to nine genes that encode the structural proteins and, at least for coronaviruses, a number of nonstructural proteins. These genes are expressed from a 3 coterminal nested set of subgenomic mRNAs. Although these mRNAs are structurally polycistronic, translation is restricted, with a few exceptions, to the unique 5 sequences not present in the next smaller RNA of the set.
The sgRNAs of corona- and arteriviruses carry a 5-leader sequence of 55 to 92 and 170 to 221 nts, respectively, which are derived from the 5-ends of the viral genomes. The mRNA synthesis thus requires a discontinuous transcription event. The torovirus mRNAs seem to lack an extensive 5-leader sequence. Both coronaviruses and arteriviruses contain conserved AU-rich sequences at the fusion sites of leader and mRNA bodies. These sequences are named intergenic (IG) sequences in coronaviruses and leader-to-body junction (LBJ) sites in arteriviruses. Cells infected by arteriviruses or coronaviruses contain negative-stranded RNAs which correspond to each mRNA and which comprise the templates for mRNA synthesis. In coronaviruses, there is strong evidence suggesting that there are replicative forms (RF) with the size of the different mRNAs corresponding to replicative intermediaries (RI), i.e., serving as templates for transcription.
Recent coronavirus and arterivirus UV transcription mapping at late times during infection showed a clear correlation between UV target size and physical size for the subgenomic RNAs. However, a quantitative analysis indicated that for both arteriviruses and coronaviruses this correlation does not justify the conclusion that in both arteriviruses and coronaviruses the subgenomic RNA transcription is fully independent from the synthesis of the genomic length RNA. Nevertheless, splicing can be ruled out as the major mechanism for coronavirus and arterivirus subgenomic RNA production late in infection.
This information has lead to the proposal of two major models for coronavirus and arterivirus mRNA synthesis. One model is leader-primer transcription. According to this model, transcription of the leader RNA would begin at the 3-end of the full-length, minus-strand template RNA and terminate with the dissociation of the leader from template, either alone or with attached polymerase proteins. The leader would then bind to intergenic sequences downstream on the minus-strand template and serve as the primer for mRNA transcription. An alternative model for coronavirus RNA synthesis postulates discontinuous transcription during minus-strand RNA synthesis. During minus-strand RNA synthesis from the genomic template, the polymerase would pause at IS or LBJ sequences and then jump to the 3-end of the leader sequence near the 5-end of the genomic RNA template, generating subgenomic minus-strand RNAs with an antisense leader sequence at its 3-end. These minus-strand sgRNAs and full-length minus strand could then serve as templates for uninterrupted transcription of the plus-strand mRNA and genomic RNA, respectively. In this model, the intergenic sequence could serve as the transcriptional termination sequence. Data available at this time do not unequivocally rule out either model of transcription. Possibly, elements of each of these models will be found to be correct.
The overlapping ORFs 1a and 1b found at the 5-end of the Nidovirales genome are frequently referred to as the “replicase gene”. The processing of the encoded polyproteins yields both proteins required for RNA synthesis as well as a number of additional products involved in other aspects of virus replication.
Comparison of the replication strategies and replicase properties of corona-, toro-, and arteriviruses has clearly distinguished the families composing the Nidovirales order from those of alpha-, picorna-, and flaviviruses. The organization of the Nidovirales replicase in two ORFs, expression of the gene by ribosomal frameshifting, and the arrangement of conserved domains within the gene product are unique.
Although arterivirus replicase genes are considerably smaller (9.5 to 12 kb) than their toro- and coronavirus counterparts, they contain a number of conserved domains that are present in the same relative positions. The sequence alignments of these domains reveal up to 30% amino acid sequence identity in the most conserved regions, a percentage that cannot be due to convergent evolution. The conservation of two of these domains (polymerase and helicase), which are common to all positive-stranded RNA viruses, is not very surprising; their presence indicates that these viruses have probably all descended from the same RNA virus prototype. It is remarkable, however that only in Nidovirales replicases is the helicase domain located downstream of the polymerase motif. The polymerase motif also carries another Nidovirales characteristic: the substitution of the classic GDD in the core motif by SDD. Also, the conservation of additional replicase domains, for example, the carboxyl-terminal ORF1b domain, for which no homologue can be found in other viral replicases, clearly indicates that the Nidovirales replicases are more related to each other than to any other group of positive-stranded RNA viruses.
Nidovirales replicase gene expression leads to the production of an ORF1a/1b fusion protein that is large in the case of arteriviruses (Mr 345-420 103) and extremely large in coronaviruses (Mr 740 to 810 103), and probably also in toroviruses. The presence of multiple protease domains in the ORF1a proteins of both coronaviruses and arteriviruses and their extensive proteolytic processing has been proven in the regulation of Nidovirales replicase function. The preliminary processing scheme of Nidovirales ORF1a/1b protein comprises between 10 to 20 proteolysis-derived cleavage products resulting from different proteases supergroups (papain-like and chymotrypsin-like) proteolytic enzymes.
Unfortunately, only the 5- and 3-sequences of Equine torovirus (EqTV) ORF1a region (about 1 kb from each end) have been determined so far and no putative proteases have been identified in these regions. The N-terminal half of the ORF1a protein is quite variable. This variability is largely responsible for the size differences in corona-, or arterivirus genomes. A comparison between the coronavirus and arterivirus N-terminal ORF1a protein sequences does not yield any significant similarities, and even within the coronavirus and arterivirus groups there is little conservation in this region.
Amino acid sequence comparisons show that the 1b polyproteins of corona-, toro-, and arteriviruses are essentially colinear. The sequence conservation between the more closely related corona- and toroviruses is clustered in six domains, four of which are also found in the arterivirus ORF1b: the “classical” RNA-dependent RNA polymerase and helicase domains, which are also present in the polymerases of most other viruses, a zinc finger motif, and a short region of 80-100 residues, which has not yet been identified in other viral polymerases and is a conserved G-terminal nidovirus.
Coronavirus, torovirus and arterivirus are not serologically related although their sequence identity may be 30% in selected domains.
Coronaviruses have four structural proteins: (1) the spike (S) protein forms trimers and is the major inducer of virus-neutralizing antibodies which are elicited by several domains located at the amino-terminus half of the molecule; (2) the membrane (M) protein has three or four intra-membrane domains being exposed at the virus surface either the amino-terminus alone or both the amino-terminus and the carboxy-terminus. Most of the antibodies elicited by the M protein are directed to the carboxy-terminus. In general, polyvalent or monovalent antibodies to the amino-terminus weakly neutralize virus infectivity, but in the presence of complement they reduce infectivity around 100-fold; (3) nucleoprotein (N) is a dominant antigen during virus infection; a N peptide is exposed on the surface of infected cells and induces protective T cell responses; (4) the envelope (E) protein is also exposed at the surface of virus and virus infected cells. In the case of MHV E protein-specific antiserum neutralizes viral infectivity in the presence of complement.
The four toroviruses described (BoTV, EqTV, Porcine torovirus, PoTV and Human torovirus, HuTV) are serologically related. Toroviruses have four structural proteins: (1) the spike (S) protein of Mr 180 103 that forms large 17 to 20 nm spikes and induce virus-neutralizing and hemagglutination inhibiting antibodies, (2) the Mr 26 103 triple spanning integral membrane protein (M), (3) a Mr 65 103 class I membrane protein (HE) exhibiting acetylesterase activity; this protein in BoTV virions forms short surface projections of 6 nm on average that acts as a prominent antigen during infection, and (4) the Mr 19 103 nucleocapsid protein.
The known arteriviruses infecting different species (EAV, LDV, PRRSV, and Simian hemorrhagic fever virus, SHFV) do not cross-react. Arteriviruses have at least four structural proteins: (1) a Mr 25 103 glycoprotein (GS) that induces virus-neutralizing antibodies; (2) a second glycoprotein (GL) which due to heterogeneous glycosylation has sizes between Mr 30 and 42 103 and also induces virus-neutralizing antibodies; (3) an unglycosylated transmembrane (M) protein; and (4) a Mr 12 103 nucleocapsid (N) protein involved in the induction of protection.
Coronaviruses infect many mammals, including humans. The main targets are epithelial cells, and consequently respiratory and gastrointestinal organ disorders result. Biological vectors are not known. Respiratory, fecal-oral and mechanical transmission are common. Swine and domestic fowl may become persistently infected with TGEV and IBV, respectively, and shed virus from the enteric tract. Hepatitis (MHV), heart and eye (Rabbit coronavirus, RbCoV) infections have also been described.
Toroviruses infect ungulates: horses (EqTV), bovines (BoTV) and swine (PoTV). Humans (HuTV) and probably carnivores (mustellids) are also hosts for toroviruses. The transmission is probably by the fecal-oral route.
Arteriviruses infect horses (EAV), mice (LDV), monkeys (SHFV) and swine (PRRSV). EAV causes necrosis in muscle cells of small arteries leading to extremely variable clinical signs. A fatal outcome of the disease has been reported in both natural and experimental infections, but most natural infections are either mild or subclinical. Primary host cells are macrophages. Persistent infections are frequently established. Spread is in general horizontal (respiratory, biting), by venereal routes and semen (EAV), and via the reproductive apparatus (PRRSV). Stallions may become persistently infected and shed virus from their reproductive tract. In pregnant animals arteriviruses (PRRSV and EAV) can cause abortions.
None reported.
Arteri, from equine arteritis, the disease caused by the type species virus.
Corona, derived from Latin corona, meaning crown, representing the appearance of surface projections of the virus particles.
Nido, from Latin nidus, meaning “nest”, representing the nested set of mRNAs.
Toro, from Latin torus, the lowest convex moulding in the base of a column, refers to the nucleocapsid morphology.
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