The severe acute respiratory syndrome coronavirus (SARS-CoV) open reading frame 3a protein has recently been shown to be a structural protein. The protein is encoded by one of the so-called group-specific genes and has no sequence homology with any of the known structural or group-specific proteins of coronaviruses. It does, however, have several similarities to the coronavirus M proteins; (i) they are triple membrane spanning with the same topology, (ii) they have similar intracellular localizations (predominantly Golgi), (iii) both are viral structural proteins, and (iv) they appear to interact with the E and S proteins, as well as with each other. The M protein plays a crucial role in coronavirus assembly and is glycosylated in all coronaviruses, either by N-linked or by O-linked oligosaccharides. The conserved glycosylation of the coronavirus M proteins and the resemblance of the 3a protein to them led us to investigate the glycosylation of these two SARS-CoV membrane proteins. The proteins were expressed separately using the vaccinia virus T7 expression system, followed by metabolic labeling. Pulse-chase analysis showed that both proteins were modified, although in different ways. While the M protein acquired cotranslationally oligosaccharides that could be removed by PNGaseF, the 3a protein acquired its modifications posttranslationally, and they were not sensitive to the N-glycosidase enzyme. The SARS-CoV 3a protein, however, was demonstrated to contain sialic acids, indicating the presence of oligosaccharides. O-glycosylation of the 3a protein was indeed confirmed using an in situ O-glycosylation assay of endoplasmic reticulum-retained mutants. In addition, we showed that substitution of serine and threonine residues in the ectodomain of the 3a protein abolished the addition of the O-linked sugars. Thus, the SARS-CoV 3a protein is an O-glycosylated glycoprotein, like the group 2 coronavirus M proteins but unlike the SARS-CoV M protein, which is N glycosylated.

ABSTRACT Viruses of the order Nidovirales encode huge replicase polyproteins. These are processed primarily by the chymotrypsin-like main proteinases (Mpros). So far, Mpros have been studied only for corona-, arteri-, and roniviruses. Here, we report the characterization of the Mpro of toroviruses, the fourth main Nidovirus branch. Comparative sequence analysis of polyprotein 1a of equine torovirus (EToV) strain Berne, identified a serine proteinase domain, flanked by hydrophobic regions. Heterologous expression of this domain resulted in autoprocessing at flanking cleavage sites. N-terminal sequence analysis of cleavage products tentatively identified FxxQ↓(S, A) as the substrate consensus sequence. EToV Mpro combines several traits of its closest relatives. It has a predicted three-domain structure, with two catalyticβ -barrel domains and an additional C-terminal domain of unknown function. With respect to substrate specificity, the EToV Mpro resembles its coronavirus homologue in its preference for P1-Gln, but its substrate-binding subsite, S1, more closely resembles that of arteri- and ronivirus Mpros, which prefer P1-Glu. Surprisingly, in contrast to the Mpros of corona- and roniviruses, but like that of arterivirus, the torovirus Mpro uses serine instead of cysteine as its principal nucleophile. Under the premise that the Mpros of corona- and toroviruses are more closely related to each other than to those of arteri- and roniviruses, the transition from serine- to cysteine-based proteolytic catalysis (or vice versa) must have happened more than once in the course of nidovirus evolution. In this respect, it is of interest that a mutant EToV Mpro with a Ser165→Cys substitution retained partial enzymatic activity.

Group 2 coronaviruses encode an accessory envelope glycoprotein species, the hemagglutinin esterase (HE), which possesses sialate-O-acetylesterase activity and which, presumably, promotes virus spread and entry in vivo by facilitating reversible virion attachment to O-acetylated sialic acids. While HE may provide a strong selective advantage during natural infection, many laboratory strains of mouse hepatitis virus (MHV) fail to produce the protein. Apparently, their HE genes were inactivated during cell culture adaptation. For this report, we have studied the molecular basis of this phenomenon. By using targeted RNA recombination, we generated isogenic recombinant MHVs which differ exclusively in their expression of HE and produce either the wild-type protein (HE+), an enzymatically inactive HE protein (HE0), or no HE at all. HE expression or the lack thereof did not lead to gross differences in in vitro growth properties. Yet the expression of HE was rapidly lost during serial cell culture passaging. Competition experiments with mixed infections revealed that this was not due to the enzymatic activity: MHVs expressing HE+ or HE0 propagated with equal efficiencies. During the propagation of recombinant MHV-HE+, two types of spontaneous mutants accumulated. One produced an anchorless HE, while the other had a Gly-to-Trp substitution at the predicted C-terminal residue of the HE signal peptide. Neither mutant incorporated HE into virion particles, suggesting that wild-type HE reduces the in vitro propagation efficiency, either at the assembly stage or at a postassembly level. Our findings demonstrate that the expression of "luxury" proteins may come at a fitness penalty. Apparently, under natural conditions the costs of maintaining HE are outweighed by the benefits.

ABSTRACT Toroviruses (order Nidovirales ) are enveloped positive-strand RNA viruses of mammals. The prototype torovirus, equine torovirus strain Berne (Berne virus [BEV]), uses two different transcription strategies to produce a 3′-coterminal nested set of subgenomic (sg) mRNAs. Its mRNA 2 carries a leader sequence derived from the 5′ end of the genome and is produced via discontinuous transcription. The remaining three sg mRNAs, 3 to 5, are colinear with the 3′ end of the genome and are made via non-discontinuous RNA synthesis. Their synthesis is supposedly regulated by short conserved sequence motifs, 5′-ACN 3-4 CUUUAGA-3′, within the noncoding intergenic regions that precede the M, HE, and N genes (A. L. van Vliet, S. L. Smits, P. J. Rottier, and R. J. de Groot, EMBO J. 21: 6571-6580, 2002). We have now studied the—for nidoviruses unusual—non-discontinuous transcription mechanism in further detail by probing the role of the postulated transcription-regulating sequences (TRSs). To this end, we constructed a synthetic defective interfering (DI) RNA, carrying a 24-nucleotide segment of the intergenic region between the HE and N genes. We demonstrate that this DI RNA, when introduced into BEV-infected cells, directs the synthesis of a sg DI RNA species; in fact, a 16-nucleotide cassette containing the TRS already proved sufficient. Synthesis of this sg DI RNA, like that of mRNAs 3 to 5 of the standard virus, initiated at the 5′-most adenylate of the TRS. An extensive mutational analysis of the TRS is presented. Our results provide first and formal experimental evidence that the conserved motifs within the BEV intergenic sequences indeed drive sg RNA synthesis.

ABSTRACT Murine hepatitis virus (MHV) infection provides a model system for the study of hepatitis, acute encephalitis, and chronic demyelinating disease. The spike glycoprotein, S, which mediates receptor binding and membrane fusion, plays a critical role in MHV pathogenesis. However, viral proteins other than S also contribute to pathogenicity. The JHM strain of MHV is highly neurovirulent and expresses a second spike glycoprotein, the hemagglutinin esterase (HE), which is not produced by MHV-A59, a hepatotropic but only mildly neurovirulent strain. To investigate a possible role for HE in MHV-induced neurovirulence, isogenic recombinant MHV-A59 viruses were generated that produced either (i) the wild-type protein, (ii) an enzymatically inactive HE protein, or (iii) no HE at all (A. Lissenberg, M. M. Vrolijk, A. L. W. van Vliet, M. A. Langereis, J. D. F. de Groot-Mijnes, P. J. M. Rottier, and R. J. de Groot, J. Virol. 79:15054-15063, 2005 [accompanying paper]). A second, mirror set of recombinant viruses was constructed in which, in addition, the MHV-A59 S gene had been replaced with that from MHV-JHM. The expression of HE in combination with A59 S did not affect the tropism, pathogenicity, or spread of the virus in vivo. However, in combination with JHM S, the expression of HE, regardless of whether it retained esterase activity or not, resulted in increased viral spread within the central nervous system and in increased neurovirulence. Our findings suggest that the properties of S receptor utilization and/or fusogenicity mainly determine organ and host cell tropism but that HE enhances the efficiency of infection and promotes viral dissemination, at least in some tissues, presumably by serving as a second receptor-binding protein.

ABSTRACT We describe the natural history, viral dynamics, and immunobiology of feline infectious peritonitis (FIP), a highly lethal coronavirus infection. A severe recurrent infection developed, typified by viral persistence and acute lymphopenia, with waves of enhanced viral replication coinciding with fever, weight loss, and depletion of CD4 + and CD8 + T cells. Our combined observations suggest a model for FIP pathogenesis in which virus-induced T-cell depletion and the antiviral T-cell response are opposing forces and in which the efficacy of early T-cell responses critically determines the outcome of the infection. Rising amounts of viral RNA in the blood, consistently seen in animals with end-stage FIP, indicate that progression to fatal disease is the direct consequence of a loss of immune control, resulting in unchecked viral replication. The pathogenic phenomena described here likely bear relevance to other severe coronavirus infections, in particular severe acute respiratory syndrome, for which multiphasic disease progression and acute T-cell lymphopenia have also been reported. Experimental FIP presents a relevant, safe, and well-defined model to study coronavirus-mediated immunosuppression and should provide an attractive and convenient system for in vivo testing of anticoronaviral drugs.

We have measured antiviral CD8 T cells responses in bovine respiratory syncytial virus (bRSV) infected calves that had been immunized with either formalin-inactivated (FI) or live-attenuated (L) bRSV, with evidence of immunopathology following challenge of calves vaccinated with FI-bRSV. In all cases, bRSV infection induced potent pulmonary CD8 T cell responses. The kinetics of the post-challenge response in L-bRSV immunized animals was accelerated compared to the FI-bRSV and PBS groups, suggesting that only the L-bRSV vaccine, and not the FI-bRSV vaccine, had primed memory T cells. The differences between primary and post-vaccination secondary infection were very minor, in terms of the proliferation status of pulmonary CD8 T cells. Functional IFN-gamma+ CD8 responses were slightly higher in the FI-bRSV vaccinated animals. Furthermore, the existence of strong IFN-gamma+ CD8 responses in FI-bRSV vaccinated animals after challenge suggests (i) that these IFN-gamma+ responses in FI-bRSV immunized animals do not protect against immunopathology, and (ii) that Th-2 biased responses during bRSV challenge after vaccination with FI-bRSV have a limited impact on the CD8 responses in the bronchoalveolar lavage fluid. Thus, several response patterns (Th-l/Th-2) seem to co-exist during bRSV infection.

We describe a three-color flow cytometry assay for the detection of virus-specific CD4+ and CD8+ T cells in the cat. The assay is based upon detection of intracellular TNFalpha using the cross-reactive mAb 6401.1111, raised against the human cytokine. Allophycocyanin-conjugated mAb 6401.1111 specifically stained feline TNFalpha-producing murine cells and also Staphylococcus aureus Enterotoxin B-stimulated feline T cells, thus providing formal evidence for cross-reactivity. By using the anti-TNFalpha mAb in combination with PE- and FITC-conjugated mAbs against feline CD4 and CD8, respectively, antiviral CD4+ and CD8+ T cells could be identified in the peripheral blood and in the spleens of feline infectious peritonitis virus-infected cats. Moreover, feline calicivirus (FCV)-specific CD4+ T cells were detected in the spleens of FCV-vaccinated cats. As antigen-presenting cells (APCs), we used immortalized autologous fibroblast cell lines, PBMC or splenocytes. A straightforward protocol, in which splenocyte preparations served both as APCs and effector cells, consistently yielded best results. The assay will permit further studies of the cellular immune responses in cats during natural and experimental viral infections. It will contribute to vaccine development against feline viruses by facilitating the identification of T cell antigens and epitopes, and by allowing the quantitative detection of virus-specific T cells after vaccination. Furthermore, the assay will add to the value of those systems in which viral infections of the cat serve as models for human disease.

ABSTRACT Toroviruses (family Coronaviridae , order Nidovirales ) are enveloped, positive-stranded RNA viruses that have been implicated in enteric disease in cattle and possibly in humans. Despite their potential veterinary and clinical relevance, little is known about torovirus epidemiology and molecular genetics. Here, we present the first study into the diversity among toroviruses currently present in European swine and cattle herds. Comparative sequence analysis was performed focusing on the genes for the structural proteins S, M, HE, and N, with fecal specimens serving as sources of viral RNA. Sequence data published for animal and human torovirus variants were included. Four genotypes, displaying 30 to 40% divergence, were readily distinguished, exemplified by bovine torovirus (BToV) Breda, porcine torovirus (PToV) Markelo, equine torovirus Berne, and the putative human torovirus. The ungulate toroviruses apparently display host species preference. In phylogenetic analyses, all PToV variants clustered, while the recent European BToVs mostly resembled the New World BToV variant Breda, identified 19 years ago. However, we found ample evidence for recurring intertypic recombination. All newly characterized BToV variants seem to have arisen from a genetic exchange, during which the 3′ end of the HE gene, the N gene, and the 3′ nontranslated region of a Breda virus-like parent had been swapped for those of PToV. Moreover, some PToV and BToV variants carried chimeric HE genes, which apparently resulted from recombination events involving hitherto unknown toroviruses. From these observations, the existence of two additional torovirus genotypes can be inferred. Toroviruses may be even more promiscuous than their closest relatives, the coronaviruses and arteriviruses.

Viruses in the families Arteriviridae and Coronaviridae have enveloped virions which contain nonsegmented, positive-stranded RNA, but the constituent genera differ markedly in genetic complexity and virion structure. Nevertheless, there are striking resemblances among the viruses in the organization and expression of their genomes, and sequence conservation among the polymerase polyproteins strongly suggests that they have a common ancestry. On this basis, the International Committee on Taxonomy of Viruses recently established a new order, Nidovirales, to contain the two families. Here, the common traits and distinguishing features of the Nidovirales are reviewed.

Arteri‐, corona‐, toro‐ and roniviruses are evolutionarily related positive‐strand RNA viruses, united in the order Nidovirales. The best studied nidoviruses, the corona‐ and arteriviruses, employ a unique transcription mechanism, which involves discontinuous RNA synthesis, a process resembling similarity‐assisted copy‐choice RNA recombination. During infection, multiple subgenomic (sg) mRNAs are transcribed from a mirror set of sg negative‐strand RNA templates. The sg mRNAs all possess a short 5′ common leader sequence, derived from the 5′ end of the genomic RNA. The joining of the non‐contiguous ‘leader’ and ‘body’ sequences presumably occurs during minus‐strand synthesis. To study whether toroviruses use a similar transcription mechanism, we characterized the 5′ termini of the genome and the four sg mRNAs of Berne virus (BEV). We show that BEV mRNAs 3–5 lack a leader sequence. Surprisingly, however, RNA 2 does contain a leader, identical to the 5′‐terminal 18 residues of the genome. Apparently, BEV combines discontinuous and non‐discontinous RNA synthesis to produce its sg mRNAs. Our findings have important implications for the understanding of the mechanism and evolution of nidovirus transcription.

The alphaherpesvirus glycoproteins gE and gI form a hetero-oligomeric complex involved in cell-to-cell transmission. The gI-deficient recombinant feline herpesvirus (FHV), FHVdeltagI-LZ, produces plaques that are only 15% the size of those of wild-type FHV. Here, we have complemented FHV(delta)gI-LZ allotopically by expressing intact gI and C-terminally truncated gI derivatives from the thymidine kinase locus. The effect on gE-gI-mediated cell-to-cell spread was assessed by plaque assay employing computer-assisted image analysis (software available at http://www.androclus.vet.uu.nl/spotter/spotter.htm+ ++). Allotopic complementation with intact gI fully restored plaque size. Deletion of the C-terminal 11 residues of gI did not affect cell-to-cell spread, whereas deletion of the complete cytoplasmic tail reduced plaque size by only 35%. Mutants expressing gI166, roughly corresponding to the N-terminal half of the ectodomain, displayed a small-plaque phenotype. Nevertheless, their plaques were reproducibly larger than those of matched gI-deficient controls, indicating that the gE-gI166 hetero-oligomer, though crippled, is still able to mediate cell-to-cell spread. Our data demonstrate that plaque analysis provides a reliable and convenient tool to measure and quantitate gE-gI function in vitro.

Recent evidence suggests that the type II feline coronavirus (FCoV) strains 79-1146 and 79-1683 have arisen from a homologous RNA recombination event between FCoV type I and canine coronavirus (CCV). In both cases, the template switch apparently took place between the S and M genes, giving rise to recombinant viruses which encode a CCV-like S protein and the M, N, 7a, and 7b proteins of FCoV type I (K. Motowaka, T. Hohdatsu, H. Hashimoto, and H. Koyama, Microbiol. Immunol. 40:425-433, 1996; H. Vennema, A. Poland, K. Floyd Hawkins, and N. C. Pedersen, Feline Pract. 23:40-44, 1995). In the present study, we have looked for additional FCoV-CCV recombination sites. Four regions in the pol gene were selected for comparative sequence analysis of the type II FCoV strains 79-1683 and 79-1146, the type I FCoV strains TN406 and UCD1, the CCV strain K378, and the TGEV strain Purdue. Our data show that the type II FCoVs have arisen from double recombination events: additional crossover sites were mapped in the ORF1ab frameshifting region of strain 79-1683 and in the 5' half of ORF1b of strain 79-1146.

A putative bovine respiratory torovirus (BRTV) was propagated in bovine fetal diploid lung and human colonic tumour cells, and fringed pleomorphic particles were detected in the culture supernatants by electron microscopy. Antisera directed against a bovine (Breda strain) and equine (Berne strain) torovirus failed to react with BRTV-infected cells in immunofluorescence assays and did not neutralise BRTV. No toroviral RNA was found in the supernatants of infected cells by means of a reverse transcriptase-polymerase chain reaction with torovirus-specific primers. On the other hand, bovine coronavirus-specific antisera and monoclonal antibodies did neutralise the cytopathic effects, and coronaviral antigen was detected in the cultures by immunofluorescence. Furthermore, bovine coronavirus RNA was detected in the supernatants of BRTV-infected cells after nucleic acid amplification. It is concluded that the cytopathic BRTV isolate is a coronavirus.

ABSTRACT A porcine torovirus (PoTV) was identified and characterized; it is a novel member of the genus Torovirus (family Coronaviridae , order Nidovirales ), closely related to but clearly distinct from the already recognized equine torovirus (ETV) and bovine torovirus (BoTV) representatives. Immunoelectron microscopy of feces from piglets revealed elongated, 120- by 55-nm particles which were recognized by a torovirus-specific antiserum. Amplification by reverse transcriptase (RT) PCR with primers designed to detect conserved regions (on the basis of the genomes of BoTV strain Breda and ETV strain Berne) resulted in the identification of the 489-bp nucleocapsid gene, encoding a 18.7-kDa protein. The sequence identity in this region between PoTV and both ETV and BoTV was only about 68%, whereas the latter two show 81% identity. Neutralizing antibodies directed against ETV were found in sera of adult and young pigs. In all 10 herds sampled, seropositive animals were present, and 81% of randomly selected adult sows possessed antibodies. A longitudinal study with RT PCR showed that piglets shed virus in the feces for 1 or more days, starting 4 to 14 days after weaning.

ABSTRACT Alphaherpesvirus glycoproteins E and I (gE and gI, respectively) assemble into a hetero-oligomeric complex which promotes cell-to-cell transmission, a determining factor of virulence. Focusing on gI of feline herpesvirus (FHV), we examined the role of disulfide bonds during its biosynthesis, its interaction with gE, and gE-gI-mediated spread of the infection in vitro. The protein’s disulfide linkage pattern was determined by single and pairwise substitutions for the four conserved cysteine residues in the ectodomain. The resulting mutants were coexpressed with gE in the vaccinia virus-based vTF7-3 system, and the formation and endoplasmic reticulum (ER)-to-Golgi transport of the hetero-oligomeric complex were monitored. The results were corroborated biochemically by performing an endoproteinase Lys-C digestion of a [ 35 S]Cys-labeled secretory recombinant form of gI followed by tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the peptides under reducing and nonreducing conditions. We found that (i) gI derivatives lacking Cys 79 (C 1 ) and/or Cys 223 (C 4 ) still assemble with gE into transport-competent complexes, (ii) mutant proteins lacking Cys 91 (C 2 ) and/or Cys 102 (C 3 ) bind to gE but are retained in the ER, (iii) radiolabeled endoproteinase Lys-C-generated peptide species containing C 1 and C 4 are linked through disulfide bonds, and (iv) peptides containing both C 2 and C 3 are not disulfide linked to any other peptide. From these findings emerges a model in which C 1 and C 4 as well as C 2 and C 3 form intramolecular disulfide bridges. Since the cysteines in the ectodomain have been conserved during alphaherpesvirus divergence, we postulate that the model applies for all gI proteins. Analysis of an FHV recombinant with a C 1 →S substitution confirmed that the C 1 -C 4 disulfide bond is not essential for the formation of a transport-competent gE-gI complex. The mutation affected the posttranslational modification of gI and caused a slight cold-sensitivity defect in the assembly or the intracellular transport of the gE-gI complex but did not affect plaque size. Thus, C 1 and the C 1 -C 4 bond are not essential for gE-gI-mediated cell-to-cell spread, at least not in vitro.

Coronaviruses display a characteristic fringe of large (17–20 nm), clubshaped peplomers, each consisting of a di- or trimer of the peplomer protein (Cavanagh et al. 1983). The peplomer protein, E2, plays an important role during the infection proces. It mediates the binding of virions to the host-cell receptors and is involved in membrane fusion. In addition, the E2 protein appears to be a major inducer of protective immunity to coronaviral infection (reviewed by Sturman and Holmes, 1983).