Genus Torovirus

Type Species	Equine torovirus	(EqTV)

Distinguishing Features

The nucleocapsid has a tubular appearance and virions are disc-, kidney- or rod-shaped (Fig. 5). Data are based mostly on the Equine torovirus (EqTV) strain Berne and on Bovine torovirus (BoTV) strain Breda. There are 4 structural proteins: N, M, S and HE. The HE protein is dispensable for replication in vitro; in EqTV, Berne virus strain most of the HE gene has been deleted and only the 3 terminal 426  nts are present. The toroviral and coronaviral HE proteins share 30% sequence identity and display a similar degree of sequence relatedness with subunit 1 of the hemagglutinin-esterase fusion protein of Influenza C virus. Although the nature of the presumed gene acquisition is uncertain, toroviruses and coronaviruses appear to have acquired their HE gene through independent heterologous RNA recombination events. So far, an RNA leader sequence has not been identified on the torovirus mRNAs.

Virion Properties

Morphology

Torovirus are pleomorphic and measure 120 to 140  nm at their largest diameter. Spherical, oval elongated and kidney-shaped particles are observed. The two most conspicuous features of toroviruses are the spikes on the envelope, which resemble the peplomers of coronaviruses, and the tubular nucleocapsid of helical symmetry, which appears to determine the shape of the virion. Toroviruses are enveloped viruses; an isometric core shell has not been identified, in contrast to coronaviruses.

Physicochemical and Physical Properties

Buoyant densities of 1.16, 1.18, and 1.14  g/cm³ in sucrose were determined for Equine torovirus (EqTV), Bovine torovirus (BoTV) serotype 2 and Human torovirus (HuTV), respectively.

Nucleic Acid

From sedimentation studies, the EqTV genome is estimated to be 20-25  kb in length and polyadenylated. It probably contains six ORFs. The 3 non-translated region (NTR) encompasses 200  nts, excluding the poly(A) tail. The characteristics of EqTV RNAs and ORFs are summarized in Figure 6.

The percentage of identical amino acids in the toro- and coronavirus polymerase and helicase domains is 40 to 45% (as compared to 70-90% amino acid identity between members of the genus Coronavirus).

Proteins

In the equine torovirus, four structural proteins have been identified: a Mr 180 10³ spike (S) protein of 1581 amino acids (which is post-translationally cleaved into two subunits, S1 and S2), a Mr 26 10³ integral membrane (M) protein of 233 amino acids, a hemagglutinin-esterase (HE) of Mr 65 10³ and a Mr 19 10³ nucleocapsid (N) protein of 160 amino acids. The S protein has an N-terminal signal sequence, a putative C-terminal transmembrane anchor, two putative heptad-repeat domains, and a possible cleavage site for a “trypsin-like” protease. The M protein is unglycosylated, accounts for about 13% of the virion protein mass and does not contain an N-terminal signal sequence. The HE is a class I membrane protein displaying 30% sequence identity with the hemagglutinin-esterase of coronavirus and Influenza C virus. The N protein is the most abundant structural protein of the EqTV particle, accounting for about 80% of its protein mass. It is a phosphorylated protein with RNA-binding properties.

Lipids

Virions contain lipid bilayer envelopes.

Carbohydrates

The S protein of EqTV is glycosylated and has 18 potential N-glycosylation sites. The M protein is not glycosylated. The BoTV HE protein has 7 potential N-glycosylation sites, all of which are probably used.

Genome Organization and Replication

The first two ORFs 1a and 1b from the 5-end are translated from genomic RNA and constitute the viral replicase. The ORF1a initiation codon is located at nucleotide position 825-827 in the genome. The four remaining ORFs 2, 3, 4, and 5, have been identified as structural genes (Fig. 6), and are expressed by the generation of a 3-co-terminal nested set of 4 mRNAs (Fig. 7).

Although the torovirus genome contains conserved AU-rich intergenic sequences (5uaUcUUUACa3) no evidence for fusion of a common leader to mRNA bodies at these positions has been observed. This appears to be an important difference between toroviruses and coronaviruses. In terms of transcription, however, the consequences of this dissimilarity between toro- and coronaviruses may be limited: direct binding of the polymerase to the various EqTV “core promoters” on a negative-stranded template could simply replace a leader-priming mechanism.

Evidence for two independent non-homologous recombination events during torovirus evolution have been obtained. The first putative recombination involves ORF4, the HE gene. The second putative recombination site involves the C-terminus of the EqTV ORF1a, which contains 31 to 36% identical amino acid residues compared with the N-terminal 190 amino acids of the non-structural coronavirus protein of Mr 30-32 10³.

In addition to the products of ORFs 2, 3, 4, and 5, which are assumed to be synthesized by monocistronic translation of a nested set of structurally polycistronic mRNAs, the 3 part of the EqTV genome may encode one more protein in ORF5, which completely overlaps 264  nts with N gene and potentially encodes a hydrophobic Mr 10 10³ protein. Although no such protein has been observed in virions or EqTV-infected cells, it is interesting to note that a similar situation, a small hydrophobic protein expressed from an ORF that completely overlaps with the N protein gene, has been reported for Bovine coronavirus (BCoV).

The composition of a 1-kb defective interfering (DI) genome in a replication competent virus suggests that the minimal sequences required for EqTV RNA replication (and probably also for packaging) are located in two small domains present at the termini of the genomic RNA.

Extensive N-glycosylation and proteolytic cleavage of the precursor are part of the post-translational processing of the torovirus S protein. The EqTV M protein accumulates in intracellular membranes and is thought to play a role in budding through intracellular membranes.

Antigenic Properties

The S protein is recognized by neutralizing and hemagglutination-inhibiting monoclonal antibodies.

Biological Properties

BoTV has been identified as a pathogen causing gastroenteritis in calves and possibly pneumonia in older cattle. BoTV infections are usually limited to the gut, although the respiratory system may be sporadically involved. No disease has been associated with EqTV. Serological evidence indicates that it infects ungulates (horses, cattle, sheep, goats, pigs), rats, rabbits, and some species of feral mice. Toroviruses have been detected by electron microscopy in humans, dogs and cats. The torovirus-like particles found in humans cross-react antigenically with BoTV and sequence similarity has been shown by using the polymerase chain reaction (PCR). No antibody to toroviruses has been found in the sera of cats. Infections by BoTV are also quite common in dairy cattle. The presence of maternal antibodies in calves does not prevent infection, but may modify the outcome of the disease.

Infections by BoTV appear to be ubiquitous, as evidence of infection has been obtained in every country where serological or virological studies have been done: Western Europe, North America, India, South Africa, and New Zealand.

Torovirus infects the epithelial cells lining the small and large intestine, with progression from areas of the mid-jejunum down to the ileum and colon. Within the small intestine, cells of the upper third of the crypt and the epithelium overlying the Peyer’s patches, including M cells, are also infected. Chronic torovirus infections may also occur.

List of Species Demarcation Criteria in the Genus

EqTV, BoTV, Porcine torovirus (PoTV) and Human torovirus (HuTV) are genetically and serologically closely related, but can be differentiated by sequence, host specificity, and pathogenesis. EqTV and BoTV share 84% sequence identity in the 3-most 3  kb of their genomes. PoTV is more distant as judged from the sequence of its nucleocapsid protein, which is only 68% identical to those of the EqTV and BoTV. PoTV can also be differentiated from BoTV and EqTV by the presence in these two viruses of a small ORF completely contained within the N gene. This ORF, encoding a polypeptide of approximately Mr 10 10³, is abrogated by a termination codon in PoTV. Sequencing of amplicons derived by PCR from C-terminus of the N gene and the 3 non-coding region have shown > 93% identity between HuTV, BoTV and EqTV. Nevertheless, small but consistent sequence differences were noted among five HuTV isolated and EqTV.

BoTV, PoTV and HuTV cause gastroenteritis and the BoTV sporadically infects the respiratory system, in contrast to EqTV that has remained as a virus in search of a disease.

List of Species in the Genus

Official virus species names are in italics. Tentative virus species names, alternative names ( ), strains or serotypes are not italicized. Virus names, genome sequence accession numbers [ ], and assigned abbreviations ( ) are:

Species in the Genus

Bovine torovirus	[Y10866]	(BoTV)
Breda virus		(BRV)
Equine torovirus		(EqTV)
Berne virus		(BEV)
Human torovirus		(HuTV)
Porcine torovirus		(PoTV)

Tentative Species in the Genus

Not reported.

List of Unassigned Viruses in the Family

None reported.

Distinguishing Features between Coronaviruses and Toroviruses

Coronavirus mRNAs contain a 5-leader sequence which has not been described in toroviruses. Coronaviruses have a helical nucleocapsid protected by a core shell while the toroviruses have a tubular nucleocapsid. Viruses in both genera have prominent spikes formed by large glycoproteins, but HE proteins only occur in some viruses. The N protein is much larger in coronaviruses than in toroviruses (Tables 2 and 3); the M protein is glycosylated only in coronaviruses. In general, there is little sequence similarity between coronavirus and torovirus proteins.

Phylogenetic Relationships within the Family

The sequence of a number of coronavirus and torovirus genes, has provided a database for the analysis of the phylogenetic relationships between the genera. This type of analysis is shown for the coronavirus S glycoprotein (Fig. 8). The results of this analysis correlate very well with that obtained using the N and the M genes, and confirm the conclusions derived from serological analysis. The viruses comprising Group 1 form one genetic cluster. However these viruses are clearly less closely related than Group 2 viruses, and an evolutionary divergence of, in particular, the HCoV-229E and the PEDV from the remainder of the group is suggested by the data. A closely related genetic cluster composed of Group 2 viruses (MHV, HCoV-OC43, BCoV, and TCoV) is evident in the analyses derived for all three proteins.

At the present time, there is insufficient sequence data to evaluate the phylogeny of toroviruses but the available data indicate that toroviruses are genetically as well as serologically closely related. Limited but convincing sequence similarities in some of the gene products between toroviruses and coronaviruses support the inclusion of these two genera in one family. Immunological evidence has shown that equine and bovine toroviruses are antigenically related to each other, and to torovirus-like particles found in human fecal specimens but not to other animal viruses, including coronaviruses.

Similarity with Other Taxa

None reported.

Derivation of Names

Corona, from the Latin corona for “crown”, representing the appearance of surface projections in negatively-stained electron micrographs of members of the Coronavirus genus.

Nidovirales was inspired from the Latin nidus, nest, because this order includes the largest nested-set arrangement of subgenomic mRNAs.

Toro, from the Latin torus, “lowest convex moulding in the base of a column”, representing the toroviruses nucleocapsid shape.