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Antibodies against the fusion (F) protein of respiratory syncytial virus (RSV) play an important role in the protective immune response to this important respiratory virus. Little is known, however, about antibody levels against multiple F-specific epitopes induced by infection or after vaccination against RSV, while this is important to guide the evaluation of (novel) vaccines. In this study, we analyzed antibody levels against RSV proteins and F-specific epitopes in human sera and in sera of vaccinated and experimentally infected cotton rats and the correlation thereof with virus neutralization. Analysis of human sera revealed substantial diversity in antibody levels against F-, G (attachment)-, and F-specific epitopes between individuals. The highest correlation with virus neutralization was observed for antibodies recognizing prefusion-specific antigenic site Ø. Nevertheless, our results indicate that high levels of antibodies targeting other parts of the F protein can also mediate a potent antiviral antibody response. In agreement, sera of experimentally infected cotton rats contained high neutralizing activity despite lacking antigenic site Ø-specific antibodies. Strikingly, vaccination with formalin-inactivated RSV (FI-RSV) exclusively resulted in the induction of poorly neutralizing antibodies against postfusion-specific antigenic site I, although antigenic sites I, II, and IV were efficiently displayed in FI-RSV. The apparent immunodominance of antigenic site I in FI-RSV likely explains the low levels of neutralizing antibodies upon vaccination and challenge and may play a role in the vaccination-induced enhancement of disease observed with such preparations. IMPORTANCE RSV is an importance cause of hospitalization of infants. The development of a vaccine against RSV has been hampered by the disastrous results obtained with FI-RSV vaccine preparations in the 1960s that resulted in vaccination-induced enhancement of disease. To get a better understanding of the antibody repertoire induced after infection or after vaccination against RSV, we investigated antibody levels against fusion (F) protein, attachment (G) protein, and F-specific epitopes in human and animal sera. The results indicate the importance of prefusion-specific antigenic site Ø antibodies as well as of antibodies targeting other epitopes in virus neutralization. However, vaccination of cotton rats with FI-RSV specifically resulted in the induction of weakly neutralizing, antigenic site I-specific antibodies, which may play a role in the enhancement of disease observed after vaccination with such preparations.

Equine arteritis virus (EAV) is an enveloped, positive-strand RNA virus. Genome replication of EAV has been associated with modified intracellular membranes that are shaped into double-membrane vesicles (DMVs). We showed by immuno-electron microscopy that the DMVs induced in EAV-infected cells contain double-strand (ds)RNA molecules, presumed RNA replication intermediates, and are decorated with the autophagy marker protein microtubule-associated protein 1 light chain 3 (LC3). Replication of EAV, however, was not affected in autophagy-deficient cells lacking autophagy-related protein 7 (ATG7). Nevertheless, colocalization of DMVs and LC3 was still observed in these knockout cells, which only contain the nonlipidated form of LC3. Although autophagy is not required, depletion of LC3 markedly reduced the replication of EAV. EAV replication could be fully restored in these cells by expression of a nonlipidated form of LC3. These findings demonstrate an autophagy-independent role for LC3 in EAV replication. Together with the observation that EAV-induced DMVs are also positive for ER degradation-enhancing α-mannosidase-like 1 (EDEM1), our data suggested that this virus, similarly to the distantly-related mouse hepatitis coronavirus, hijacks the ER-derived membranes of EDEMosomes to ensure its efficient replication.

International audience; The tremendous pandemic potential of coronaviruses was demonstrated twice in the last 15 years by two global outbreaks of deadly pneumonia. Entry of coronaviruses into cells is mediated by the transmembrane spike glycoprotein S, which forms a trimer carrying receptor-binding and membrane fusion functions. Despite their biomedical importance, coronavirus S glycoproteins have proven difficult targets for structural characterization, precluding high-resolution studies of the biologically relevant trimer. Recent technological developments in single particle cryo-electron microscopy allowed us to determine the first structure of a coronavirus S glycoprotein trimer which provided a framework to understand the mechanisms of viral entry and suggested potential inhibition strategies for this family of viruses. Here, we describe the key factors that enabled this breakthrough.

International audience; The tremendous pandemic potential of coronaviruses was demonstrated twice in the past few decades by two global outbreaks of deadly pneumonia. Entry of coronaviruses into cells is mediated by the transmembrane spike glycoprotein S, which forms a trimer carrying receptor-binding and membrane fusion functions. S also contains the principal antigenic determinants and is the target of neutralizing antibodies. Here we present the structure of a mouse coronavirus S trimer ectodomain determined at 4.0 Å resolution by single particle cryo-electron microscopy. It reveals the metastable pre-fusion architecture of S and highlights key interactions stabilizing it. The structure shares a common core with paramyxovirus F proteins, implicating mechanistic similarities and an evolutionary connection between these viral fusion proteins. The accessibility of the highly conserved fusion peptide at the periphery of the trimer indicates potential vaccinology strategies to elicit broadly neutralizing antibodies against coronaviruses. Finally, comparison with crystal structures of human coronavirus S domains allows rationalization of the molecular basis for species specificity based on the use of spatially contiguous but distinct domains.

Some Newcastle disease virus (NDV) variants isolated from pigeons (pigeon paramyxovirus type 1; PPMV-1) do not show their full virulence potential for domestic chickens but may become virulent upon spread in these animals. In this study we examined the molecular changes responsible for this gain of virulence by passaging a low-pathogenic PPMV-1 isolate in chickens. Complete genome sequencing of virus obtained after 1, 3 and 5 passages showed the increase in virulence was not accompanied by changes in the fusion protein – a well known virulence determinant of NDV – but by mutations in the L and P replication proteins. The effect of these mutations on virulence was confirmed by means of reverse genetics using an infectious cDNA clone. Acquisition of three amino acid mutations, two in the L protein and one in the P protein, significantly increased virulence as determined by intracerebral pathogenicity index tests in day-old chickens. The mutations enhanced virus replication in vitro and in vivo and increased the plaque size in infected cell culture monolayers. Furthermore, they increased the activity of the viral replication complex as determined by an in vitro minigenome replication assay. Our data demonstrate that PPMV-1 replication in chickens results in mutations in the polymerase complex rather than the viral fusion protein, and that the virulence level of pigeon paramyxoviruses is directly related to the activity of the viral replication complex.

Rift Valley fever virus (RVFV) is an emerging mosquito-borne virus causing significant morbidity and mortality in livestock and humans. Rift Valley fever is endemic in Africa, but also outside this continent outbreaks have been reported. Here we report the evaluation of two vaccine candidates based on the viral Gn and Gc envelope glycoproteins, both produced in a Drosophila insect cell expression system. Virus-like particles (VLPs) were generated by merely expressing the Gn and Gc glycoproteins. In addition, a soluble form of the Gn ectodomain was expressed and affinity-purified from the insect cell culture supernatant. Both vaccine candidates fully protected mice from a lethal challenge with RVFV. Importantly, absence of the nucleocapsid protein in either vaccine candidate facilitates the differentiation between infected and vaccinated animals using a commercial recombinant nucleocapsid protein-based indirect ELISA.

One of the most striking and dramatic genomic changes observed in the severe acute respiratory syndrome coronavirus (SARS-CoV) isolated from humans soon after its zoonotic transmission from palm civets was the acquisition of a characteristic 29-nucleotide deletion. This occurred in open reading frame 8 (ORF8), one of the accessory genes unique to the SARS-CoV. The function of ORF8 and the significance of the deletion are unknown. The intact ORF8 present in animal and some early human isolates encodes a 122-amino-acid polypeptide (8ab + ), which we expressed in cells using the vaccinia virus T7 expression system. It was found to contain a cleavable signal sequence, which directs the precursor to the endoplasmic reticulum (ER) and mediates its translocation into the lumen. The cleaved protein became N-glycosylated, assembled into disulfide-linked homomultimeric complexes, and remained stably in the ER. The 29-nucleotide deletion splits ORF8 into two ORFs, 8a and 8b, encoding 39- and 84-residue polypeptides. The 8a polypeptide is likely to remain in the cytoplasm, as it is too small for its signal sequence to function and will therefore be directly released from the ribosome. However, we could not confirm this experimentally due to the lack of proper antibodies. ORF8b appeared not to be expressed in SARS-CoV-infected cells or when expressed from mRNA's mimicking mRNA8. This was due to the context of the internal AUG initiation codon, as we demonstrated after placing the ORF8b immediately behind the T7 promoter. A soluble, unmodified and monomeric 8b protein was now expressed in the cytoplasm, which was highly unstable and rapidly degraded. Clearly, the 29-nucleotide deletion disrupts the proper expression of the SARS-CoV ORF8, the implications of which are discussed.

Infection by the coronavirus mouse hepatitis virus strain A59 (MHV-A59) requires the release of the viral genome by fusion with the respective target membrane of the host cell. Fusion is mediated by the viral S protein. Here, the entry pathway of MHV-A59 into murine fibroblast cells was studied by independent approaches. Infection of cells assessed by plaque reduction assay was strongly inhibited by lysosomotropic compounds and substances that interfere with clathrin-dependent endocytosis, suggesting that MHV-A59 is taken up via endocytosis and delivered to acidic endosomal compartments. Infection was only slightly reduced in the presence of substances inhibiting proteases of endosomal compartments, precluding that the endocytic uptake is required to activate the fusion potential of the S protein by its cleavage. Fluorescence confocal microscopy of labeled MHV-A59 confirmed that virus is taken up via endocytosis. Bright labeling of intracellular compartments suggests their fusion with the viral envelope. No fusion with the plasma membrane was observed at neutral pH conditions. However, when virus was bound to cells and the pH was lowered to 5.0, we observed a strong labeling of the plasma membrane. Electron microscopy revealed low pH triggered conformational alterations of the S ectodomain. Very likely, these alterations are irreversible because low-pH treatment of viruses in the absence of target membranes caused an irreversible loss of the fusion activity. The results imply that endocytosis plays a major role in MHV-A59 infection and the acidic pH of the endosomal compartment triggers a conformational change of the S protein mediating fusion.

Over the last 2 decades, yeast two-hybrid became an invaluable technique to decipher protein-protein interaction networks. In the field of virology, it has proven instrumental to identify virus-host interactions that are involved in viral embezzlement of cellular functions and inhibition of immune mechanisms. Here, we present a yeast two-hybrid protocol that has been used in our laboratory since 2006 to search for cellular partners of more than 300 viral proteins. Our aim was to develop a robust and straightforward pipeline, which minimizes false-positive interactions with a decent coverage of target cDNA libraries, and only requires a minimum of equipment. We also discuss reasons that motivated our technical choices and compromises that had to be made. This protocol has been used to screen most non-structural proteins of murine hepatitis virus (MHV), a member of betacoronavirus genus, against a mouse brain cDNA library. Typical results were obtained and are presented in this report.

The 5(-terminal leader sequence of the equine arteritis virus (EAV) genome contains an open reading frame (ORF) with an AUG codon in a suboptimal context for initiation of protein synthesis. To investigate the significance of this intraleader ORF (ILO), an expression plasmid was generated carrying a DNA copy of the subgenomic mRNA7 behind a T7 promoter. Capped RNA transcribed from this construct was shown to direct, in an in vitro translation system, the synthesis of leader peptide as well as N protein. Site-directed mutations aimed to either optimize or weaken the sequence context of the ILO start codon affected leader peptide synthesis as predicted; no peptide was detected when the initiation codon was incapacitated. Translation of the downstream N gene was inversely affected by leader peptide production, consistent with a ribosomal leaky scanning mechanism. To investigate the role of the leader peptide in the EAV replication life cycle we generated, using an infectious EAV cDNA clone, two mutant viruses in one of which the ILO start codon was in an optimal Kozak context for translation initiation while in the other the codon was again incapacitated. Surprisingly, both mutant viruses were equally viable and exhibited similar phenotypes in BHK-21 cells. However, their replication kinetics and viral yields were reduced relative to that of the wild-type parental virus, as were their plaque sizes. Importantly, the mutations introduced into the viruses appeared to be rapidly and precisely repaired upon passaging. Already after one viral passage a significant fraction of the viruses had regained the wild-type sequence as well as its phenotype. The results demonstrate that EAV replication is not dependent on the synthesis of the intraleader peptide. Rather, the leader peptide does not seem to have any function in the EAV life cycle. As we discuss, the available data indicate that the ILO 5( nucleotide sequence per se, not its functioning in translation initiation, is of critical importance for EAV replication.

Immunization against gonadotrophin releasing hormone (GnRH) was studied as an alternative for the commonly used surgical castration in stallions. Two GnRH vaccines comprising non-mineral oil adjuvants were evaluated for their potential to induce high antibody titers directed against GnRH and subsequent effects on reproductive characteristics. Twelve sexually mature male hemicastrated Shetland ponies were assigned to three groups. Group 1 and 2 were injected with 1mg peptide equivalent of G6k-GnRH-tandem-dimer conjugated to ovalbumin (OVA) in CoVaccine HT adjuvant (GnRH/CoVaccine) and in Carbopol (GnRH/Carbopol), respectively, and group 3 was injected with CoVaccine HT adjuvant without antigen (controls). After immunization no adverse effects were observed with respect to the injections sites or general health. Two weeks after the second vaccination antibody titers against GnRH increased rapidly in all animals of the GnRH/CoVaccine group, at the same time reducing serum testosterone levels maximally for the further duration of the experiment. In the GnRH/Carbopol group antibody responses and effects on testosterone levels were intermediate in two stallions and not apparent in the remaining stallions of this group. Semen evaluation showed that from 2 weeks after the second immunization onwards, sperm motility was affected in all stallions treated with GnRH/CoVaccine and one stallion treated with GnRH/Carbopol. Seven weeks after the second immunization, no semen could be collected from two stallions, one of each group, due to suppressed libido. Histological examination of the testes, 15 weeks after the initial immunization, demonstrated reduction in seminiferous tubuli diameters in all stallions of the GnRH/CoVaccine group and one stallion of the GnRH/Carbopol group. Furthermore, spermatogenesis was extremely disorganized in these stallions, as indicated by absence of the lumen in the seminiferous tubules, the absence of spermatozoa and spermatids in the tubular cross-sections and the impossibility to determine the stage of the tubular cross-sections. Testis size was also substantially reduced in three out of four stallions treated with GnRH/CoVaccine. The results demonstrate that two immunizations with G6k-GnRH-tandem-dimer-OVA conjugate in a suitable adjuvant such as CoVaccine HT caused a rapid and complete reduction of serum testosterone levels in sexually mature stallions, subsequently leading to reduced sperm motility and affected testis function, while no adverse reactions were observed after immunizations.

A reverse transcriptase polymerase chain reaction (RT-PCR) for the detection of feline coronavirus (FCoV) messenger RNA in peripheral blood mononuclear cells (PBMCs) is described. The assay is evaluated as a diagnostic test for feline infectious peritonitis (FIP). It is based on a well-documented key event in the development of FIP: the replication of virulent FCoV mutants in monocytes/macrophages. To detect most feline coronavirus field strains, the test was designed to amplify subgenomic mRNA of the highly conserved M gene. The test was applied to 1075 feline blood samples (424 from healthy, 651 from sick cats suspected of FIP) and returned 46% of the diseased cats as positive for feline coronavirus mRNA in their peripheral blood cells; of the healthy cats, 5% tested positive. Of a group of 81 animals in which FIP had been confirmed by post-mortem examination, 75 (93%) tested positive, whereas 17 cats with different pathologies (non-FIP cases) all tested negative. In view of the low rate of false-positive results (high specificity) the mRNA RT-PCR may be a valuable addition to the diagnostic arsenal for FIP.

Infectious bursal disease virus (IBDV), a member of the family Birnaviridae, is a non-enveloped, double-stranded RNA virus. Viral protein 1 (VP1), the putative RNA-dependent RNA polymerase, occurs in virions both as a free polypeptide and as a genome-linked protein, called VPg. To gain more insight in its function, we initiated a yeast two-hybrid screen. With this approach we identified the carboxy-terminal domain of eukaryotic translation initiation factor 4AII (eIF4AII) as an interactor for VP1. The association between these molecules was confirmed by co-immunoprecipitation analyses. eIF4A plays an essential role in the initiation of translation of both capped and uncapped mRNAs. Its association with IBDV VP1 suggests an involvement of this viral protein in IBDV mRNA translation. An interaction between VP1 and full-length eIF4AII was, however, not observed. In view of the known two-domain structure of eIF4AII it is conceivable that the interaction of VP1 with full-length eIF4AII requires collaborating proteins that open up its structure and expose the VP1-binding site in the carboxy-terminal domain. The biological relevance of the potential VP1-eIF4AII interaction is discussed.

Equine arteritis virus (EAV) is an enveloped, positive-strand RNA virus belonging to the family Arteriviridae of the order Nidovirales . EAV virions contain six different envelope proteins. The glycoprotein GP 5 (previously named G L ) and the unglycosylated membrane protein M are the major envelope proteins, while the glycoproteins GP 2b (previously named G S ), GP 3 , and GP 4 are minor structural proteins. The unglycosylated small hydrophobic envelope protein E is present in virus particles in intermediate molar amounts compared to the other transmembrane proteins. The GP 5 and M proteins are both essential for particle assembly. They occur as covalently linked heterodimers that constitute the basic protein matrix of the envelope. The GP 2b , GP 3 , and GP 4 proteins occur as a heterotrimeric complex in which disulfide bonds play an important role. The function of this complex has not been established yet, but the available data suggest it to be involved in the viral entry process. Here we investigated the role of the four cysteine residues of the mature GP 2b protein in the assembly of the GP 2b /GP 3 /GP 4 complex. Open reading frames encoding cysteine-to-serine mutants of the GP 2b protein were expressed independently or from a full-length infectious EAV cDNA clone. The results of these experiments support a model in which the cysteine residue at position 102 of GP 2b forms an intermolecular cystine bridge with one of the cysteines of the GP 4 protein, while the cysteine residues at positions 48 and 137 of GP 2b are linked by an intrachain disulfide bond. In this model, another cysteine residue in the GP 4 protein is responsible for the covalent association of GP 3 with the disulfide-linked GP 2b /GP 4 heterodimer. In addition, our data highlight the importance of the correct association of the minor EAV envelope glycoproteins for their efficient incorporation into viral particles and for virus infectivity.

Equine arteritis virus (EAV) is an enveloped plus-strand RNA virus of the family Arteriviridae (order Nidovirales) that causes respiratory and reproductive disease in equids. Protective, virus-neutralizing antibodies (VNAb) elicited by infection are directed predominantly against an immunodominant region in the membrane-proximal domain of the viral envelope glycoprotein G L , allowing recently the establishment of a sensitive peptide enzyme-linked immunosorbent assay (ELISA) based on this particular domain (J. Nugent et al., J. Virol. Methods 90: 167-183, 2000). By using an infectious cDNA we have now generated, in the controlled background of a nonvirulent virus, a mutant EAV from which this immunodominant domain was deleted. This virus, EAV-G L Δ, replicated to normal titers in culture cells, although at a slower rate than wild-type EAV, and caused an asymptomatic infection in ponies. The antibodies induced neutralized the mutant virus efficiently in vitro but reacted poorly to wild-type EAV strains. Nevertheless, when inoculated subsequently with virulent EAV, the immunized animals, in contrast to nonvaccinated controls, were fully protected against disease; replication of the challenge virus occurred briefly at low though detectable levels. The levels of protection achieved suggest that an immune effector mechanism other than VNAb plays an important role in protection against infection. As expected, infection with EAV-G L Δ did not induce a measurable response in our G L -peptide ELISA while the challenge infection of the animals clearly did. EAV-G L Δ or similar mutants are therefore attractive marker vaccine candidates, enabling serological discrimination between vaccinated and wild-type virus-infected animals.

Equine arteritis virus (EAV) is an enveloped, positive-stranded RNA virus belonging to the family Arteriviridae of the order Nidovirales . Six transmembrane proteins have been identified in EAV particles: the nonglycosylated membrane protein M and the glycoprotein GP 5 (previously named G L ), which occur as disulfide-bonded heterodimers and are the major viral envelope proteins; the unglycosylated small envelope protein E; and the minor glycoproteins GP 2b (formerly designated G S ), GP 3 , and GP 4 . Analysis of the appearance of the GP 2b , GP 3 , and GP 4 proteins in viral particles by gel electrophoresis under reducing and nonreducing conditions revealed the occurrence of two different covalently linked oligomeric complexes between these proteins, i.e., heterodimers of GP 2b and GP 4 and heterotrimers of GP 2b , GP 3 , and GP 4 . Shortly after their release from infected cells, virions contained mainly cystine-linked GP 2b /GP 4 heterodimers, which were subsequently converted into disulfide-bonded GP 2b /GP 3 /GP 4 trimers through the covalent recruitment of GP 3 . This process occurred faster at a higher pH but was arrested at 4°C. Furthermore, the conversion was almost instantaneous in the presence of the thiol oxidant diamide. In contrast, the sulfhydryl-modifying agent N -ethylmaleimide inhibited the formation of disulfide-bonded GP 2b /GP 3 /GP 4 trimers. Using sucrose density gradients, we could not demonstrate a noncovalent association of GP 3 with the cystine-linked GP 2b /GP 4 dimer in freshly released virions, nor did we observe higher-order structures of the GP 2b /GP 4 or GP 2b /GP 3 /GP 4 complexes. Nevertheless, the instantaneous diamide-induced formation of disulfide-bonded GP 2b /GP 3 /GP 4 heterotrimers at 4°C suggests that the three minor glycoproteins of EAV are assembled as trimeric complexes. The existence of a noncovalent interaction between the cystine-linked GP 2b /GP 4 dimer and GP 3 was also inferred from coexpression experiments showing that the presence of GP 3 increased the electrophoretic mobility of the disulfide-bonded GP 2b /GP 4 dimers. Our study reveals that the minor envelope proteins of arteriviruses enter into both covalent and noncovalent interactions, the function of which has yet to be established.

Summary. The aim of this study was to identify the receptor(s) for PRRSV on porcine alveolar macrophages (PAMs) by producing monoclonal antibodies (MAbs) against these cells. Hybridoma supernatants were selected for their ability to block PRRSV infection. Four MAbs, 1-8D2, 9.4C7, 9.9F2, and 3-3H2 inhibited infection and recognised cell surface, PAM-specific antigens as shown by immunofluorescence and immunoperoxidase monolayer assay. These MAbs were then used to identify cellular proteins involved in PRRSV infection by radioimmunoprecipitation assays (RIPAs). MAbs 1-8D2 and 9.9F2 each recognised a 150 kDa-polypeptide doublet, while MAbs 9.4C7 and 3-3H2 both recognised a 220 kDa-polypeptide. Glycosidase treatment demonstrated all these polypeptides to be N-glycosylated. Thus, multiple glycoproteins appear to be involved in infection of PAMs by PRRSV.

Equine arteritis virus (EAV) is an enveloped, positive-stranded RNA virus belonging to the family Arteriviridae of the order Nidovirales . Four envelope proteins have hitherto been identified in EAV particles: the predominant membrane proteins M and G L , the unglycosylated small envelope protein E, and the nonabundant membrane glycoprotein G S . In this study, we established that the products of EAV open reading frame 3 (ORF3) and ORF4 (designated GP 3 and GP 4 , respectively) are also minor structural glycoproteins. The proteins were first characterized by various analyses after in vitro translation of RNA transcripts in a rabbit reticulocyte lysate in the presence and absence of microsomal membranes. We subsequently expressed ORF3 and -4 in baby hamster kidney cells by using the vaccinia virus expression system and, finally, analyzed the GP 3 and GP 4 proteins synthesized in EAV-infected cells. The results showed that GP 4 is a class I integral membrane protein of 28 kDa with three functional N-glycosylation sites and with little, if any, of its carboxy terminus exposed. Both after independent expression and in EAV-infected cells, the protein localizes in the endoplasmic reticulum (ER), as demonstrated biochemically by analysis of its oligosaccharide side chains and as visualized directly by immunofluorescence studies. GP 3 , on the other hand, is a heavily glycosylated protein whose hydrophobic amino terminus is not cleaved off. It is an integral membrane protein anchored by either or both of its hydrophobic terminal domains and with no parts detectably exposed cytoplasmically. Also, GP 3 localizes in the ER when expressed independently and in the context of an EAV infection. Only a small fraction of the GP 3 and GP 4 proteins synthesized in infected cells ends up in virions. Most, but not all, of the oligosaccharides of these virion glycoproteins are biochemically mature. Our results bring the number of EAV envelope proteins to six.

Arteriviruses are enveloped, positive-strand RNA viruses for which the two major envelope proteins GP(5) and M occur as disulfide-linked heterodimers. These were assumed to serve the viral targeting functions, but recent ectodomain swapping studies with equine arteritis virus (EAV) indicate that the GP(5) protein does not determine arteriviral tropism. Here, we focused on the short, 13- to 18-residue ectodomain of the M protein. Using an infectious cDNA clone of the Lelystad virus isolate of porcine reproductive and respiratory syndrome virus (PRRSV), we substituted the genomic sequence encoding the M ectodomain by that of murine lactate dehydrogenase-elevating virus, EAV, and the US PRRSV-isolate, VR2332. Viable viruses with a chimeric M protein were obtained in all three cases, but for the latter two only after removal of the genomic overlap between the M and GP(5) genes. Characterization of the chimeric viruses revealed that they could be distinguished immunologically from wild-type virus, that they were genetically stable in vitro, but that they were impaired in their growth, reaching lower titers than the parental virus. The latter appeared to be due to an increased particle-to-infectivity ratio of the chimeric virus particles. Interestingly, the chimeric viruses had retained their ability to infect porcine cells and had not acquired tropism for cells susceptible to the viruses from which the foreign ectodomains were derived. We conclude that the surface structures composed by the arterivirus M and GP(5) ectodomains do not determine viral tropism.

We used an infectious cDNA clone of Porcine reproductive and respiratory syndrome virus (PRRSV) to investigate the presence of essential replication elements in the region of the genome encoding the structural proteins. Deletion analysis showed that a stretch of 34 nucleotides (14653 to 14686) within ORF7, which encodes the nucleocapsid protein, is essential for RNA replication. Strand-specific reverse transcription-PCR analysis of viral RNA isolated from transfected BHK-21 cells revealed that this region is required for negative-strand genomic RNA synthesis. The 34-nucleotide stretch is highly conserved among PRRSV isolates and folds into a putative hairpin. A 7-base sequence within the loop of this structure was suggested to base-pair with a sequence present in the loop of a hairpin located in the 3′ noncoding region, resulting in a kissing interaction. Mutational analyses confirmed that this kissing interaction is required for RNA replication.

Cell-mediated immunity is thought to play a decisive role in protecting cats against feline infectious peritonitis (FIP), a progressive and lethal coronavirus disease. In view of the potential of DNA vaccines to induce cell-mediated responses, their efficacy to induce protective immunity in cats was evaluated. The membrane (M) and nucleocapsid (N) proteins were chosen as antigens, because antibodies to the spike (S) protein of FIP virus (FIPV) are known to precipitate pathogenesis. However, vaccination by repeated injections of plasmids encoding these proteins did not protect kittens against challenge infection with FIPV. Also, a prime–boost protocol failed to afford protection, with priming using plasmid DNA and boosting using recombinant vaccinia viruses expressing the same coronavirus proteins. Because of the role of IL-12 in initiating cell-mediated immunity, the effects of co-delivery of plasmids encoding the feline cytokine were studied. Again, IL-12 did not meet expectations – on the contrary, it enhanced susceptibility to FIPV challenge. This study shows that DNA vaccination failed to protect cats against FIP and that IL-12 may yield adverse effects when used as a cytokine adjuvant.

To the Editor: Feline coronaviruses (FCoVs) occur as 2 pathotypes, feline infectious peritonitis virus (FIPV) and feline enteric coronavirus (FECV). FECV is common in cats, causing mild transient enteritis in kittens, but is asymptomatic in adult cats. In contrast, FIPV occurs sporadically but is lethal. It replicates in monocytes and macrophages and rapidly disseminates throughout the body causing systemic immunopathologic disease (1–4). The relationship between FECV and FIPV has become a matter of debate. Genetic and animal experimental evidence indicates that FIPV arises by mutation from FECV in the intestinal tract of a persistently infected cat; the virus thereby acquires the monocyte or macrophage tropism that enables it to spread systemically and cause FIP (5–7,8). According to another view, the 2 pathotypes circulate independently in the field. This circulating virulent/avirulent FCoV theory recently was advocated by Brown et al. (9). Their conclusion was based on sequence analyses of parts of the viral genome including the matrix (M) gene, phylogenetic analysis of which revealed reciprocal monophyly of the sequences obtained from FIP cases versus those of asymptomatic FECV-infected animals. In addition, the authors suggested 5 aa residues in the M protein to represent potential diagnostic markers for distinguishing virulent FIPV from avirulent FECV (9). To try to verify the findings of Brown et al. (9), we determined and analyzed M genes from 43 FCoV genomes, 20 of which came from cats in single-cat households, and 23 from cattery animals. The latter group consisted of 10 asymptomatic healthy cats (FECV; test specimens: feces) and 13 dead cats with FIP confirmed through pathology (FIPV; test specimens: organs, ascites). These animals came from 8 catteries. FECV and FIPV cases were found in 7 (designated A to G); the remaining cattery (H) provided 2 cats with FIP. The genomes from individually living cats were from 15 FIPV- and 5 FECV-infected animals. Using specific primers (sense 5′-CGTCTCAATCAAGGCATATAATCCCGACGAAG-3′, antisense 5′-CAGTTGACGCGTTGTCCCTGTG-3′), we amplified the same 575-bp M gene fragment as studied by Brown et al. (9). GenBank accession numbers for the FCoV M gene sequences determined in this study are {"type":"entrez-nucleotide-range","attrs":{"text":"HQ738691-HQ738733","start_term":"HQ738691","end_term":"HQ738733","start_term_id":"321530205","end_term_id":"321530289"}}HQ738691-HQ738733. When compared by phylogenetic analysis, the nucleotide sequences of FIPV and FECV M genes distributed into paraphyletic patterns rather than in monophyletic clusters (Figure, panel A). Figure A) Phylogenetic relationships of feline coronaviruses (FCoVs) detected in feces of healthy cats and in organs/ascites of cats with feline infectious peritonitis. Alignment of the matrix (M) gene sequences was used to generate a rooted neighbor-joining ... Thus, as we observed earlier for the 3c gene (10), M gene sequences generally clustered according to the cattery from where they originated, irrespective of their pathotype (e.g., FECV 586 and FIPVs 584 and 585 from cattery A; FECV 620 and FIPVs 615 and 622 from cattery G; FECV 10 and FIPV 8 from cattery F). Such a distribution pattern is consistent with the mutation theory, according to which FIPVs originate from FECVs and are thus closely related (7,9). Exceptions in this picture were FIPV 9 in cattery F and FECVs 406 and 407 in cattery D, presumably caused by multiple FCoV lineages in these open catteries (an open cattery is one in which cats can move in and out, usually for breeding purposes). We also examined the 5 aa sites in the M protein identified by Brown et al. (9) as being potentially diagnostic of FIP. An alignment of the relevant part of the polypeptide sequence, comprising the presumed signature residues at positions 108, 120, 138, 163 and 199, is shown in the Figure, panel B, for all FIPV and FECV genomes sequenced in this study. Within this sample collection, we observed complete sequence conservation at positions 108 and 199, virtually complete conservation (1 difference) at position 163. The 2 aa identities (Val and Ile) found at position 120 and 138 occurred with similar frequencies in FIPV and FECV (position 120: Ile in 16/36 [44%] FIPVs and in 6/14 [43%] FECVs; position 138: Ile in 29/36 [81%] FIPVs and in 12/14 [86%] FECVs). These observations do not indicate the slightest tendency of sequence segregation among the 2 pathotypes. In the alignment of the Figure, panel B, we also included M protein sequences translated from several FCoV genomes from the Americas, 7 FIPV, and 1 FECV (8). The comparison does not reveal peculiarities indicative of geographic segregation. Hence, our data do not confirm the diagnostic potential of the M protein sequence nor do they support the suggested role of the membrane protein in FIP pathogenesis (9). Informative as it may be, comparative sequence analysis will eventually not be sufficient to answer the FECV/FIPV question. What will be needed is a reverse genetics system to generate and manipulate the FCoV genome as well as a cell culture system to propagate the viruses, both of which have thus far not been achieved.

This chapter presents an overview of cell entry by nidoviruses, and focuses on the functioning of the coronavirus fusion protein, in particular, on its proteolytic cleavage activation and on its conformation rearrangements in the process of membrane fusion. The actual fusion reaction is primed by interaction with the target cell where any of a number of triggers-receptor binding, proteolytic cleavage, or acidic pH-can spark off a cascade of conformational changes by which the metastable trimer eventually ends up in a trimer-of-hairpins conformation that is characterized by two main features: a six-helix bundle structure formed by the antiparallel association of the HR1 and HR2 domains, and the juxtaposed fusion peptides and transmembrane domains anchored together in the fused lipid bilayer. With regard to the intermediate conformational states, experimental support has only been obtained for the occurrence of a prehairpin intermediate in which the fusion peptides, originally hidden away in the native trimeric structure, have become exposed, pointing away from the viral membrane and into the target membrane. The authors describe how coronavirus membrane fusion fits in this picture by discussing the triggers, the key structural elements in the spike protein, its conformational states, and the actual fusion process. Membrane fusion is currently viewed as a process with defined events: merging first of the apposing leaflets (lipid stalk formation or hemifusion) and then of the distal leaflets (fusion pore formation), followed by fusion pore enlargement.

The glycoprotein precursor (G1/G2) gene of tomato spotted wilt virus (TSWV) was expressed in BHK cells using the Semliki Forest virus expression system. The results reveal that in this cell system, the precursor is efficiently cleaved and the resulting G1 and G2 glycoproteins are transported from the endoplasmic reticulum (ER) to the Golgi complex, where they are retained, a process that could be blocked by tunicamycin. Expression of G2 alone resulted in transport to and retention in the Golgi complex, albeit less efficient, suggesting that G2 contains a Golgi retention signal. G1 alone was retained in the ER, irrespective of whether it contained the precursor's signal sequence or its own N-terminal hydrophobic sequence. Coexpression of G1 and G2 from separate gene constructs resulted in rescue of efficient G1 transport, as the proteins coaccumulated in the Golgi complex, indicating that their interaction is essential for proper targeting to this organelle. The results demonstrate that transport and targeting of the plant TSWV glycoproteins in mammalian BHK cells are strikingly similar to those of animal-infecting bunyavirus glycoproteins in mammalian cells. The observations are likely to reflect the dual tropism of TSWV, which replicates both in its plant host and in its animal (thrips) vector.

The potential of porcine reproductive and respiratory syndrome virus (PRRSV) as a viral vector was explored by the insertion of a sequence encoding a foreign antigen into the infectious cDNA clone of the Lelystad virus isolate. An epitope of the hemagglutinin (HA) protein of human influenza A virus was introduced at the 5′ end and at the 3′ end of ORF7, in each case resulting in a fusion protein between the HA epitope and the nucleocapsid (N) protein. Furthermore, in the construct carrying the HA sequences at the 5′ end of ORF7, additional in-frame insertions encoding the autoprotease 2A of foot-and-mouth disease virus were made between the HA and ORF7 sequences to ensure the generation of a functional N protein from its hybrid precursor. When RNA transcripts from these full-length cDNA clones were transfected into BHK-21 cells, they were each found to replicate, to express the HA epitope, and to produce progeny virus. However, fusion of the HA epitope directly to the nucleocapsid protein either at the N terminus or at the C terminus adversely affected both the viability and the genetic stability of the recombinant PRRS viruses. Serial passage of the recombinant viruses on porcine alveolar macrophages demonstrated that these viruses had lost (part of) the HA epitope at passage four. In contrast, in the PRRS viruses expressing the HA epitope from a precursor cleavable by the autoprotease 2A peptide, the HA epitope was still intact after four passages, and no effect on the viability of these viruses was observed. Immunoprecipitation and pulse chase experiments revealed the efficient and presumably cotranslational cleavage of the HA epitope from the N protein by the 2A protease. Our results demonstrate the feasibility of using PRRSV as a viral vector that might be suitable for the delivery of antigens from other pathogens to the immune system of the pig.

Enzyme-linked immunosorbant assays (ELISAs) were developed for the detection of antibodies against the major envelope glycoprotein (G(L)) of equine arteritis virus (EAV). A 6-Histidine tagged recombinant protein expressing the complete G(L) ectodomain (G(L)-6His), a glutathione-S-transferase recombinant protein expressing amino acids 55-98 of G(L) (G(L)-GST) and an ovalbumin-conjugated synthetic peptide representing amino acids 81-106 of G(L) (G(L)-OVA) were used as diagnostic antigens. An ELISA procedure was developed and optimised for each antigen. The G(L)-OVA and G(L)-6His assays showed the greatest specificity while the G(L)-GST assay was slightly more sensitive that the G(L)-OVA and G(L)-6His assays; results based on the analysis of 50 virus neutralisation positive and 50 virus neutralisation negative sera. The G(L)-OVA ELISA was selected for further evaluation since it was simpler to use than ELISAs based on recombinant antigens and did not suffer from background reactivity. The final sensitivity and specificity of the G(L)-OVA ELISA were 96.75 and 95.6%, respectively, results based on the analysis of 400 virus neutralisation positive and 400 virus neutralisation negative sera. It also detected EAV antibody (100% efficiency) in seropositive shedding stallions and, in ponies infected experimentally with the UK93 isolate of EAV, the appearance of virus neutralising antibodies and G(L)-OVA ELISA-specific immunoglobulins coincided.

Epithelial cells are the first host cells to be infected by incoming c oronaviruses. Recent observations in vitro show that coronaviruses are released from a specific side of these polarized cells, and this polarized release might be important for the spread of the infection in vivo. Mechanisms for the directional sorting of coronaviruses might be similar to those governing the polar release of secretory proteins.

We recently developed a highly effective immunization procedure for the generation of monoclonal antibodies (MAbs) directed against the porcine reproductive and respiratory syndrome virus (E. Weiland, M. Wieczorek-Krohmer, D. Kohl, K. K. Conzelmann, and F. Weiland, Vet. Microbiol. 66:171–186, 1999). The same method was used to produce a panel of 16 MAbs specific for the equine arteritis virus (EAV). Ten MAbs were directed against the EAV nucleocapsid (N) protein, and five MAbs recognized the major viral envelope glycoprotein (G L ). Two of the EAV G L -specific MAbs and one antibody of unknown specificity neutralized virus infectivity. A comparison of the reactivities of the MAbs with 1 U.S. and 22 newly obtained European field isolates of EAV demonstrated that all N-specific MAbs, the three nonneutralizing anti-G L MAbs, and the weakest neutralizing MAb (MAb E7/d15-c9) recognized conserved epitopes. In contrast, the two MAbs with the highest neutralization titers bound to 17 of 23 (MAb E6/A3) and 10 of 23 (MAb E7/d15-c1) of the field isolates. Ten of the virus isolates reacted with only one of these two MAbs, indicating that they recognized different epitopes. The G L -specific MAbs and the strongly neutralizing MAb of unknown specificity (MAb E6/A3) were used for the selection of neutralization-resistant (NR) virus variants. The observation that the E6/A3-specific NR virus variants were neutralized by MAb E7/d15-c1 and that MAb E6/A3 blocked the infectivity of the E7/d15-c1-specific NR escape mutant confirmed that these antibodies reacted with distinct antigenic sites. Immunoelectron microscopy revealed for the first time that the antigenic determinants recognized by the anti-G L MAbs were localized on the virion surface. Surprisingly, although the immunofluorescence signal obtained with the neutralizing antibodies was relatively weak, they mediated binding of about three times as much gold granules to the viral envelope than the nonneutralizing anti-G L MAbs.

Equine arteritis virus (EAV) is an enveloped, positive-stranded RNA virus belonging to the family Arteriviridae of the order Nidovirales. The unsegmented, infectious genome of EAV is 12,704 nt in length [exclusive of the poly(A) tail] and contains eight overlapping genes that are expressed from a 3′-coterminal nested set of seven leader-containing mRNAs. To investigate the importance of the overlapping gene arrangement in the viral life-cycle and to facilitate the genetic manipulation of the viral genome, a series of mutant full-length cDNA clones was constructed in which either EAV open reading frames (ORFs) 4 and 5 or ORFs 5 and 6 or ORFs 4, 5, and 6 were separated by newly introduced AflII restriction endonuclease cleavage sites. RNA transcribed from each of these plasmids was infectious, demonstrating that the overlapping gene organization is not essential for EAV viability. Moreover, the recombinant viruses replicated with almost the same efficiency, i.e., reached nearly the same infectious titers as the wildtype virus, and stably maintained the mutations that were introduced. The AflII site engineered between ORFs 5 and 6 was subsequently used to generate a virus in which the ectodomain of the ORF 6-encoded M protein was extended with nine amino acids derived from the extreme N-terminus of the homologous protein of mouse hepatitis virus (MHV; family Coronaviridae, order Nidovirales). This nonapeptide contains a functional O-glycosylation signal as well as an epitope recognized by an MHV-specific monoclonal antibody, both of which were expressed by the recombinant virus. Although the hybrid virus had a clear growth disadvantage in comparison to the parental virus, three serial passages did not result in the loss of the foreign genetic material.

Feline coronaviruses are widespread and come in different flavors. There are two main serotypes both of which occur in two pathotypes, the avirulent enteric viruses and the virulent, usually fatal peritonitis viruses, the latter in turn occurring either in a 'wet' or exudative form or in a 'dry' or proliferative form. In this paper a concise overview is given of the molecular features of these viruses. Special attention is given to the genetic dynamics of the viruses as these now allow us to begin to understand the origin of the different phenotypes, in particular the genesis of virulence during persistent infection. As discussed, the surprising new insights obtained over the last few years call for a critical reevaluation of strategies for protection.

Antibody-mediated targeting of viral vectors to the Fc receptor expressed on acute myeloid leukemia cells

Coronaviruses infect humans and animals through epithelial cells of the gastrointestinal and respiratory tracts that serve as their primary target. When studying infections in cultured polarized epithelial cells, we found previously that coronaviruses are released from specific plasma-membrane domains; thus, mouse hepatitis virus (strain A59; MHV-A59) leaves murine epithelial kidney cells from the basolateral surface, whereas release of transmissible gastroenteritis virus from porcine epithelial kidney cells is confined to the apical membrane. This observation begged the question whether a particular coronavirus is consistently shed through the same membrane, irrespective of the nature of the epithelial cell. We therefore extended our studies with MHV-A59 to Madin-Darby canine kidney (MDCK) strain I and human colon carcinoma (Caco-2) cells, both of which are naturally refractory to MHV-A59 but were made susceptible to infection by transfection with recombinant MHV receptor cDNA. The release of MHV-A59 from Caco(MHVR) cells occurred preferentially from the basolateral side, consistent with our previous observations. In contrast, release from MDCK(MHVR) cells occurred almost exclusively from the apical surface. Because of this difference, we studied MHV-A59 infection of MDCK(MHVR) cells in more detail. The virus entered the cells preferentially from the apical side, a situation similar to that in murine epithelial cells, where the highest density of MHV receptor glycoprotein was found. The results from this and previous studies show that targeting of vesicles containing MHV-A59 to a specific side of epithelial cells may vary in different epithelial cell types.

[Background]: A new betacoronavirus-Middle East respiratory syndrome coronavirus (MERS-CoV)-has been identified in patients with severe acute respiratory infection. Although related viruses infect bats, molecular clock analyses have been unable to identify direct ancestors of MERS-CoV. Anecdotal exposure histories suggest that patients had been in contact with dromedary camels or goats. We investigated possible animal reservoirs of MERS-CoV by assessing specific serum antibodies in livestock. [Methods]: We took sera from animals in the Middle East (Oman) and from elsewhere (Spain, Netherlands, Chile). Cattle (n=80), sheep (n=40), goats (n=40), dromedary camels (n=155), and various other camelid species (n=34) were tested for specific serum IgG by protein microarray using the receptor-binding S1 subunits of spike proteins of MERS-CoV, severe acute respiratory syndrome coronavirus, and human coronavirus OC43. Results were confirmed by virus neutralisation tests for MERS-CoV and bovine coronavirus. [Findings]: 50 of 50 (100%) sera from Omani camels and 15 of 105 (14%) from Spanish camels had protein-specific antibodies against MERS-CoV spike. Sera from European sheep, goats, cattle, and other camelids had no such antibodies. MERS-CoV neutralising antibody titres varied between 1/320 and 1/2560 for the Omani camel sera and between 1/20 and 1/320 for the Spanish camel sera. There was no evidence for cross-neutralisation by bovine coronavirus antibodies. [Interpretation]: MERS-CoV or a related virus has infected camel populations. Both titres and seroprevalences in sera from different locations in Oman suggest widespread infection., RDS was funded by the European Public Health Training Program (Euphem), ECDC, Stockholm, Sweden. Contributions to the study were funded through the European Union FP7 projects EMPERIE (contract number 223498; to BLH, SLS, AO, CD) and ANTIGONE (contract number 278976; to CG, CD, MPGK, AO). Work in Bonn was also funded by Deutsche Forschungsgemeinschaft (DFG grant DR772/3-1 to CD).

A nested reverse transcriptase PCR (RT-nPCR) was developed for the detection of feline coronavirus (FCoV) RNA in the feces, tissues, and body fluids of infected cats. The RT-nPCR was targeted to the highly conserved 3'-untranslated region of the viral genome and will detect most, if not all, feline coronaviruses in the field. With the RT-nPCR, FCoV RNA was detected in plasma samples from experimentally infected cats as early as 2 days postinoculation. FCoV RNA was also detected in serum, plasma, or ascitic fluid samples from 14 of 18 cats (78%) with naturally occurring feline infectious peritonitis (FIP). The use of RT-PCR for FIP diagnosis is limited because of the occurrence of apparently healthy FCoV carriers. These asymptomatic cats shed the virus in the feces and, in a number of cases, also had detectable virus in the plasma. Because of the nature of FCoV infections, our RT-PCR assay with plasma or serum cannot be used to establish a definite diagnosis of FIP. However, this assay does provide a new means to identify asymptomatic FCoV carriers. As such, RT-nPCR will be of use to screen cats before their introduction into FCoV-free catteries. Moreover, this assay provides an important tool to study the epidemiology of FCoV.

This special issue of the journal is dedicated to the recent progress on coronaviruses and covers the topics of viral epidemiology, virus replication and the interactions between the coronaviruses and their hosts.

The entry of the enveloped Rift Valley fever virus (RVFV) into its host cell is mediated by the viral glycoproteins Gn and Gc. We investigated the RVFV entry process and, in particular, its pH-dependent activation mechanism using our recently developed nonspreading-RVFV-particle system. Entry of the virus into the host cell was efficiently inhibited by lysosomotropic agents that prevent endosomal acidification and by compounds that interfere with dynamin- and clathrin-dependent endocytosis. Exposure of plasma membrane-bound virions to an acidic pH (

Immunological checkpoints, such as the inhibitory CD200 receptor (CD200R), play a dual role in balancing the immune system during microbial infection. On the one hand these inhibitory signals prevent excessive immune mediated pathology but on the other hand they may impair clearance of the pathogen. We studied the influence of the inhibitory CD200-CD200R axis on clearance and pathology in two different virus infection models. We find that lack of CD200R signaling strongly enhances type I interferon (IFN) production and viral clearance and improves the outcome of mouse hepatitis corona virus (MHV) infection, particularly in female mice. MHV clearance is known to be dependent on Toll like receptor 7 (TLR7)-mediated type I IFN production and sex differences in TLR7 responses previously have been reported for humans. We therefore hypothesize that CD200R ligation suppresses TLR7 responses and that release of this inhibition enlarges sex differences in TLR7 signaling. This hypothesis is supported by our findings that in vivo administration of synthetic TLR7 ligand leads to enhanced type I IFN production, particularly in female Cd200−/− mice and that CD200R ligation inhibits TLR7 signaling in vitro. In influenza A virus infection we show that viral clearance is determined by sex but not by CD200R signaling. However, absence of CD200R in influenza A virus infection results in enhanced lung neutrophil influx and pathology in females. Thus, CD200-CD200R and sex are host factors that together determine the outcome of viral infection. Our data predict a sex bias in both beneficial and pathological immune responses to virus infection upon therapeutic targeting of CD200-CD200R., Author Summary Immune responses need to be carefully orchestrated to prevent disease due to an overactive immune system. Immunological checkpoints are provided by immune inhibitory receptors, which set a threshold for activation and dampen the immune system. In the case of a viral infection, this prevents pathology induced by the immune system, but on the other hand may prevent adequate removal of the virus. In this paper, we show that removal of such an immunological checkpoint in mice leads to rapid removal of corona virus, but also to more immune-induced disease symptoms in case of influenza virus infection. We observe this predominantly in female mice. We demonstrate that this particular checkpoint inhibits anti-viral responses that are naturally stronger in females. Release of this checkpoint enlarges these sex differences. Our findings have major implications for therapeutic use of blockers of this pathway, which are currently in clinical trials for the treatment of cancer, as we predict that female patients will have a stronger response to such therapeutics.

The M protein of mouse hepatitis virus (MHV) is a triple-spanning membrane glycoprotein that is exclusively O-glycosylated. When expressed independently, it accumulates in late Golgi and the trans-Golgi network (TGN) (Locker, J. K., Griffiths, G., Horzinek, M. C., and Rottier, P. J. M. (1992) (J. Biol. Chem. 267, 14094-14101). To analyze the domains of this protein responsible for its localization, we have generated deletion mutants by site-directed mutagenesis and analyzed their intracellular transport. The intracellular distribution of the mutant proteins was determined by following the extent of O-glycosylation in pulse-chase experiments, by electron microscopic immunocytochemistry, and by surface immunoprecipitation. Mutant proteins lacking the first or the first and second transmembrane domains were not efficiently retained in the Golgi complex or TGN. The latter mutant proteins also localized to endocytic compartments but were not subject to rapid lysosomal degradation. Deletion of the COOH-terminal 22 amino acids, including a tyrosine residue in the context of a potential internalization signal, resulted in plasma membrane exposure of the respective mutant protein. We show that the wild-type MHV-M protein does not recycle between the plasma membrane and the TGN, but rather behaves as a late Golgi/TGN resident in our assays. We propose that the MHV-M protein is retained in the Golgi by two signals, one contained in the cytoplasmic tail and the other determined by the transmembrane domains.

Mouse hepatitis coronavirus (MHV) buds into pleomorphic membrane structures with features expected of the intermediate compartment between the ER and the Golgi complex. Here, we characterize the MHV budding compartment in more detail in mouse L cells using streptolysin O (SLO) permeabilization which allowed us to better visualize the membrane structures at the ER-Golgi boundary. The MHV budding compartment shares membrane continuities with the rough ER as well as with cisternal elements on one side of the Golgi stack. It also labeled with p58 and rab2, two markers of the intermediate compartment, and with PDI, usually considered to be a marker of the rough ER. The membranes of the budding compartment, as well as the budding virions themselves, but not the rough ER, labeled with the N-acetyl-galactosamine (GalNAc)-specific lectin Helix pomatia. When the SLO-permeabilized cells were treated with guanosine 5'-(3-O-thio)triphosphate (GTP gamma S), the budding compartment accumulated a large number of beta-cop-containing buds and vesicular profiles. Complementary biochemical experiments were carried out to determine whether vesicular transport was required for the newly synthesized M protein, that contains only O-linked oligosaccharides, to acquire first, GalNAc and second, the Golgi modifications galactose and sialic acid. The results from both in vivo studies and from the use of SLO-permeabilized cells showed that, while GalNAc addition occurred under conditions which block vesicular transport, both cytosol and ATP were prerequisites for the M protein oligosaccharides to acquire Golgi modifications. Collectively, our data argue that transport from the rough ER to the Golgi complex requires only one vesicular transport step and that the intermediate compartment is a specialized domain of the endoplasmatic reticulum that extends to the first cisterna on the cis side of the Golgi stack.

In the last decade many studies have been performed on the virulence of Newcastle disease virus (NDV). This is mainly due to the development of reverse genetics systems which made it possible to genetically modify NDV and to investigate the contribution of individual genes and genome regions to its virulence. However, the available information is scattered and a comprehensive overview of the factors and conditions determining NDV virulence is lacking. This review summarises, compares and discusses the available literature and shows that virulence of NDV is a complex trait determined by multiple genetic factors.

Eel herpesvirus or anguillid herpesvirus 1 (AngHV1) frequently causes disease in freshwater eels. The complete genome sequence of AngHV1 and its taxonomic position within the family Alloherpesviridae were determined. Shotgun sequencing revealed a 249 kbp genome including an 11 kbp terminal direct repeat that contains 7 of the 136 predicted protein-coding open reading frames. Twelve of these genes are conserved among other members of the family Alloherpesviridae and another 28 genes have clear homologues in cyprinid herpesvirus 3. Phylogenetic analyses based on amino acid sequences of five conserved genes, including the ATPase subunit of the terminase, confirm the position of AngHV1 within the family Alloherpesviridae, where it is most closely related to the cyprinid herpesviruses. Our analyses support a recent proposal to subdivide the family Alloherpesviridae into two sister clades, one containing AngHV1 and the cyprinid herpesviruses and the other containing Ictalurid herpesvirus 1 and the ranid herpesviruses.

We have demonstrated that influenza A virus (IAV) RNA synthesis depends on the ubiquitin-proteasome system. IAV replication was reduced both by proteasome inhibitors and in E36ts20 cells, which contain the thermolabile ubiquitin-activating enzyme E1. While virus entry was not affected in E36ts20 cells, the proteasome inhibitor MG132 retained viral particles in the cytoplasm. Addition-removal experiments of MG132 in combination with bafilomycin A1, a well-established inhibitor of IAV entry and fusion, showed that MG132 affected IAV infection at a postfusion step. This was confirmed by the lack of inhibition of IAV entry by proteasome inhibitors in a virus-like particle fusion assay.

The genomic organization at the 3' end of canine coronavirus (CCV) and feline enteric coronavirus (FECV) was determined by sequence analysis and compared to that of feline infectious peritonitis virus (FIPV) and transmissible gastroenteritis virus (TGEV) of swine. Comparison of the latter two has previously revealed an extra open reading frame (ORF) at the 3' end of the FIPV genome, lacking in TGEV, which is currently designated ORF 6b. Both CCV and FECV possess 6b-related ORFs at the 3' ends of their genomes. The presence of ORF 6b in three of four viruses in this antigenic cluster strongly suggests that TGEV has lost this ORF by deletion. The CCV ORF 6b is collinear with that of FIPV, but the predicted amino acid sequences are only 58% identical. The FECV ORF 6b contains a large deletion compared to that of FIPV, reducing the collinear part to 60%. The sequence homologies were highest between CCV and TGEV on the one hand and between FECV and FIPV on the other. Previously, we showed that the expression product of the FIPV ORF 6b can be detected in infected cells by immunoprecipitation (Vennema et al., 1992). In the present study we have performed similar experiments with CCV and FECV. In infected cells both viruses produced proteins related to but different from the FIPV 6b protein.

In this review we discuss five groups of viruses that bud into, or assemble from, different compartments along the biosynthetic pathway. These are herpes-, rota-, corona-, bunya- and pox-viruses. Our main emphasis will be on the virally-encoded membrane glycoproteins that are responsible for determining the site of virus assembly. In a number of cases these proteins have been well characterized and appear to serve as resident markers of the budding compartments. The assembly and dissemination of these viruses raises many questions of cell biological interest.

Targeted RNA recombination has proven to be a powerful tool for the genetic engineering of the coronavirus genome, particularly in its 3' part. Here we describe procedures for the generation of recombinant and mutant mouse hepatitis virus and feline infectious peritonitis virus. Key to the two-step method is the efficient selection of recombinant viruses based on host cell switching. The first step consists of the preparation---using this selection principle--of an interspecies chimeric coronavirus. In this virus the ectodomain of the spike glycoprotein is replaced by that of a coronavirus with a different species tropism. In the second step this chimeric virus is used as the recipient for recombination with synthetic donor RNA carrying the original spike gene. Recombinant viruses are then isolated on the basis of their regained natural (e.g., murine or feline) cell tropism. Additional mutations created in the donor RNA can be co-incorporated into the recombinant virus in order to generate mutant viruses.

Coronaviruses are positive-strand RNA viruses of extraordinary genetic complexity and diversity. In addition to a common set of genes for replicase and structural proteins, each coronavirus may carry multiple group-specific genes apparently acquired through relatively recent heterologous recombination events. Here we describe an accessory gene, ORF3, unique to canine coronavirus type I (CCoV-I) and characterize its product, glycoprotein gp3. Whereas ORF3 is conserved in CCoV-I, only remnants remain in CCoV-II and CCoV-II-derived porcine and feline coronaviruses. Our findings provide insight into the evolutionary history of coronavirus group 1a and into the dynamics of gain and loss of accessory genes.