key: cord-0823560-yilv9z2m
authors: nan
title: Coronaviridae
date: 2011-11-23
journal: Virus Taxonomy
DOI: 10.1016/b978-0-12-384684-6.00068-9
sha: b02fff00f306111d355bf3f5d1affab899cd066d
doc_id: 823560
cord_uid: yilv9z2m

This chapter focuses on Coronaviridae family whose two subfamilies include Coronavirinae and Torovirinae. The member genera include Alphacoronavirus, Betacoronavirus, Gammacoronavirus, Torovirus, and Bafinivirus. The members of the family Coronaviridae are enveloped and positive stranded RNA viruses of three classes of vertebrates, which include corona- and toroviruses for mammals, coronaviruses for birds, and bafiniviruses for fishes. The nucleocapsids are helical and can be released from the virion by treatment with detergents. Where the coronavirus nucleocapsid appears to be loosely wound, those of the Torovirinae are distinctively tubular. The entire replication cycle takes place in the cytoplasm and involves the production of full-length and subgenome-sized (sg) minus-strand RNA intermediates with the viral genome serving both as mRNA for the replicase polyproteins and as a template for minus-strand synthesis. Members of the family Coronaviridae all seem to share two envelope protein species, the membrane (M) and spike (S) proteins. Similarities in size, predicted structures and presumed function(s) suggest a common ancestry, and the remote, but significant sequence similarities observed for toro-, bafini-, and (to lesser extent) coronavirus S proteins lend further support to this view. The replicase polyproteins of the Coronaviridae comprise a number of characteristic domains arranged in a conserved order.

In terms of genome size and genetic complexity, the Coronaviridae are the largest RNA viruses identified so far, rivaled only by the okaviruses, large nidoviruses of invertebrates assigned to the family Roniviridae. Replication has been studied in detail only for coronaviruses, but the limited data available for toro-and bafiniviruses suggest that the latter viruses use essentially similar strategies. Virions attach to dedicated host cell surface receptors via their spikes (Table 1 ) and release their genome into the target cell via fusion of the viral envelope with the plasma membrane and/or the limiting membrane of an endocytic vesicle. The entire replication cycle takes place in the cytoplasm and involves the production of full-length and subgenome-sized (sg) minus-strand RNA intermediates with the viral genome serving both as mRNA for the replicase polyproteins and as a template for minus-strand synthesis. RNA synthesis is catalyzed by an as yet poorly characterized replication-transcription complex, composed of viral and host proteins and associated (at least in coronaviruses) with an interconnected network of modified intracellular membranes and doublemembrane vesicles that are presumably endoplasmic reticulum (ER)-derived.

The genome contains multiple ORFs. Its 5-most two-thirds are occupied by the replicase gene, which is comprised of two overlapping ORFs called 1a and 1b (Figure 1 ). The replicase gene is translated to produce polyprotein pp1a and, subject to programmed 1 ribosomal frameshifting, a C-terminally extended product, pp1ab. The polyproteins are co-and post-translationally processed by a set of virus-encoded proteinases and, thus, are not detectable as full-length proteins in virus-infected cells. The N-termini of pp1a and pp1ab are processed by one or two papain-like proteinases, whereas the C-terminal half of coronavirus pp1a and the ORF1b-encoded part of pp1ab are cleaved at 11 well-conserved sites by the main proteinase (M pro or 3CL pro ), a nidovirus-wide conserved enzyme with a chymotrypsin-like fold, a poliovirus 3C proteinase-like substrate specificity and either a serine (torovirus, bafinivirus) or a cysteine (coronavirus) as active site nucleophile. In coronaviruses, proteolytic processing results in the production of 15 (in viruses belonging to the species Avian coronavirus) or 16 mature products, commonly referred to as non-structural proteins (nsp's) and numbered according to their position -from N-to C-terminus -in the viral polyproteins ( Figure 1 ). Many nsp's are unique enzymes involved in one or more essential step(s) in viral replication. Others appear to be exclusively involved in virus-host interactions (including immune evasion) and are dispensable for virus propagation in vitro (Table 2) . Polyprotein processing in toroand bafiniviruses has not been studied in detail.

The 3-proximal genes (3 in bafiniviruses and up to at least 12 in some coronaviruses) code for the structural proteins and, in the case of coronaviruses, a variable number of "accessory" or "nichespecific" proteins. These genes are expressed -as is typical for nidoviruses -from a 3-coterminal nested set of sg mRNAs that are thought to be transcribed not from the full-length minus-strand anti-genome, but from a mirror copy set of sg minus-strand templates.

Members of the family Coronaviridae all seem to share two envelope protein species, the membrane (M) and spike (S) proteins. Similarities in size, predicted structures and presumed function(s) suggest a common ancestry, and the remote, but significant sequence similarities observed for toro-, bafini-and (to lesser extent) coronavirus S proteins lend further support to this view. Presumably, progenitors of the S and M proteins were encoded in the last common ancestor of the Corona-and Torovirinae lineages. Virus assembly involves budding of preformed nucleocapsids at membranes of the endoplasmic reticulum and early Golgi compartment and the completed virions are released via the exocytotic pathway. Nidovirus replication is discussed in more detail in paragraphs below and also in Chapter Nidovirales.

All members of the Coronaviridae family share the following characteristics: l Virions: enveloped and decorated with large (15-20 nm) surface projections. l Nucleocapsid: helical, comprised of genome and multiple copies of a single basic phosphoprotein species (N). l Envelope: contains a variable number of viral membrane protein species, two of which seem to be conserved family-wide and are essential for virion morphogenesis and/or infectivity (at least in coronaviruses): l a 200-to 250-aa triple-spanning N exo C endo integral membrane protein M l an extensively N-glycosylated, 1100-to 1600-aa class I fusion protein S which forms peplomers. l Genome: positive sense RNA, linear, unimolecular, infectious, 26-32 kb in length, capped, polyadenylated and structurally polycistronic. l General genome organization: 5-UTR-replicase-S-M-N-UTR-3 (genes named after their product), with the genome functioning as mRNA for the replicase gene. Replicase gene: comprised of overlapping ORFs 1a and 1b that code for two huge polyproteins, pp1a and pp1ab, production of the latter requiring a programmed 1 ribosomal frameshift; pp1a and pp1ab are processed autoproteolytically.

l ORFs downstream of the replicase gene: expression from a 3 co-terminal nested set of two or more subgenomic mRNAs that are capped and polyadenylated.

l Morphogenesis: virion assembly through budding of preformed nucleocapsids at smooth intracellular membranes of endoplasmic reticulum/early Golgi compartments.

The replicase polyproteins of the Coronaviridae comprise a number of characteristic domains arranged in a conserved order (see Chapter Nidovirales; see also this Chapter Figures 1, 9 and 12 and  Table 2 ). Two ORF1a-encoded replicase domains, an ADP-ribose-1-phosphatase (ADRP, also called macrodomain; located in coronavirus nsp3) and a noncanonical "secondary" RdRp with possible primase activity (coronavirus nsp8) may represent diagnostic markers that distinguish members of the family Coronaviridae from viruses in other nidovirus taxa. 

Figure 1: Coronavirus genome organization and expression. (Upper panel) Schematic representation of the genome of mouse hepatitis virus (MHV) shown as an example. ORFs are represented by boxes, indicated by number (above) and encoded protein (acronyms below). Regions encoding key domains in replicase polyproteins pp1a and pp1ab are colour-coded with hydrophobic segments shown in dark grey. The 5-leader sequence is depicted by a small red box. The arrow between ORF 1a and 1b represents the ribosomal frameshifting site. The poly(A) tail is indicated by "A(n)". Red arrowheads indicate the locations of transcription-regulating sequences (TRSs). PL (green) papain-like proteinase 1 (PL1 pro ); PL (red), papain-like proteinase 2 (PL2 pro ); A, ADP-ribose-1phosphatase (macrodomain); M pro , 3C-like main protease; Pr, noncanonical RNA-dependent RNA polymerase, putative primase; RdRp, RNA-dependent RNA polymerase; Z, zinc-binding domain; Hel, helicase domain; Exo, 3 to-5 exoribonuclease domain; N7, guanine-N7-methyltransferase; U, nidoviral uridylate-specific endoribonuclease (NendoU); MT, ribose-2-O-methyltransferase domain; HE, hemagglutinin-esterase; S, spike protein; E, envelope protein; M, membrane protein, N, nucleocapsid protein; I, internal ORF. (Lower panel) Processing of the replicase polyproteins and structural relationship between the genomic RNA and subgenomic mRNAs of coronaviruses. Arrows indicate cleavage sites for PL1 pro (green), PL2 pro (red) and M pro (blue). The locations of the non-structural proteins (nsp's) are indicated by their number (see also Table 2 ). mRNA species are numbered as by convention on the basis of their size, from large to small, with the genome designated as RNA1. For the sg mRNAs only ORF(s) that are translated are shown.

Only viruses for which a complete genome sequence is available (see Supplementary Table 1 available online on Science Direct®, www.sciencedirect.com) are to be considered for taxonomy and the following demarcation criteria are used. l Established and newly identified members of the family Coronaviridae are assigned to a subfamily and genus on the basis of rooted phylogeny and calculation of pair-wise evolutionary distances for the following Coronaviridae-wide conserved domains in replicase polyprotein pp1ab: ADRP, nsp5 (3CL pro ), nsp12 (RdRp), nsp13 (Hel), nsp14 (ExoN), nsp15 (NendoU) and nsp16 (O-MT). This procedure, developed by Lauber and Gorbalenya (in preparation) , at present unambiguously identifies 20 distinct non-overlapping clusters (with the largest intra-cluster distance being smaller than the smallest inter-cluster distance): 17 coronaviruses, 2 toroviruses, 1 bafinivirus). Likewise, the higher-rank clusters corresponding to genus and subfamily levels are recognized. l Phylogenetic outliers assigned to the family Coronaviridae may be considered representatives of a new genus when they do not cluster with any of the current genera and share less On the basis of rooted and unrooted phylogenetic trees estimated for different regions of the genome, four coronavirus (CoV) clusters can be distinguished, three of which (corresponding to the former nonofficial "groups" 1, 2 and 3) have been recognized and classified as genera (Alpha-, Betaand Gammacoronavirus, respectively). The fourth cluster comprises a number of recently identified coronaviruses of birds and by all standards appears to represent a novel (but yet to be approved) genus, provisionally named Deltacoronavirus. In the genus Betacoronavirus, four separate lineages can be discerned, designated A through D, that correspond to former subgroups 2A through D, respectively ( Figure 2 ). 

By conventional negative-staining electron microscopy, virions appear pleiomorphic, roughly spherical, 120-160 nm in diameter, with a characteristic fringe of large (ca. 20 nm), petal-shaped surface projections that are comprised of trimers of the spike (S) glycoprotein ( Figure 3 ). Group A betacoronaviruses ( Figure 2 ) display a second type of surface projection, 5-7 nm in length, comprised of the homodimeric hemagglutinin-esterase (HE) glycoprotein. Coronavirions as studied by cryo-electron tomography are homogeneous in size and spherical (envelope outer diameter 85  5 nm). The envelope is exceptionally thick (7.8  0.7 nm) in comparison to typical biological membranes (average thickness ca. 4 nm). The nucleocapsid, a loosely-wound helix, seems to be tightly folded to form a compact core that appears to be separated from the envelope by a gap of about 4 nm ( Figure 3 ).

The estimated Mr of the virion is 400  10 6 , its buoyant density in sucrose and CsCl is 1.15-1.20 g cm 3 and 1.23-1.24 g cm 3 , respectively, and its S 20,W is 300 to 500S. Particles are sensitive to heat, lipid solvents, non-ionic detergents, formaldehyde, oxidizing agents and UV irradiation. ectodomain is decorated with N-or O-linked glycans. The long C-terminal endodomain, comprising an amphiphilic region and a hydrophilic tail, is believed to associate with the inner leaflet of the membrane to form a matrix-like lattice, which would explain the remarkable thickness of the coronavirus envelope ( Figure 3 ). In transmissible gasteroenteritis virus of swine (TGEV, sp. Alphacoronavirus 1), a second population of tetra-spanning M proteins, adopting an N exo -C exo topology in the viral envelope, has been described; l the envelope protein (E), a small (74-109 aa) pentameric integral membrane protein with ion channel and/or membrane permeabilizing (viroporin) activities. With around 20 copies per particle, the E protein is only a minor structural component. Although its precise function remains to be defined, the E protein plays a role in virion assembly and morphogenesis and has been identified as a virulence factor for the severe acute respiratory syndrome-coronavirus (SARS-CoV); l the nucleocapsid protein N, a 349 to 470 aa RNA-binding phosphoprotein. Besides its obvious function in genome encapsidation, the N protein also is involved in RNA synthesis and translation, displays RNA chaperone activity, and acts as a type I interferon antagonist.

Depending on the coronavirus species, additional accessory proteins may be incorporated into the virion. Group A betacoronaviruses (Betacoronavirus 1, Murine coronavirus and Human coronavirus HKU1) code for an accessory homo-dimeric type I envelope glycoprotein, the hemagglutininesterase (HE). It mediates reversible virion attachment to O-acetylated sialic acids by acting both as a lectin and as a sialate-O-acetylesterase. The coronavirus HE shares about 30% aa sequence identity with the torovirus HE protein and is equally related to subunit 1 of the influenza C virus hemagglutinin-esterase fusion protein (HEF). In SARS-CoV, proteins 3a, 6 and 7 have been described as structural proteins and nsp2 through 5 and nsp9 were all detected in purified virion preparations.

In virions of murine coronavirus, the stoichiometric ratio of N, M and HE proteins is approximately 1:2.6:0.4; in TGEV, N and M occur at a ratio of 1:3. There are no reliable estimates for the S protein as it is present in small quantities in virus particles, may occur both in cleaved and uncleaved forms, and is easily lost during virus purification.

Coronaviruses acquire their lipid envelopes by budding at membranes of the endoplasmic reticulum, intermediate compartment and/or Golgi complex. The S and E proteins are palmitoylated.

Coronavirus S and HE proteins are heavily glycosylated and contain multiple N-linked glycans (20-35 and 5-11, respectively). The M protein of coronaviruses contains a small number of either N-or O-linked glycans, depending on the virus species, located near the amino-terminus. Coronavirus E proteins are not glycosylated.

Coronavirus genomes contain 5 and 3 UTRs ranging in size from 200 to 600 and from 200 to 500 nt, respectively. Signals for genome replication and encapsidation reside not only in these UTRs, but also in adjacent and more internal coding regions. Six ORFs are conserved subfamily-wide and arranged in a fixed order: (as listed in the 5 to-3 direction) ORFs 1a and 1b, together comprising the replicase gene, and the ORFs for the structural proteins S, E, M and N. Downstream of ORF1b and interspersed between the structural protein genes, there may be up to at least eight accessory (also called "group" or "niche-specific") genes, the products of which are generally dispensable for replication in vitro, but key to efficient replication during natural infection ( Figure 1 ).

Apparently, these accessory genes were acquired through horizontal gene transfer and occasionally also lost again as the different coronaviruses evolved and diverged while adapting to new hosts and niches. The diversity of accessory genes, most of which are specific only to a distinct CoV lineage species or strain (see also Figures 5-7) , attest to the plasticity and highly dynamic nature of the coronavirus genome.

While the genome serves as an mRNA for the replicase polyproteins, the 3 proximal genes are expressed from a nested set of sg mRNAs the coding regions of which (the "body" sequences) are

3-coterminal with the genome. Each of these mRNAs is provided with a short 5 leader sequence identical to the 5-terminal end of the genome. Leader and body sequences are not contiguous on the genome (they may in fact be separated by more than 20,000 nts), but become joined in a process of discontinuous minus-strand RNA synthesis (detailed below). Although all except the smallest mRNAs are structurally polycistronic, translation is restricted to the 5-proximal ORF(s) not present in the next smaller mRNA of the set (Figure 1 ).

On the genome, each transcription unit (one or more ORFs expressed from a single RNA species) is preceded by a short conserved sequence element, commonly called the transcription-regulating sequence (TRS). A TRS copy is also found at the 5 end of the genome, immediately downstream of the leader sequence. According to the prevailing model for transcription, leader-body fusion occurs during the synthesis of genome-templated sg minus-strand RNAs by 3-discontinuous extension via a mechanism resembling homology-assisted RNA recombination. This process apparently is driven by sequence complementarity between the anti-TRS at the 3 end of the nascent minusstrand and the 5 genomic TRS (Figure 4 ). In support of this model, the production of a 5-terminal nested set of transcriptionally-active sg minus-strand RNAs with a 3-terminal anti-leader sequence (in effect a mirror copy set of the mRNAs) has been demonstrated in coronavirus-infected cells. It is believed that each mRNA is transcribed from its corresponding sg minus-strand RNA template via a process of "continuous" RNA synthesis. For more information about other aspects of coronavirus replication, please see the preceding paragraphs and Chapter Nidovirales.

Cross-reactivity among coronaviruses is limited to (closely-related) species within the same genus. The S protein is the major inducer of virus-neutralizing antibodies that are elicited mainly by epitopes in the amino terminal half of the molecule. The surface-exposed amino-terminus of the M protein induces antibodies that neutralize virus infectivity in the presence of complement, while the HE protein of group A betacoronaviruses induces antibodies that prevent virion binding Figure 4 : Coronavirus mRNA synthesis: the discontinuous 3-extension model. Minus-strand synthesis initiates at the 3 end of the genome and proceeds until a TRS is copied (1). The nascent minus-strand RNA may then be transferred to the 5 end of the genome (2). Base complementarity allows the minus-strand RNA to anneal to the leader TRS (3) after which RNA synthesis resumes and body (in blue) and leader sequences (in red) become fused (4). The chimeric sg minus-strand RNA in turn serves as a template for "continuous" synthesis of sg mRNAs (5).

to O-acetylated sialic acids or inhibit sialate-O-acetylesterase activity. The N protein is a dominant antigen during the natural infection and while N-specific antibodies may provide little immune protection, they are of serodiagnostic relevance.

The ectodomains of the S and HE proteins are highly variable, suggestive of extensive antigenic drift. There are also indications for the occurrence of antigenic shifts as there are several examples of intra-and possibly interspecies exchange through RNA recombination of coding sequences of S (for Avian coronavirus, Murine coronavirus and the Alphacoronavirus 1 subspecies feline and canine coronavirus) and HE ectodomains (Murine coronavirus) sometimes with as yet unidentified coronaviruses serving as donors. Studies performed with murine and feline coronaviruses indicate that both structural and non-structural (replicase) proteins serve as CD4  and CD8  T cell antigens.

There is no serologic cross-reactivity between corona-, toro-and bafiniviruses.

Coronaviruses infect birds and mammals and include several pathogens of clinical, veterinary and economic interest. Transmission is not by biological vectors, but -depending on the virus speciesvia fomites or via aerogenic and/or fecal-oral routes. As CoVs primarily target epithelial cells, they are generally associated with gastrointestinal and respiratory infections that may be acute or become chronic with prolonged shedding of virus. In general, these infections are mild and often asymptomatic. Some coronaviruses, however, cause severe, even lethal disease. Murine coronavirus (genus Betacoronavirus) may cause hepatitis and severe neurologic infection, resulting in paralysis and demyelination, providing a rodent model for the study of the neuropathogenesis of human multiple sclerosis. Some members of the species Alphacoronavirus 1 (feline, canine and ferret coronavirus) cause fatal immune-mediated systemic infections in their respective hosts, presumably through the infection of cells of the macrophage/monocyte lineage, with widespread inflammatory lesions in multiple organs. The human coronaviruses that were identified early on (Betacoronavirus-1 subspecies HCoV-OC43 and Alphacoronavirus HCoV-229E) mostly cause common colds and have long been considered of modest clinical importance. It is now recognized that these viruses may also cause severe lower respiratory tract infections (LRTI) in infants and elderly, and apparently are responsible for about 5% of infant hospitalizations from LRTI, globally.

In 2002-2003, a previously unknown coronavirus, SARS-CoV, caused an epidemic in human populations of a severe pulmonary disease with a mortality rate of 10% that rapidly spread to four continents, infecting 8,096 individuals and claiming 774 victims before it was contained. Epidemiological evidence indicates that this novel human virus originated in bats, spread to Himalayan palm civets, Chinese ferret badgers and raccoon dogs at the wet markets of Guangdong, China, to enter the human population through handling or consumption of these exotic species. Although SARS has since vanished, the episode does underline the pathogenic potential of coronaviruses and the possibility of novel emerging coronavirus infections arising from cross-species transmissions. Similar incidents, though with a less dramatic outcome, seem to have given rise to human coronavirus OC43 (a single cross-species transmission of bovine coronavirus from cattle to humans), to human coronavirus 229E (transmitted from bats?) and, more recently, to canine respiratory coronavirus (transmission of bovine coronavirus to dogs). In the wake of the SARS epidemic, molecular surveillance and virus discovery studies have yielded evidence for at least 60 novel coronaviruses among which are two new human respiratory coronaviruses, HCoV-HKU1 and HCoV-NL63. The latter is considered an important cause of (pseudo)croup and bronchiolitis in children. These studies also revealed a new lineage of predominantly avian viruses (Thrush, Bulbul and Munia coronavirus), with possible relatives in mammals (Asian leopard cat, Chinese ferret badger), that on the basis of rooted phylogeny appear to belong to a new genus (Figure 2 ). Bats harbor an exceptionally wide diversity of coronaviruses and have been proposed to play a vital role in coronavirus ecology and evolution, maybe even as the original hosts from which many if not all alpha-and betacoronavirus lineages were derived. Bat population densities and their roosting and migration habits would all favor such a role. Although this hypothesis has its merits and the recent virus discovery studies that prompted this view have been of truly Herculean proportions, it is of note that the actual coronavirus sampling size remains in fact limited and as efforts so far focused mainly on bats, our present perceptions may be biased. Further surveillance studies of similar extent must be performed in other host species (rodents, birds) before final conclusions can be drawn.

Type species Alphacoronavirus 1

The viruses in this genus form a distinct monophyletic group within the Coronavirinae subfamily. Apart from their relatively close phylogenetic relationship, the only general characteristics that would set them apart from other coronaviruses are (i) a unique type of nsp1, distinct in size and sequence from betacoronavirus nsp1 and without apparent counterpart in the gammacoronaviruses, and (ii) the presence of a commonly-shared accessory gene (designated ORF3 in most alphacoronavirus species, ORF3b and 3c in TGEV and in FCoV/CCoV, respectively) for a dispensable multi-spanning alphacoronavirus membrane protein (αmp). While for some alphacoronaviruses, αmp is the only accessory protein, others may carry up to at least six accessory genes (e.g. members of the subspecies canine coronavirus in the species Alphacoronavirus 1; note that "subspecies" is not an officially recognized level in virus taxonomy; the term is used here and throughout this chapter to indicate well-defined monophyletic groups of viruses within a coronavirus species that are genetically and biologically distinct from other members of the same species). A comparison of the genome organization of alphacoronaviruses is presented in Figure 5 . ORFs for accessory proteins are named as by convention according to number (referring to the mRNA species from which they are expressed) and, in the case of multiple ORFs in one transcription unit, alphabetically. Conservation of genes is indicated by identical colouring. Accessory genes of different viruses that are located in the same genomic location but believed to encode non-related products are coloured differently. For the abbreviations of virus names, please see list of species in the genus Alphacoronavirus below. 1b, ORF1b; mp, alphacoronavirus-specific accessory membrane protein αmp; all other acronyms as in Figure 1 . 

Betacoronaviruses form a distinct monophyletic group in the Coronavirinae subfamily. Except for their relatively close phylogenetic relationship, the only known general characteristic that would set them apart from other coronaviruses is their unique nsp1, distinct in size and sequence from alphacoronavirus nsp1 and without obvious counterpart in the gammacoronaviruses. Four betacoronavirus lineages can be distinguished (A through D; Figure 2 ) each with a unique set of accessory genes ( Figure 6 ). 

Gammacoronaviruses form a distinct monophyletic group in the Coronavirinae subfamily. Except for their relatively close phylogenetic relationship, there are no known common characteristics in terms of virion morphology, genome organization and gene composition, replication or biology that would set them apart from other coronaviruses. Viruses of the species Avian coronavirus lack an nsp1 moiety. Whether this is also the case for members of the other gammacoronavirus species, Beluga whale coronavirus, remains to be determined. For the genome organization of gammacoronaviruses see Figure 7 . 

None reported. 

The members of the bigeneric subfamily Torovirinae (family Coronaviridae) form a distinct monophyletic cluster that is well-separated from the "true" coronaviruses united in the subfamily Coronavirinae (for a phylogram depicting the relationships among the Coronaviridae, see Chapter Nidovirales, Figure 4 ). Apart from their relatively close phylogenetic relationship, bafini-and toroviruses can be distinguished from their closest relatives, the true coronaviruses, by the following common features: Remarkably, in coronaviruses, related CPD and HE proteins have been identified, but exclusively in one subset of betacoronaviruses (group A) and in completely different genome locations; here, the CPD protein is encoded by an accessory gene, located immediately downstream of ORF1b (see Figure 6 ). The torovirus CPD and HE coding sequences are believed to have been acquired by horizontal gene transfer from as yet unknown donors presumably after the toro-bafinivirus split.

In conventional negative-staining electron micrographs, toroviruses appear as a mixture of spherical, rod-and kidney-shaped particles ( Figure 8A ). Native torovirus particles presumably are bacilliform with rounded ends, measuring 100-140 nm in length and 35-42 nm in width (envelope outer rim). Virions carry two types of surface projections that in size and shape resemble those of the (beta) coronaviruses: multimers of the S protein comprising large 15-20 nm peplomers and homo-dimers of the HE protein forming an inner ring of smaller (5-7 nm) spikes. The most distinctive virion element, the core, is a flexible and seemingly hollow tube of helical symmetry (periodicity ca. 4.5 nm),

about 100 nm in length and about 23 nm across with a central channel of about 10 nm in diameter. In crescent-shaped and spherical (disk-shaped?) particles, the nucleocapsid is bent into an open toroid, from which the name "torovirus" was derived (Figure 8 ).

Virions have a buoyant density in sucrose of 1.14-1.18 g cm 3 . Particles are sensitive to heat, lipid solvents, non-ionic detergents, formaldehyde, oxidizing agents and UV irradiation, but highly resistant to bile salts (0.1% deoxycholate) and extreme pH conditions (infectivity not affected by exposure to pH values between 2.5 and 10.3).

The torovirus genome is a positive-stranded, capped and polyadenylated RNA molecule of about 28 kb in length that is infectious when transfected into mammalian cells. At present, complete genomes are available only for bovine torovirus (BToV) strain Breda and equine torovirus (EToV) strain Berne. Partial sequences, mostly for the genes for the structural proteins, are available for various Eurasian BToV and porcine torovirus (PToV) field strains.

Virions of torovirus field strains contain the following protein species: (i) the spike protein S, a large (1562-1584 aa), presumably trimeric envelope glycoprotein with features typical for class I fusion proteins (bioinformatical analysis revealed heptad repeat regions and a putative fusion peptide); (ii) the hemagglutinin-esterase protein HE, a homo-dimeric 416 to 430 aa type I membrane glycoprotein that mediates reversible virion attachment to O-acetylated sialic acids by acting both as a lectin and as a receptor-destroying enzyme; (iii) the membrane protein M, a highly conserved 233-aa nonglycosylated integral membrane protein with three predicted transmembrane regions and a N exo C endo topology; (iv) the nucleocapsid protein N, a 159 to 167-aa basic RNA-binding phosphoprotein. The HE protein is dispensable for replication in vitro and its expression has been lost in EToV strain Berne presumably as a result of adaptation to replication in cultured cells. carbohydrateS respectively) .

During natural infection, toroviruses presumably attach to their host cells by binding to 9-mono-O-(PToV) or 7,9-di-O-acetylated sialic acids (BToV) via their HE protein. Entry, however, would require the S protein to bind to a main receptor, most likely a specific glycoprotein, and to mediate fusion between the viral envelope and a cellular membrane. Whether entry occurs at the plasma membrane or via endocytosis is not known nor has any torovirus main receptor been identified so far.

The viral genome contains 5 and 3 UTRs of 821-857 and 200 nt, respectively, and six ORFs called ORF1a and 1b (together comprising the replicase gene), -2, -3, -4 and -5, the latter four encoding the structural proteins (S, M, HE and N, respectively; Figure 9 ). Signals for EToV genome replication and possibly also for encapsidation apparently reside in the 5-terminal 604 and 3-terminal 200 residues as suggested by studies with defective interfering RNAs.

Replication is believed to occur largely as described for coronaviruses with the genes for the structural proteins being expressed from a nested set of four sg mRNAs (designated (m)RNAs 2 through 5) that are 3-coterminal with the genome (RNA 1). However, in striking contrast to corona-and bafiniviruses, toroviruses employ a mixed transcription strategy and combine discontinuous and continuous RNA synthesis to produce their complement of mRNAs. mRNAs 3, 4 and 5 lack a common leader sequence and are fully co-linear with the viral genome. The genes for M, HE and N, expressed from these RNAs, are each preceded by a conserved 13-14 nt sequence element, conforming to consensus (C)ACN 3-4 CUUUAGA, a copy of which (but without the 5-terminal C residue) is also found at the extreme 5 end of the genome. This sequence element is thought to act as a premature termination signal of genome-templated minus-strand synthesis and, in the resulting sg minus-strand RNAs, as a promoter for mRNA synthesis with transcription initiating at the 5-most adenine residue. The S gene lacks such an internal putative terminator/promoter (TP) element and apparently is expressed via a process similar to, yet distinct from, discontinuous RNA synthesis in coronaviruses. Its mRNA (mRNA 2) is the only sg mRNA species to carry a short 15-18 nt leader identical to the genomic 5 end (i.e. the genomic TP). A conserved hairpin structure in ORF1b is believed to attenuate minus-strand synthesis to allow a subsequent similarity-assisted template switching event facilitated by sequence complementarity between the 3 end of the nascent minus strand RNA and residues 16 through 38 of the genome that are located immediately downstream of the 5-terminal genomic TP copy. This would result in a chimeric sg minus-strand RNA that, because of its acquisition of a complementary copy of the genomic TP, can serve as template to direct 'continuous' synthesis of mRNA 2 ( Figure 9 ).

Limited information is available about torovirus morphogenesis. The M and S proteins are produced in the endoplasmic reticulum and largely retained in premedial-Golgi compartments. Assembly of torovirus virions occurs through budding of preformed nucleocapsids at smooth membranes consistent with the ER-to-Golgi intermediate compartment and/or Golgi complex. Mature particles egress by exocytosis.

EToV, PToV and BToV are serologically related. During natural infection, antibodies arise against each of the four structural proteins (S, HE, M and N) . The spike (S) protein induces virus-neutralizing antibodies; sera from BToV-or PToV-infected animals cross-neutralize EToV.

So far, torovirus infection has been conclusively demonstrated only in ungulates: horse (EToV), bovine (BToV) and swine (PToV). Evidence is based on classical virological studies (isolation and Pos. ssRNA propagation in vitro of EToV and BToV strains; experimental infection of cattle with BToV Breda), serology and molecular genetic analysis (RT-PCR amplification of torovirus sequences from fecal samples of infected animals). There is serological evidence for torovirus infections also in goats and sheep. Among the non-ungulate species that have been proposed as potential hosts for toroviruses are human (HToV), turkey and carnivores, including dog, cat, mustelids, but these claims are supported only by EM detection of torovirus-like particles and/or limited genetic analysis and would require further experimental confirmation. *** ** * ********* *** Figure 1 . The 5-leader sequence present in the genome and sg mRNA 2 is depicted by a small red box. Green and red arrowheads/ boxes indicate the locations of the internal and the 5-terminal putative terminator/promoter (TP) elements, respectively. Blue arrows indicate established M pro cleavage sites. The location of the discontinuous transcription element (DTE) driving mRNA 2 synthesis is shown by a hairpin. PL, papain-like proteinase; C, torovirus-specific ORF1a-encoded cyclic nucleotide phosphodiesterase domain. All other acronyms as in Figure 1 . (inset) Structure of the mRNA 2 discontinuous transcription element, showing the hairpin structure and downstream "homology region" with sequence identity to the 5 end of the genome indicated by asterisks. A hairpin residue-pair displaying co-variation in BToV and PToV is highlighted in green. The site of mRNA 2 leader-body fusion is indicated by an arrowhead. The 5-terminal genomic TP copy is highlighted by a red box. (Lower panel) Models for discontinuous (left) and non-discontinuous sg RNA synthesis (right) in toroviruses. The hairpin indicates the mRNA 2 DTE. Red boxes correspond to the 5-terminal genomic TP copy and complementary sequences, blue boxes to the DTE homology region and the corresponding 5 genomic acceptor sequence. The TP consensus sequence is shown and highlighted by a green box. Internal TPs and complementary sequences are shown in green. The models show (from top to bottom) synthesis of genome-templated minus-strand RNA (minus-strand RNAs indicated by a wiggly line), attenuation and 3-discontinuous extension directed by the DTE element and premature termination directed by internal TPs, and subsequent mRNA synthesis from sg minus-strand templates. Details are described in the text.

Toroviruses of horses, swine and cattle have a world-wide distribution and are evidently ubiquitous as seroprevalence in host populations may exceed 80%. Transmission is probably via the oral/ nasal route through contact with contaminated feces or nasopharyngeal secretions. Infected animals shed virus in the feces; in the case of BToV, nasal shedding has also been reported. Toroviruses are likely to cause both acute and chonic infections. Equine and porcine toroviruses are associated with asymptomatic enteric infections and remain viruses in search of a disease. Bovine torovirus, an established respiratory and enteric pathogen of cattle, may cause mild to profuse diarrhoea. The virus, originally designated Breda virus, was first isolated during an outbreak of severe neonatal gastroenteritis with 56.5% morbidity and 8.7% mortality in cattle from dairy farms round the township Breda, Iowa, and duly identified as the etiological agent. In experimentally-infected animals, BToV 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, also become infected. Neonatal calves appear to be most susceptible to clinical infection. Maternal antibodies do not prevent infection, but modify the outcome of the disease as colostrum-deprived animals are more prone to develop severe diarrhoea.

Bovine and porcine toroviruses display host species preference at least to a certain degree. In phylogenetic analyses, all PToVs cluster, while extant BToVs mostly resemble the New World BToV isolate Breda, identified 30 years ago. However, there is evidence for recurring intergenotypic/ interspecies recombination, suggesting that cross-species transmission may occur at least incidentally. Currently circulating Eurasian BToVs seem to have arisen from a genetic exchange, during which the 3 end of the HE gene, the N gene, and the 3 UTR of a Breda virus-like parent were exchanged for those of PToV. Moreover, some PToV and BToV variants carry chimeric HE genes, which apparently resulted from recombination events involving hitherto unknown toroviruses as donors. For the provisional nomenclature of BToV and PToV genotypes and a comparison of their genome organization, see Figure 10 . 

Only a modest number of toroviruses has been characterized and complete genome sequences are available solely for BToV strain Breda and EToV strain Berne. Thus far, host preference has been the main criterion for torovirus species demarcation, but future taxonomic classifications should follow the general criteria as outlined at the beginning of this chapter. According to these criteria, bovine and equine toroviruses justify their current status as distinct species.

For PToV, sequences are available only for the 3-terminal region of the genome, containing the genes for the structural proteins. In phylogenetic trees, constructed for S, HE and M genes, all PToV field strains cluster and appear to be separated from EToV and BToV by an evolutionary distance larger than that between the latter two viruses, supporting the notion that PToV represents a distinct species. The limitations of taxonomy based solely upon phylogenetic analysis of genes for the structural proteins, however, are poignantly illustrated by the occurrence of toroviruses that have acquired novel HE and N genes via interspecies recombination.

Although HToV is designated as a species, evidence for the existence of the virus remains tenuous. Available sequence data are open to varying interpretation and, in any case, are insufficient to justify classification. Future taxonomic proposals are planned to resolve the issue. 

None reported.

Type species

Virions are enveloped and bacilliform in shape (130-160  37-45 nm, excluding the surface projections; Figure 11 ). The most conspicuous virion elements are the large coronavirus-like spikes (20-25 nm) and a seemingly rigid tubular nucleocapsid of presumably helical symmetry (120-150  19-22 nm, with a central channel of 2-5 nm in diameter).

Virions have a buoyant density in sucrose of 1.17-1.19 g cm 3 and are sensitive to lipid solvents.

White bream virus DF24/00, the sole bafinivirus described to date, possesses a 26.6 kb RNA genome, which is capped, polyadenylated and infectious.

Virions contain the following protein species: (i) the spike protein S, a 1220-aa type I membrane glycoprotein, with features typical for class I fusion proteins (bioinformatical analysis revealed heptad repeat regions and a putative fusion peptide); (ii) a 227-aa integral membrane protein M with three predicted transmembrane regions; (iii) a 161-aa basic nucleocapsid protein.

WBV acquires its lipid envelope primarily by budding at intracellular membranes and, only rarely, at the plasma membrane.

From carbohydrate-specific labelling experiments, the S and M proteins appear to be glycosylated. Glycans on the S protein are recognized by the lectin concanavalin-A and thus are likely to contain α-mannose.

The WBV genome contains five ORFs called ORF 1a, 1b (together comprising the replicase gene) and -2, -3 and -4 (for the spike (S), membrane (M) and nucleocapsid protein (N), respectively). The structural proteins are expressed from three sg mRNA species that are 3 co-terminal with the genome and believed to be produced via a process of discontinuous minus-strand RNA synthesis similar to that of coronaviruses ( Figure 12 ). Each sg mRNA carries a 42-nt leader sequence identical to the 5-terminal end of the genome. A conserved nonanucleotide sequence, CA(G/A)CACUAC, located upstream of each structural protein gene and immediately downstream of the genomic 5-leader sequence, presumably represents the WBV equivalent of the coronavirus TRS core element. Bafinivirus virions assemble through the budding of preformed nucleocapsids at membranes of the ER and/or Golgi complex ( Figure 11E ) and subsequently egress via exocytosis. 

None reported.

White bream virus is the first nidovirus to be isolated from a teleost, white bream (Blicca bjoerkna L.), a species of fresh water fish (family Cyprinidae). No information is available on its ecology, biology and pathogenic properties.

Newly identified viruses are to be assigned to (or excluded from) the genus Bafinivirus on the basis of rooted phylogeny and pair-wise comparisons of Coronaviridae-wide conserved domains in replicase polyprotein pp1ab as outlined at the beginning of this chapter. 

None reported.

None reported.

None reported.

In rooted and unrooted phylogenetic trees constructed for the main replicative enzymes, members of the family Coronaviridae consistently form a monophyletic cluster that is separate from the Arteriand Roniviridae (see Chapter Nidovirales, Figure 4 ). The relatively close relationship between members of the family Coronaviridae is supported (i) by the presence of unique ORF1a-encoded enzyme domains in the replicase polyproteins that might be considered diagnostic molecular markers, i.e. the ADRP and the non-canonical RdRp/putative primase (coronavirus nsp8), and (ii) by similarities in their structural proteins (S and M for all Coronaviridae, S, M and N in the Torovirinae). On the basis of rooted phylogeny and pair-wise comparisons of Coronaviridae-wide conserved replicase domains, four well-separated monophyletic clusters can be distinguished within the subfamily Coronavirinae, three of which are established genera (Alpha-, Beta-and Gammacoronavirus). It is anticipated that the remaining cluster, comprised of recently identified avian coronaviruses (Thrush, Bulbul and Munia coronavirus) and related viruses in mammals, will be classified as a new genus (Figure 2) . Viruses in the subfamily Torovirinae (genera Bafini-and Torovirus) are phylogenetically more related to each other than to those in the subfamily Coronavirinae.

For features shared with other members of the order Nidovirales and with non-nidovirus taxa, please see Chapter Nidovirales.

Corona: from Latin corona, "halo"; refers to the characteristic appearance of surface projections that create an image reminiscent of the solar corona. Toro: from Latin torus, a term used in architecture for the convex molding at the base of a column and in geometry for a three-dimensional structure in the shape of a hollow donut; refers to the nucleocapsid morphology in a subset of particles. Bafini: from bacilliform fish nidoviruses, refers to the virion morphology and host tropism.

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