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Four canine coronavirus type II (CCoV-II) strains were identified in the guts and internal organs of pups which had died of acute gastroenteritis. The CCoV-II strains were strictly related to porcine transmissible gastroenteritis virus (TGEV) in the N-terminal domain of the spike protein, whereas in the other parts of the genome, a higher genetic relatedness to recent CCoV-II isolates was observed. Experimental infection of dogs with a TGEV-like isolate induced mild gastroenteritis without any systemic involvement. By virus neutralization tests, antigenic differences between reference and TGEV-like CCoVs were found. Our data support the potential recombinant origin of the TGEV-like CCoVs. Copyright © 2009, American Society for Microbiology. All Rights Reserved., This work was supported by grants from the Italian Ministry of Health, Ricerca finalizzata 2008, project Mammalian Coronaviruses: Molecular Epidemiology, Vaccine Development and Implications for Animal and Human Health (Nicola Decaro), and from the Italian Ministry of Education, University and Research, PRIN 2006, project Infezione del Cane da Coronavirus Pantropico: Aspetti Epidemiologici, Patogenetici e Molecolari (Canio Buonavoglia).

The immunogenicity and protective efficacy of a live attenuated vaccine consisting of a recombinant severe acute respiratory syndrome (SARS) coronavirus lacking the E gene (rSARS-CoV-ΔE) were studied using hamsters. Hamsters immunized with rSARS-CoV-ΔE developed high serum-neutralizing antibody titers and were protected from replication of homologous (SARS-CoV Urbani) and heterologous (GD03) SARS-CoV in the upper and lower respiratory tract. rSARS-CoV-ΔE-immunized hamsters remained active following wild-type virus challenge, while mock-immunized hamsters displayed decreased activity. Despite being attenuated in replication in the respiratory tract, rSARS-CoV-ΔE is an immunogenic and efficacious vaccine in hamsters., This research was supported in part by the Intramural Research Program of the NIH, NIAID; by NIH AID AI059136; and by the European Community (projects DISSECT SP22-CT-2004-511060 and Rivigene SSPE-CT-2005-022639).

Recombinant severe acute respiratory virus (SARS-CoV) variants lacking the group specific genes 6, 7a, 7b, 8a, 8b and 9b (rSARS-CoV-Δ[6-9b]), the structural gene E (rSARS-CoV-ΔE), and a combination of both sets of genes (rSARS-CoV-Δ[E,6-9b]) have been generated. All these viruses were rescued in monkey (Vero E6) cells and were also infectious for human (Huh-7, Huh7.5.1 and CaCo-2) cell lines and for transgenic (Tg) mice expressing the SARS-CoV receptor human angiotensin converting enzyme-2 (hACE-2), indicating that none of these proteins is essential for the viral cycle. Furthermore, in Vero E6 cells, all the viruses showed the formation of particles with the same morphology as the wt virus, indicating that these proteins do not have a high impact in the final morphology of the virions. Nevertheless, in the absence of E protein, release of virus particles efficacy was reduced. Viruses lacking E protein grew about 100-fold lower than the wt virus in lungs of Tg infected mice but did not grow in the brains of the same animals, in contrast to the rSARS-CoV-Δ[6-9b] virus, which grew almost as well as the wt in both tissues. Viruses lacking E protein were highly attenuated in the highly sensitive hACE-2 Tg mice, in contrast to the minimal rSARS-CoV-Δ[6-9b] and wt viruses. These data indicate that E gene might be a virulence factor influencing replication level, tissue tropism and pathogenicity of SARS-CoV, suggesting that ΔE attenuated viruses are promising vaccine candidates. © 2008 Elsevier Inc. All rights reserved., This work was supported by grants from the Ministry of Education and Science of Spain (BIO2004-00636), the European Community (DISSECT PROJECT, SP22-CT-2004-511060 and RiViGene PROJECT, SSPE-CT-2005-022639), Fort Dodge Veterinaria and the National Institutes of Health, USA (PO1 AI 060699).

Coronavirus (CoV) transcription includes a discontinuous mechanism during the synthesis of subgenome-length minus-strand RNAs leading to a collection of mRNAs in which the 5′ terminal leader sequence is fused to contiguous genome sequences. It has been previously shown that transcription-regulating sequences (TRSs) preceding each gene regulate transcription. Base pairing between the leader TRS (TRS-L) and the complement of the body TRS (cTRS-B) in the nascent RNA is a determinant factor during CoV transcription. In fact, in transmissible gastroenteritis CoV, a good correlation has been observed between subgenomic mRNA (sg mRNA) levels and the free energy (ΔG) of TRS-L and cTRS-B duplex formation. The only exception was sg mRNA N, the most abundant sg mRNA during viral infection in spite of its minimum ΔG associated with duplex formation. We postulated that additional factors should regulate transcription of sg mRNA N. In this report, we have described a novel transcription regulation mechanism operating in CoV by which a 9-nucleotide (nt) sequence located 449 nt upstream of the N gene TRS core sequence (CS-N) interacts with a complementary sequence just upstream of CS-N, specifically increasing the accumulation of sg mRNA N. Alteration of this complementarity in mutant replicon genomes showed a correlation between the predicted stability of the base pairing between 9-nt sequences and the accumulation of sg mRNA N. This interaction is exclusively conserved in group 1a CoVs, the only CoV subgroup in which the N gene is not the most 3′ gene in the viral genome. This is the first time that a long-distance RNA-RNA interaction regulating transcriptional activity specifically enhancing the transcription of one gene has been described to occur in CoVs. Copyright © 2008, American Society for Microbiology. All Rights Reserved., This work was supported by grants from the European Union (Frame VI, DISSECT PROJECT, SP22-CT-2004-511060, and RiViGene PROJECT, SSPE-CT-2005-022639) and a grant (CPE03-021-C4-3) from the National Institute of Agronomics Research (INIA), Spain. J.L.M., S.Z., and I.S.G. received fellowships from Consejo Superior de Investigaciones Científicas (CSIC).

An important effort has been performed after the emergence of severe acute respiratory syndrome (SARS) epidemic in 2003 to diagnose and prevent virus spreading. Several types of vaccines have been developed including inactivated viruses, subunit vaccines, virus-like particles (VLPs), DNA vaccines, heterologous expression systems, and vaccines derived from SARS-CoV genome by reverse genetics. This review describes several aspects essential to develop SARS-CoV vaccines, such as the correlates of protection, virus serotypes, vaccination side effects, and bio-safeguards that can be engineered into recombinant vaccine approaches based on the SARS-CoV genome. The production of effective and safe vaccines to prevent SARS has led to the development of promising vaccine candidates, in contrast to the design of vaccines for other coronaviruses, that in general has been less successful. After preclinical trials in animal models, efficacy and safety evaluation of the most promising vaccine candidates described has to be performed in humans.

A recombinant transmissible gastroenteritis coronavirus (rTGEV) in which E gene was deleted (rTGEV-ΔE) has been engineered. This deletion mutant only grows in cells expressing E protein (E+ cells) indicating that E was an essential gene for TGEV replication. Electron microscopy studies of rTGEV-ΔE infected BHK-pAPN-E- cells showed that only immature intracellular virions were assembled. These virions were non-infectious and not secreted to the extracellular medium in BHK-pAPN-E- cells. RNA and protein composition analysis by RNase-gold and immunoelectron microscopy showed that rTGEV-ΔE virions contained RNA and also all the structural TGEV proteins, except the deleted E protein. Nevertheless, full virion maturation was blocked. Studies of the rTGEV-ΔE subcellular localization by confocal and immunoelectron microscopy in infected E- cells showed that in the absence of E protein virus trafficking was arrested in the intermediate compartment. Therefore, the absence of E protein in TGEV resulted in two actions, a blockade of virus trafficking in the membranes of the secretory pathway, and prevention of full virus maturation. © 2007 Elsevier Inc. All rights reserved.

A SARS-CoV lacking the full-length E gene (SARS-CoV-∆E) was attenuated and an effective vaccine. Here, we show that this mutant virus regained fitness after serial passages in cell culture or in vivo, resulting in the partial duplication of the membrane gene or in the insertion of a new sequence in gene 8a, respectively. The chimeric proteins generated in cell culture increased virus fitness in vitro but remained attenuated in mice. In contrast, during SARS-CoV-∆E passage in mice, the virus incorporated a mutated variant of 8a protein, resulting in reversion to a virulent phenotype. When the full-length E protein was deleted or its PDZ-binding motif (PBM) was mutated, the revertant viruses either incorporated a novel chimeric protein with a PBM or restored the sequence of the PBM on the E protein, respectively. Similarly, after passage in mice, SARS-CoV-∆E protein 8a mutated, to now encode a PBM, and also regained virulence. These data indicated that the virus requires a PBM on a transmembrane protein to compensate for removal of this motif from the E protein. To increase the genetic stability of the vaccine candidate, we introduced small attenuating deletions in E gene that did not affect the endogenous PBM, preventing the incorporation of novel chimeric proteins in the virus genome. In addition, to increase vaccine biosafety, we introduced additional attenuating mutations into the nsp1 protein. Deletions in the carboxy-terminal region of nsp1 protein led to higher host interferon responses and virus attenuation. Recombinant viruses including attenuating mutations in E and nsp1 genes maintained their attenuation after passage in vitro and in vivo. Further, these viruses fully protected mice against challenge with the lethal parental virus, and are therefore safe and stable vaccine candidates for protection against SARS-CoV., Author Summary Zoonotic coronaviruses, including SARS-CoV, Middle East respiratory syndrome (MERS-CoV), porcine epidemic diarrhea virus (PEDV) and swine delta coronavirus (SDCoV) have recently emerged causing high morbidity and mortality in human or piglets. No fully protective therapy is still available for these CoVs. Therefore, the development of efficient vaccines is a high priority. Live attenuated vaccines are considered most effective compared to other types of vaccines, as they induce a long-lived, balanced immune response. However, safety is the main concern of this type of vaccines because attenuated viruses can eventually revert to a virulent phenotype. Therefore, an essential feature of any live attenuated vaccine candidate is its stability. In addition, introduction of several safety guards is advisable to increase vaccine safety. In this manuscript, we analyzed the mechanisms by which an attenuated SARS-CoV reverted to a virulent phenotype and describe the introduction of attenuating deletions that maintained virus stability. The virus, engineered with two safety guards, provided full protection against challenge with a lethal SARS-CoV. Understanding the molecular mechanisms leading to pathogenicity and the in vivo evaluation of vaccine genetic stability contributed to a rational design of a promising SARS-CoV vaccine.

Modification of host-cell ionic content is a significant issue for viruses, as several viral proteins displaying ion channel activity, named viroporins, have been identified. Viroporins interact with different cellular membranes and self-assemble forming ion conductive pores. In general, these channels display mild ion selectivity, and, eventually, membrane lipids play key structural and functional roles in the pore. Viroporins stimulate virus production through different mechanisms, and ion channel conductivity has been proved particularly relevant in several cases. Key stages of the viral cycle such as virus uncoating, transport and maturation are ion-influenced processes in many viral species. Besides boosting virus propagation, viroporins have also been associated with pathogenesis. Linking pathogenesis either to the ion conductivity or to other functions of viroporins has been elusive for a long time. This article summarizes novel pathways leading to disease stimulated by viroporin ion conduction, such as inflammasome driven immunopathology., The work done by the authors was supported by grants from the government of Spain (BIO2013-42869-R, FIS2013-40473-P), Generalitat Valenciana (Prometeo 2012/069), Fundació Caixa Castelló-Bancaixa (P1-1B2012-03) and a U.S. National Institutes of Health (NIH) project (5P01AI060699). JLN-T received a contract from NIH. CC-R received a contract from Fundacion La Caixa. We acknowledge support by the CSIC Open Access Publication Initiative through its Unit of Information Resources for Research (URICI).

Severe acute respiratory syndrome coronavirus (SARS-CoV) causes a respiratory disease with a mortality rate of 10%. A mouseadapted SARS-CoV (SARS-CoV-MA15) lacking the envelope (E) protein (rSARS-CoV-MA15-ΔE) is attenuated in vivo. To identify E protein regions and host responses that contribute to rSARS-CoV-MA15-ΔE attenuation, several mutants (rSARS-CoVMA15- E*) containing point mutations or deletions in the amino-terminal or the carboxy-terminal regions of the E protein were generated. Amino acid substitutions in the amino terminus, or deletion of regions in the internal carboxy-terminal region of E protein, led to virus attenuation. Attenuated viruses induced minimal lung injury, diminished limited neutrophil influx, and increased CD4+ and CD8+ T cell counts in the lungs of BALB/c mice, compared to mice infected with the wild-type virus. To analyze the host responses leading to rSARS-CoV-MA15-E* attenuation, differences in gene expression elicited by the native and mutant viruses in the lungs of infected mice were determined. Expression levels of a large number of proinflammatory cytokines associated with lung injury were reduced in the lungs of rSARS-CoV-MA15-E*-infected mice, whereas the levels of anti-inflammatory cytokines were increased, both at the mRNA and protein levels. These results suggested that the reduction in lung inflammation together with a more robust antiviral T cell response contributed to rSARS-CoV-MA15-E* attenuation. The attenuated viruses completely protected mice against challenge with the lethal parental virus, indicating that these viruses are promising vaccine candidates., This work was supported by grants from the Ministry of Science and Innovation of Spain (MICINN BIO2010-16705 and MINECO BIO2013- 42869-R), the European Community’s Seventh Framework Programme (FP7/2007–2013) under the project “EMPERIE” EC grant agreement number 223498, and a U.S. National Institutes of Health (NIH) project (2P01AI060699). J.A.R.-N. and C.C.-R. received a fellowship from the Fundacion La Caixa.

A 32-nucleotide (nt) RNA motif located at the 3= end of the transmissible gastroenteritis coronavirus (TGEV) genome was found to specifically interact with the host proteins glutamyl-prolyl-tRNA synthetase (EPRS) and arginyl-tRNA synthetase (RRS). This RNA motif has high homology in sequence and secondary structure with the gamma interferon-activated inhibitor of translation (GAIT) element, which is located at the 3= end of several mRNAs encoding proinflammatory proteins. The GAIT element is involved in the translation silencing of these mRNAs through its interaction with the GAIT complex (EPRS, heterogeneous nuclear ribonucleoprotein Q, ribosomal protein L13a, and glyceraldehyde 3-phosphate dehydrogenase) to favor the resolution of inflammation. Interestingly, we showed that the viral RNA motif bound the GAIT complex and inhibited the in vitro translation of a chimeric mRNA containing this RNA motif. To our knowledge, this is the first GAIT-like motif described in a positive RNA virus. To test the functional role of the GAIT-like RNA motif during TGEV infection, a recombinant coronavirus harboring mutations in this motif was engineered and characterized. Mutations of the GAIT-like RNA motif did not affect virus growth in cell cultures. However, an exacerbated innate immune response, mediated by the melanoma differentiation-associated gene 5 (MDA5) pathway, was observed in cells infected with the mutant virus compared with the response observed in cells infected with the parental virus. Furthermore, the mutant virus was more sensitive to beta interferon than the parental virus. All together, these data strongly suggested that the viral GAIT-like RNA motif modulates the host innate immune response IMPORTANCE The innate immune response is the first line of antiviral defense that culminates with the synthesis of interferon and proinflammatory cytokines to limit virus replication. Coronaviruses encode several proteins that interfere with the innate immune response at different levels, but to date, no viral RNA counteracting antiviral response has been described. In this work, we have characterized a 32-nt RNA motif located at the 3= end of the TGEV genome that specifically interacted with EPRS and RRS. This RNA motif presented high homology with the GAIT element, involved in the modulation of the inflammatory response. Moreover, the disruption of the viral GAIT-like RNA motif led to an exacerbated innate immune response triggered by MDA5, indicating that the GAIT-like RNA motif counteracts the host innate immune response. These novel findings may be of relevance for other coronaviruses and could serve as the basis for the development of novel antiviral strategies., This work was supported by grants from the Ministry of Science and Innovation of Spain (MCINN) (BIO2010-16705), the European Community’s Seventh Framework Programme (FP7/2007-2013) under the project “EMPERIE” (HEALTH-F3-2009-223498), and the National Institutes of Health (NIH) of The United States (2P01AI060699-06A1). S.M.-J. received a predoctoral fellowship from the National Institutes of Health (ISCIII) of Spain and a contract from the National Institutes of Health (NIH) of The United States. A.N. and S.Z. received contracts from EU and NIH

The large size of the coronavirus (CoV) genome (around 30 kb) and the instability in bacteria of plasmids carrying CoV replicase sequences, represent serious restrictions for the development of CoV infectious clones using reverse genetic systems similar to those used for smaller positive sense RNA viruses. To overcome these problems, several approaches have been established in the last thirteen years. Here we describe the engineering of CoV full-length cDNA clones as bacterial artificial chromosomes (BACs), using the Middle East respiratory syndrome CoV (MERS-CoV) as a model., This work was supported by grants from the Ministry of Science and Innovation of Spain (MCINN) (BIO2010-16705), the European Community's Seventh Framework Programme (FP7/2007-2013) under the project “EMPERIE” (HEALTH-F3-2009-223498), and the National Institute of Health (NIH) of USA (2P01AI060699-06A1). S. M. received a predoctoral fellowship from the National Institute of Health (ISCIII) of Spain.

Proteins secreted to mammalian cell supernatants are usually in a low concentration and purity, due to the limitation of the expression systems or the presence of a large amount of contaminant proteins from the cell medium. So, initial protein recovery from cell supernatants requires of a highly specific chromatography step. We compared several purification methods based on affinity chromatography for purification of proteins from cell culture supernatants: metal chelate affinity, strep-tag and immunopurification with a monoclonal antibody. Soluble receptor glycoproteins were engineered with the corresponding peptide tag at their C-terminal end. The proteins were expressed in 293T cells and secreted to the cell supernatant, as monitored by sandwich ELISA. Supernatants were run through the different chromatography columns and several purification-related parameters determined. While all column-retained proteins were easily eluted, the chelating and immunopurification chromatography gave the highest yield and the latest method provided a sample with the highest purity. So, in spite of its cost, immunopurification chromatography gave optimal results for purification of a low abundance protein from a cell supernatant. Finally, we applied a protein expression system together with immunopurification chromatography for preparation of a glycoprotein for crystallization.

This review focuses on the monophyletic group of animal RNA viruses united in the order Nidovirales. The order includes the distantly related coronaviruses, toroviruses, and roniviruses, which possess the largest known RNA genomes (from 26 to 32 kb) and will therefore be called ‘large’ nidoviruses in this review. They are compared with their arterivirus cousins, which also belong to the Nidovirales despite having a much smaller genome (13–16 kb). Common and unique features that have been identified for either large or all nidoviruses are outlined. These include the nidovirus genetic plan and genome diversity, the composition of the replicase machinery and virus particles, virus-specific accessory genes, the mechanisms of RNA and protein synthesis, and the origin and evolution of nidoviruses with small and large genomes. Nidoviruses employ single-stranded, polycistronic RNA genomes of positive polarity that direct the synthesis of the subunits of the replicative complex, including the RNA-dependent RNA polymerase and helicase. Replicase gene expression is under the principal control of a ribosomal frameshifting signal and a chymotrypsin-like protease, which is assisted by one or more papain-like proteases. A nested set of subgenomic RNAs is synthesized to express the 3′-proximal ORFs that encode most conserved structural proteins and, in some large nidoviruses, also diverse accessory proteins that may promote virus adaptation to specific hosts. The replicase machinery includes a set of RNA-processing enzymes some of which are unique for either all or large nidoviruses. The acquisition of these enzymes may have improved the low fidelity of RNA replication to allow genome expansion and give rise to the ancestors of small and, subsequently, large nidoviruses.

Highlights • The four different reverse genetic systems developed for CoVs are reviewed and discussed. • These systems were based on the use of BACs, in vitro ligation and vaccinia virus as a vector. • CoV reverse genetics is an essential tool for the study of CoV replication, virus–host interaction and pathogenesis. • These reverse genetic systems allow the development of genetically defined vaccines and the analysis of antiviral drugs., Coronaviruses (CoVs) infect humans and many animal species, and are associated with respiratory, enteric, hepatic, and central nervous system diseases. The large size of the CoV genome and the instability of some CoV replicase gene sequences during its propagation in bacteria, represent serious obstacles for the development of reverse genetic systems similar to those used for smaller positive sense RNA viruses. To overcome these limitations, several alternatives to more conventional plasmid-based approaches have been established in the last 13 years. In this report, we briefly review and discuss the different reverse genetic systems developed for CoVs, paying special attention to the severe acute respiratory syndrome CoV (SARS-CoV).

Humanized mice are crucial tools for studying human pathogens in systemic situations. An animal model of human coronavirus infectious disease has been generated by gene transfer of the human receptor for virus-cell interaction (aminopeptidase N, APN, CD13) into mice. We showed that in vitro and in vivo infections across the species barrier differ in their requirements. Transgenic cells were susceptible to human coronavirus HCoV-229E infection demonstrating the requirement of hAPN for viral cell entry. Transgenic mice, however, could not be infected suggesting additional requirements for in vivo virus susceptibility. Crossing hAPN transgenic mice with interferon unresponsive Stat1(-/- )mice resulted in markedly enhanced virus replication in vitro but did not result in detectable virus replication in vivo. Adaptation of the human virus to murine cells led to successful infection of the humanized transgenic mice. Future genetic engineering approaches are suggested to provide animal models for the better understanding of human infectious diseases.

The coronavirus nucleocapsid (N) protein is a viral RNA-binding protein with multiple functions in terms of virus replication and modulating cell signalling pathways. N protein is composed of three distinct regions containing RNA-binding motif(s), and appropriate signals for modulating cell signalling. The subcellular localization of severe acute respiratory syndrome coronavirus (SARS-CoV) N protein was studied. In infected cells, SARS-CoV N protein localized exclusively to the cytoplasm. In contrast to the avian coronavirus N protein, overexpressed SARS-CoV N protein remained principally localized to the cytoplasm, with very few cells exhibiting nucleolar localization. Bioinformatic analysis and deletion mutagenesis coupled to confocal microscopy and live-cell imaging, revealed that SARS-CoV N protein regions I and III contained nuclear localization signals and region II contained a nucleolar retention signal. However, cytoplasmic localization was directed by region III and was the dominant localization signal in the protein.

During the construction of the transmissible gastroenteritis virus (TGEV) full-length cDNA clone, a point mutation at position 637 that was present in the defective minigenome DI-C was maintained as a genetic marker. Sequence analysis of the recovered viruses showed a reversion at this position to the original virus sequence. The effect of point mutations at nucleotide 637 was analyzed by reverse genetics using a TGEV full-length cDNA clone and cDNAs from TGEV-derived minigenomes. The replacement of nucleotide 637 of TGEV genome by a T, as in the DI-C sequence, or an A severely affected virus recovery from the cDNA, yielding mutant viruses with low titers and small plaques compared to those of the wild type. In contrast, T or A at position 637 was required for minigenome rescue in trans by the helper virus. No relationship between these observations and RNA secondary-structure predictions was found, indicating that mutations at nucleotide 637 most likely had an effect at the protein level. Nucleotide 637 occupies the second codon position at amino acid 108 of the pp1a polyprotein. This position is predicted to map in the N-terminal polyprotein papain-like proteinase (PLP-1) cleavage site at the p9/p87 junction. Replacement of G-637 by A, which causes a drastic amino acid change (Gly to Asp) at position 108, affected PLP-1-mediated cleavage in vitro. A correlation was found between predicted cleaving and noncleaving mutations and efficient virus rescue from cDNA and minigenome amplification, respectively.

The nucleocapsid (N) protein is the only phosphorylated structural protein of the coronavirus Transmissible gastroenteritis virus (TGEV). The phosphorylation state and intracellular distribution of TGEV N protein in infected cells were characterized by a combination of techniques including: (i) subcellular fractionation and analysis of tryptic peptides by two-dimensional nano-liquid chromatography, coupled to ion-trap mass spectrometry; (ii) tandem mass-spectrometry analysis of N protein resolved by SDS-PAGE; (iii) Western blotting using two specific antisera for phosphoserine-containing motifs; and (iv) confocal microscopy. A total of four N protein-derived phosphopeptides were detected in mitochondria–Golgi–endoplasmic reticulum–Golgi intermediate compartment (ERGIC)-enriched fractions, including N-protein phosphoserines 9, 156, 254 and 256. Confocal microscopy showed that the N protein found in mitochondria–Golgi–ERGIC fractions localized within the Golgi–ERGIC compartments and not with mitochondria. Phosphorylated N protein was also present in purified virions, containing at least phosphoserines 156 and 256. Coronavirus N proteins showed a conserved pattern of secondary structural elements, including six β-strands and four α-helices. Whilst serine 9 was present in a non-conserved domain, serines 156, 254 and 256 were localized close to highly conserved secondary structural elements within the central domain of coronavirus N proteins. Serine 156 was highly conserved, whereas no clear homologous sites were found for serines 254 and 256 for other coronavirus N proteins.

Human coronavirus (HCoV) 229E is a group 1 coronavirus and is specific to humans. So far, no animal model is available to study the pathogenesis of infection by HCoV-229E. We show here that the expression of aminopeptidase N (APN, also termed CD13), the receptor for HCoV-229E, is required but not sufficient to confer susceptibility in vivo . HCoV-229E infection was facilitated by crossing APN transgenic mice into signal transducers and activators of transcription ( Stat ) 1 null mice and by adaptation of HCoV-229E to grow in primary APN transgenic, Stat1 null fibroblasts. Double transgenic mice allow the study of human coronavirus group 1 infections in an animal model, in particular, viral tropism, replication, recombination, and spread in an immunocompromised situation. Furthermore, these mice provide an important tool for the evaluation of biosafety and efficacy of coronavirus-based vectors.

The generation of subgenomic mRNAs in coronavirus involves a discontinuous mechanism of transcription by which the common leader sequence, derived from the genome 5′ terminus, is fused to the 5′ end of the mRNA coding sequence (body). Transcription-regulating sequences (TRSs) precede each gene and include a conserved core sequence (CS) surrounded by relatively variable sequences (5′ TRS and 3′ TRS). Regulation of transcription in coronaviruses has been studied by reverse-genetics analysis of the sequences immediately flanking a unique CS in the Transmissible gastroenteritis virus genome (CS-S2), located inside the S gene, that does not lead to detectable amounts of the corresponding mRNA, in spite of its canonical sequence. The transcriptional inactivity of CS-S2 was genome position independent. The presence of a canonical CS was not sufficient to drive transcription, but subgenomic synthesis requires a minimum base pairing between the leader TRS (TRS-L) and the complement of the body TRS (cTRS-B) provided by the CS and its adjacent nucleotides. A good correlation was observed between the free energy of TRS-L and cTRS-B duplex formation and the levels of subgenomic mRNA S2, demonstrating that base pairing between the leader and body beyond the CS is a determinant regulation factor in coronavirus transcription. In TRS mutants with increasing complementarity between TRS-L and cTRS-B, a tendency to reach a plateau in ΔG values was observed, suggesting that a more precise definition of the TRS limits might be proposed, specifically that it consists of the central CS and around 4 nucleotides flanking 5′ and 3′ the CS. Sequences downstream of the CS exert a stronger influence on the template-switching decision according to a model of polymerase strand transfer and template switching during minus-strand synthesis., This work was supported by grants from the Comision Interministerial de Ciencia y Tecnología (CICYT), la Consejería de Educación y Cultura de la Comunidad de Madrid, Fort-Dodge Veterinaria, and the European Communities (Frame V, Key Action 2, Control of Infectious Disease Projects). I.S., J.L.M., and S.Z. received fellowships from the Community of Madrid and the European Union (Frame V, Key Action 2, Control of Infectious Disease Projects QLRT-2000-00874, QLRT-2001-00825, and QLRT-2001-01050).

The construction of a set of transmissible gastroenteritis coronavirus (TGEV)-derived replicons as bacterial artificial chromosomes is reported. These replicons were generated by sequential deletion of nonessential genes for virus replication, using a modified TGEV full-length cDNA clone containing unique restriction sites between each pair of consecutive genes. Efficient activity of TGEV replicons was associated with the presence of the nucleoprotein provided either in cis or in trans . TGEV replicons were functional in several cell lines, including the human cell line 293T, in which no or very low cytopathic effect was observed, and expressed high amounts of heterologous protein.

A protocol for obtaining small amounts of highly pure virus preparations starting from reduced volumes (

Normal piglets weaned onto soy- or egg-based diets generated antibody responses to fed protein. Concurrent infection with transmissible gastroenteritis virus (TGEV) did not affect the responses to dietary antigens at weaning, nor did it affect the subsequent development of tolerance. However, TGEV infection did enhance the primary immunoglobulin M (IgM) and IgG1, but not IgG2, antibody responses to injected soy in comparison to those of uninfected animals. Paradoxically, TGEV-infected animals showed an enhanced primary IgG1 antibody response to injected soy at 4 weeks of age, but they subsequently showed a reduced secondary response after an intraperitoneal challenge at 9 weeks of age in comparison to uninfected animals. The results suggest that an enteric virus, either used as a vaccine vector or present as a subclinical infection, may not have significant effects on the development of dietary allergies but may have effects both on the primary response and on the subsequent recall response to systemic antigens to which the animal is exposed concurrently with virus antigens.

The antigenic homology of 26 coronavirus isolates, of which 22 were antigenically related to transmissible gastroenteritis virus (TGEV), was determined with 42 monoclonal antibodies. Type, group, and interspecies specific epitopes were defined. Two group specific MAbs distinguished the enteric TGEV isolates from the respiratory variants. An antigenic subsite involved in neutralization was conserved in porcine, feline, and canine coronavirus. The classification of the human coronavirus 229E in a taxonomic cluster distinct from TGEV group is suggested. © 1990.

Summary. The Coronaviridae family, comprising the Coronavirus and Torovirus genera, is part of the Nidovirales order that also includes two other families, Arteriviridae and Roniviridae. Based on genetic and serological relationships, groups 1, 2 and 3 were previously recognized in the Coronavirus genus. In this report we present results of comparative sequence analysis of the spike (S), envelope (E), membrane (M), and nucleoprotein (N) structural proteins, and the two most conserved replicase domains, putative RNA-dependent RNA polymerase (RdRp) and RNA helicase (HEL), aimed at a revision of the Coronaviridae taxonomy. The results of pairwise comparisons involving structural and replicase proteins of the Coronavirus genus were consistent and produced percentages of sequence identities that were distributed in discontinuous clusters. Inter-group pairwise scores formed a single cluster in the lowest percentile. No homologs of the N and E proteins have been found outside coronaviruses, and the only (very) distant homologs of S and M proteins were identified in toroviruses. Intragroup sequence conservation was higher, although for some pairs, especially those from the most diverse group 1, scores were close or even overlapped with those from the intergroup comparisons. Phylogenetic analysis of six proteins using a neighbor-joining algorithm confirmed three coronavirus groups. Comparative sequence analysis of RdRp and HEL domains were extended to include arterivirus and ronivirus homologs. The pairwise scores between sequences of the genera Coronavirus and Torovirus (22–25% and 21–25%) were found to be very close to or overlapped with the value ranges (12 to 22% and 17 to 25%) obtained for interfamily pairwise comparisons, but were much smaller than values derived from pairwise comparisons within the Coronavirus genus (63–71% and 59–67%). Phylogenetic analysis confirmed toroviruses and coronaviruses to be separated by a large distance that is comparable to those between established nidovirus families. Based on comparison of these scores with those derived from analysis of separate ranks of several multi-genera virus families, like the Picornaviridae, a revision of the Coronaviridae taxonomy is proposed. We suggest the Coronavirus and Torovirus genera to be re-defined as two subfamilies within the Coronavirdae or two families within Nidovirales, and the current three informal coronavirus groups to be converted into three genera within the Coronaviridae.

Transmissible gastroenteritis coronavirus (TGEV) contains eight overlapping genes that are expressed from a 3'-coterminal nested set of leader-containing mRNAs. To facilitate the genetic manipulation of the viral genome, genes were separated by duplication of transcription regulating sequences (TRSs) and introduction of unique restriction endonuclease sites at the 5' end of each gene using an infectious cDNA clone. The recombinant TGEV (rTGEV) replicated in cell culture with similar efficiency to the wild-type virus and stably maintained the modifications introduced into the genome. In contrast, the rTGEV replication level in the lungs and gut of infected piglets and virulence were significantly reduced. rTGEV in which gene 7 expression was abrogated (rTGEV-delta7) were recovered from cDNA constructs, indicating that TGEV gene 7 was a nonessential gene for virus replication. Interestingly, in vivo infections with rTGEV-delta7 showed an additional reduction in virus replication in the lung and gut, and in virulence, indicating that TGEV gene 7 influences virus pathogenesis.

The antigenic structure of transmissible gastroenteritis (TGE) virus E2 glycoprotein has been defined at three levels: antigenic sites, antigenic subsites and epitopes. Four antigenic sites (A, B, C and D) were defined by competitive radioimmunoassay (RIA) using monoclonal antibodies (MAbs) selected from 9 fusions. About 20% (197) of the hybridomas specific for TGE virus produced neutralizing MAbs specific for site A, which was one of the antigenically dominant determinants. Site A was differentiated in three antigenic subsites: a, b and c, by characterization of 11 MAb resistant (mar) mutants, that were defined by 8, 3, and 3 MAbs, respectively. These subsites were further subdivided in epitopes. A total of 11 epitopes were defined in E2 glycoprotein, eight of which were critical for virus neutralization. Neutralizing MAbs were obtained only when native virus was used to immunize mice, although to produce hybridomas mice immunizations were made with antigen in the native, denatured, or mixtures of native and denatured form. All neutralizing MAbs reacted to conformational epitopes. The antigenic structure of the E2-glycoprotein has been defined with murine MAbs, but the antigenic sites were relevant in the swine, the natural host of the virus, because porcine sera reacted against these sites. MAbs specific for TGE virus site C reacted to non-immune porcine sera. This reactivity was not directed against porcine immunoglobulins. These results indicated that TGE virus contains epitope(s) also present in some non-immunoglobulin component of porcine serum.

The surface glycoprotein S of transmissible gastroenteritis virus (TGEV) has two binding activities. (i) Binding to porcine aminopeptidase N (pAPN) is essential for the initiation of infection. (ii) Binding to sialic acid residues on glycoproteins is dispensable for the infection of cultured cells but is required for enteropathogenicity. By comparing parental TGEV with mutant viruses deficient in the sialic acid binding activity, we determined the contributions of both binding activities to the attachment of TGEV to cultured cells. In the presence of a functional sialic acid binding activity, the amount of virus bound to two different porcine cell lines was increased sixfold compared to the binding of the mutant viruses. The attachment of parental virus was reduced to levels observed with the mutants when sialic acid containing inhibitors was present or when the cells were pretreated with neuraminidase. In virus overlay binding assays with immobilized cell surface proteins, the mutant virus only recognized pAPN. In addition, the parental virus bound to a high-molecular-mass sialoglycoprotein. The recognition of pAPN was sensitive to reducing conditions and was not dependent on sialic acid residues. On the other hand, binding to the sialic acid residues of the high-molecular-mass glycoprotein was observed regardless of whether the cellular proteins had been separated under reducing or nonreducing conditions. We propose that binding to a surface sialoglycoprotein is required for TGEV as a primary attachment site to initiate infection of intestinal cells. This concept is discussed in the context of other viruses that use two different receptors to infect cells.

The stable propagation of a full-length transmissible gastroenteritis coronavirus (TGEV) cDNA in Escherichia coli cells as a bacterial artificial chromosome has been considerably improved by the insertion of an intron to disrupt a toxic region identified in the viral genome. The viral RNA was expressed in the cell nucleus under the control of the cytomegalovirus promoter and the intron was efficiently removed during translocation of this RNA to the cytoplasm. The insertion in two different positions allowed stable plasmid amplification for at least 200 generations. Infectious TGEV was efficiently recovered from cells transfected with the modified cDNAs., This work was supported by grants from the Comisión Interministerial de Ciencia y Tecnología (CICYT), La Consejería de Educación y Cultura de la Comunidad de Madrid, Fort Dodge Veterinaria, and the European Communities (Frame V, Key Action 2, Control of Infectious Disease Projects QLRT-1999-00002, QLRT-1999-30739, and QLRT2000-00874). J.M.G. received a fellowship from the Spanish Department of Education and Culture. Z.P., F.A., and E.C. received fellowships from the Biotechnology and Fishery and Agro-Industrial Research Programs of the European Community.

A recombinant severe acute respiratory syndrome coronavirus (SARS-CoV) lacking the envelope (E) protein is attenuated in vivo. Here we report that E protein PDZ-binding motif (PBM), a domain involved in protein-protein interactions, is a major determinant of virulence. Elimination of SARS-CoV E protein PBM by using reverse genetics caused a reduction in the deleterious exacerbation of the immune response triggered during infection with the parental virus and virus attenuation. Cellular protein syntenin was identified to bind the E protein PBM during SARS-CoV infection by using three complementary strategies, yeast two-hybrid, reciprocal coimmunoprecipitation and confocal microscopy assays. Syntenin redistributed from the nucleus to the cell cytoplasm during infection with viruses containing the E protein PBM, activating p38 MAPK and leading to the overexpression of inflammatory cytokines. Silencing of syntenin using siRNAs led to a decrease in p38 MAPK activation in SARS-CoV infected cells, further reinforcing their functional relationship. Active p38 MAPK was reduced in lungs of mice infected with SARS-CoVs lacking E protein PBM as compared with the parental virus, leading to a decreased expression of inflammatory cytokines and to virus attenuation. Interestingly, administration of a p38 MAPK inhibitor led to an increase in mice survival after infection with SARS-CoV, confirming the relevance of this pathway in SARS-CoV virulence. Therefore, the E protein PBM is a virulence domain that activates immunopathology most likely by using syntenin as a mediator of p38 MAPK induced inflammation., This work was supported by grants from the government of Spain (BIO2010-16705), the European Community’s Seventh Framework Programme (FP7/ 2007-2013) under the project ‘‘EMPERIE’’ EC Gran Agreement number 223498, and U.S. National Institutes of Health (NIH) (2P01AI060699, 0258-3413/ HHSN266200700010C). JMJG received a JAE fellowship from the CSIC-JAE Program co-funded by the European Social Fund. JARN and CCR received a contract from Fundación La Caixa.

This chapter provides a brief overview of the state of the art on coronavirus (CoV) vaccines, with special attention paid to severe acute respiratory syndrome CoV (SARS-CoV), as this virus has had the largest impact on humans. First, the chapter considers basic aspects affecting vaccine development, such as correlates of protection, antigenic variability, and role of B- and T-cell responses. Then, it discusses different types of vaccines under development, including inactivated viruses, subunit vaccines, virus-like particles (VLPs), DNA vaccines, heterologous expression systems, and vaccines derived by reverse genetics. Finally, the chapter considers potential side effects of these vaccines.

In this era of continued emergence of zoonotic virus infections, the rapid development of rodent models represents a critical barrier to public health preparedness, including the testing of antivirus therapy and vaccines. The Middle East respiratory syndrome coronavirus (MERS-CoV) was recently identified as the causative agent of a severe pneumonia. Given the ability of coronavirus to rapidly adapt to new hosts, a major public health concern is that MERS-CoV will further adapt to replication in humans, triggering a pandemic. No small-animal model for this infection is currently available, but studies suggest that virus entry factors can confer virus susceptibility. Here, we show that mice were sensitized to MERS-CoV infection by prior transduction with adenoviral vectors expressing the human host-cell receptor dipeptidyl peptidase 4. Mice developed a pneumonia characterized by extensive inflammatorycell infiltration with virus clearance occurring 6-8 d after infection. Clinical disease and histopathological changes were more severe in the absence of type-I IFN signaling whereas the T-cell response was required for virus clearance. Using these mice, we demonstrated the efficacy of a therapeutic intervention (poly I:C) and a potential vaccine [Venezuelan equine encephalitis replicon particles expressing MERS-CoV spike protein]. We also found little protective cross-reactivity between MERS-CoV and the severe acute respiratory syndrome-CoV. Our results demonstrate that this system will be useful for MERS-CoV studies and for the rapid development of relevant animal models for emerging respiratory viral infections., This research was supported in part by National Institutes of Health Grants RO1AI091322 (to S.P.), PO106099 (to S.P., P.B.M., T.G., and L.E.), AI074973 and AI083019 (to M.J.G.), and AI057157 (to R.S.B.).

Coronaviruses (CoVs) infect humans and many animal species, and are associated with respiratory, enteric, hepatic, and central nervous system diseases. The large size of the CoV genome and the instability of some CoV replicase gene sequences during its propagation in bacteria, represent serious obstacles for the development of reverse genetic systems similar to those used for smaller positive sense RNA viruses. To overcome these limitations, several alternatives to more conventional plasmid-based approaches have been established in the last 13 years. In this report, we briefly review and discuss the different reverse genetic systems developed for CoVs, paying special attention to the severe acute respiratory syndrome CoV (SARS-CoV)., This work was supported by grants from the Ministry of Science and Innovation of Spain (MCINN) (BIO2010-16705), the European Community’s Seventh Framework Programme (FP7/2007–2013) under the project “EMPERIE” (HEALTH-F3-2009-223498), and the National Institute of Health (NIH) of USA (2P01AI060699-06A1).

Both helper dependent expression systems, based on two components, and single genomes constructed by targeted recombination, or by using infectious cDNA clones, have been developed. The sequences that regulate transcription have been characterized mainly using helper dependent expression systems and it will now be possible to validate them using single genomes. The genome of coronaviruses has been engineered by modification of the infectious cDNA leading to an efficient (20 microg ml(-1)) and stable (20 passages) expression of the foreign gene. The possibility of engineering the tissue and species tropism to target expression to different organs and animal species, including humans, increases the potential of coronaviruses as vectors. Thus, coronaviruses are promising virus vectors for vaccine development and, possibly, for gene therapy.

The complete sequence (28580 nt) of the PUR46-MAD clone of the Purdue cluster of transmissible gastroenteritis coronavirus (TGEV) has been determined and compared with members of this cluster and other coronaviruses. The computing distances among their S gene sequences resulted in the grouping of these coronaviruses into four clusters, one of them exclusively formed by the Purdue viruses. Three new potential sequence motifs with homology to the alpha-subunit of the polymerase-associated nucleocapsid phosphoprotein of rinderpest virus, the Bowman-Birk type of proteinase inhibitors, and the metallothionein superfamily of cysteine rich chelating proteins have been identified. Comparison of the TGEV polymerase sequence with that of other RNA viruses revealed high sequence homology with the A-E domains of the palm subdomain of nucleic acid polymerases.

The construction of cDNA clones encoding large-size RNA molecules of biological interest, like coronavirus genomes, which are among the largest mature RNA molecules known to biology, has been hampered by the instability of those cDNAs in bacteria. Herein, we show that the application of two strategies, cloning of the cDNAs into a bacterial artificial chromosome and nuclear expression of RNAs that are typically produced within the cytoplasm, is useful for the engineering of large RNA molecules. A cDNA encoding an infectious coronavirus RNA genome has been cloned as a bacterial artificial chromosome. The rescued coronavirus conserved all of the genetic markers introduced throughout the sequence and showed a standard mRNA pattern and the antigenic characteristics expected for the synthetic virus. The cDNA was transcribed within the nucleus, and the RNA translocated to the cytoplasm. Interestingly, the recovered virus had essentially the same sequence as the original one, and no splicing was observed. The cDNA was derived from an attenuated isolate that replicates exclusively in the respiratory tract of swine. During the engineering of the infectious cDNA, the spike gene of the virus was replaced by the spike gene of an enteric isolate. The synthetic virus replicated abundantly in the enteric tract and was fully virulent, demonstrating that the tropism and virulence of the recovered coronavirus can be modified. This demonstration opens up the possibility of employing this infectious cDNA as a vector for vaccine development in human, porcine, canine, and feline species susceptible to group 1 coronaviruses.

Transmissible gastroenteritis coronavirus (TGEV) agglutinates erythrocytes of several species by virtue of sialic acid binding activity of the surface protein S. We have isolated and characterized five haemagglutination-defective (HAD) mutants. In contrast to the parental virus, the mutants were unable to bind to porcine submandibulary mucin, a substrate rich in sialic acid. Each of the mutants was found to contain a single point mutation in the S protein (Cys155Phe, Met195Val, Arg196Ser, Asp208Asn or Leu209Pro), indicating that these amino acids are affecting the sialic acid binding site. In four of the HAD mutants a nearby antigenic site is affected in addition to the sialic acid binding site, as indicated by reactivity with monoclonal antibodies. The parental virus was found to have an increased resistance to the detergent octylglucoside compared to the HAD mutants. This effect depended on cellular sialoglycoconjugates bound to the virion. If the binding of sialylated macromolecules was prevented by neuraminidase treatment, the parental virus was as sensitive to octylglucoside as were the HAD mutants. We discuss the possibility that the sialic acid binding activity helps TGEV to resist detergent-like substances encountered during the gastrointestinal passage and thus facilitates the infection of the intestinal epithelium. An alternative function of the sialic acid binding activity – accessory binding to intestinal tissues – is also discussed.

To study the molecular basis of TGEV tropism, a collection of recombinants between the PUR46-MAD strain of transmissible gastroenteritis coronavirus (TGEV) infecting the enteric and respiratory tracts and the PTV strain, which only infects the respiratory tract, was generated. The recombinant isolation frequency was about 10−9recombinants per nucleotide and was 3.7-fold higher at the 5′-end of the S gene than in other areas of the genome. Thirty recombinants were plaque purified and characterized phenotypically and genetically. All recombinant viruses had a single crossover and had inherited the 5′- and 3′-halves of their genome from the enteric and respiratory parents, respectively. Recombinant viruses were classified into three groups, named 1 to 3, according to the location of the crossover. Group 1 recombinants had the crossover in the S gene, while in Groups 2 and 3 the crossovers were located in ORF1b and ORF1a, respectively. The tropism of the recombinants was studied. Recombinants of Group 1 had enteric and respiratory tropism, while Group 2 recombinants infected the respiratory, but not the enteric, tract. Viruses of both groups differed by two nucleotide changes at positions 214 and 655. Both changes may be in principle responsible for the loss of enteric tropism but only the change in nucleotide 655 was specifically found in the respiratory isolates and most likely this single nucleotide change, which leads to a substitution in amino acid 219 of the S protein, was responsible for the loss of enteric tropism in the closely related PUR-46 isolates. The available data indicate that in order to infect enteric tract cells with TGEV, two different domains of the S protein, mapping between amino acids 522 and 744 and around amino acid 219, respectively, are involved. The first domain binds to porcine aminopeptidase N, the cellular receptor for TGEV. In the other domain maps a second factor of undefined nature but which may be the binding site for a coreceptor essential for the enteric tropism of TGEV.

Middle East respiratory syndrome coronavirus (MERS-CoV) is an emerging coronavirus infecting humans that is associated with acute pneumonia, occasional renal failure, and a high mortality rate and is considered a threat to public health. The construction of a full-length infectious cDNA clone of the MERS-CoV genome in a bacterial artificial chromosome is reported here, providing a reverse genetics system to study the molecular biology of the virus and to develop attenuated viruses as vaccine candidates. Following transfection with the cDNA clone, infectious virus was rescued in both Vero A66 and Huh-7 cells. Recombinant MERS-CoVs (rMERS-CoVs) lacking the accessory genes 3, 4a, 4b, and 5 were successfully rescued from cDNA clones with these genes deleted. The mutant viruses presented growth kinetics similar to those of the wild-type virus, indicating that accessory genes were not essential for MERS-CoV replication in cell cultures. In contrast, an engineered mutant virus lacking the structural E protein (rMERS-CoV-¿E) was not successfully rescued, since viral infectivity was lost at early passages. Interestingly, the rMERS-CoV-¿E genome replicated after cDNA clone was transfected into cells. The infectious virus was rescued and propagated in cells expressing the E protein in trans, indicating that this virus was replication competent and propagation defective. Therefore, the rMERS-CoV-¿E mutant virus is potentially a safe and promising vaccine candidate to prevent MERS-CoV infection. IMPORTANCE Since the emergence of MERS-CoV in the Arabian Peninsula during the summer of 2012, it has already spread to 10 different countries, infecting around 94 persons and showing a mortality rate higher than 50%. This article describes the development of the first reverse genetics system for MERS-CoV, based on the construction of an infectious cDNA clone inserted into a bacterial artificial chromosome. Using this system, a collection of rMERS-CoV deletion mutants has been generated. Interestingly, one of the mutants with the E gene deleted was a replication-competent, propagation-defective virus that could only be grown in the laboratory by providing E protein in trans, whereas it would only survive a single virus infection cycle in vivo. This virus constitutes a vaccine candidate that may represent a balance between safety and efficacy for the induction of mucosal immunity, which is needed to prevent MERS-CoV infection., This work was supported by grants from the Ministry of Science and Innovation of Spain (MCINN) (BIO2010-16705), the European Community’s Seventh Framework Programme (FP7/2007-2013) under the project “EMPERIE” (HEALTH-F3-2009-223498), and the U.S. National Institutes of Health (NIH) (2P01AI060699-06A1). G.A. was supported by the Amarouto Program for senior researchers from the Comunidad Auónoma de Madrid. S.M.-J. received a predoctoral fellowship from the National Institute of Health (ISCIII) of Spain. J.L.N.-T. and M.L.D. received contracts from the U.S. National Institutes of Health (NIH) (2P01AI060699-06A1) and the European Community (EMPERIE project HEALTH-F3-2009-223498), respectively

Preferential RNA packaging in coronaviruses involves the recognition of viral genomic RNA, a crucial process for viral particle morphogenesis mediated by RNA specific sequences, known as packaging signals. An essential packaging signal component of transmissible gastroenteritis coronavirus (TGEV) has been further delimited to the first 598 nt from 5' end of its RNA genome, using recombinant viruses transcribing subgenomic mRNA that included potential packaging signals. The integrity of entire sequence domain was necessary because deletion of any of the five structural motifs defined within this region abrogated specific packaging of this viral RNA. One of these RNA motifs was the stem-loop SL5, a highly conserved motif in coronaviruses located at position 106-136 nt. Partial deletion or point mutations within this motif also abrogated packaging. Using TGEV-derived defective minigenomes replicated in trans by a helper virus, we have shown that TGEV RNA packaging is a replication-independent process. Furthermore, the last 494 nt of genome 3' end were not essential for packaging, although this region increased packaging efficiency. TGEV RNA sequences identified as necessary for viral genome packaging, were not sufficient to direct packaging of an heterologous sequence derived from the green fluorescent protein gene. These results indicated that TGEV genome packaging is a complex process involving many factors in addition to the identified RNA packaging signal. The identification of well defined RNA motifs within TGEV RNA genome that are essential for packaging will be useful in the design of packaging-deficient biosafe coronavirus derived-vectors and to provide new targets for antiviral therapies., This work was supported by grants from the Ministry of Science and Innovation of Spain (BIO2010-167075) and the European Community's Seventh Framework Programme (FP7/2007-2013), under the project PoRRSCon (EC grant agreement 245141). L.M. and P.A.M.-G. received predoctoral fellowships from the Ministry of Science and Innovation of Spain (BES-2011-043489 and BES-2008-001932, respectively). L.D.P. received a predoctoral fellowship from the National Institute of Health (ISCIII) of Spain (FI08/00109).

Highlights • We cloned a DENV-2 infectious cDNA into a BAC under the control of the CMV promoter. • We assessed the production of infectious particles. • We rescued infectious viruses after serial passages in C6/36 cells. • Parental and recombinant viruses were similar in plaque and syncytia phenotypes., Major progress in Dengue virus (DENV) biology has resulted from the use of infectious clones obtained through reverse genetics. The construction of these clones is commonly based on high- or low-copy number plasmids, yeast artificial chromosomes, yeast-Escherichia coli shuttle vectors, and bacterial artificial chromosomes (BACs). Prokaryotic promoters have consistently been used for the transcription of these clones. The goal of this study was to develop a novel DENV infectious clone in a BAC under the control of the cytomegalovirus immediate-early promoter and to generate a virus with the fusion envelope-green fluorescent protein in an attempt to track virus infection. The transfection of Vero cells with a plasmid encoding the DENV infectious clone facilitated the recovery of infectious particles that increased in titer after serial passages in C6/36 cells. The plaque size and syncytia phenotypes of the recombinant virus were similar to those of the parental virus. Despite the observation of autonomous replication and the detection of low levels of viral genome after two passages, the insertion of green fluorescent protein and Renilla luciferase reporter genes negatively impacted virus rescue. To the best of our knowledge, this is the first study using a DENV infectious clone under the control of the cytomegalovirus promoter to facilitate the recovery of recombinant viruses without the need for in vitro transcription. This novel molecular clone will be useful for establishing the molecular basis of replication, assembly, and pathogenesis, evaluating potential antiviral drugs, and the development of vaccine candidates for attenuated recombinant viruses.

A partial characterization of the ion channels formed by the SARS coronavirus (CoV) envelope (E) protein was previously reported (C. Verdiá-Báguena et al., 2012 [12]). Here, we provide new significant insights on the involvement of lipids in the structure and function of the CoV E protein channel on the basis of three series of experiments. First, reversal potential measurements over a wide range of pH allow the dissection of the contributions to channel selectivity coming from ionizable residues of the protein transmembrane domain and also from the negatively charged groups of diphytanoyl phosphatidylserine (DPhPS) lipid. The corresponding effective pKas are consistent with the model pKas of the acidic residue candidates for titration. Second, the change of channel conductance with salt concentration reveals two distinct regimes (Donnan-controlled electrodiffusion and bulk-like electrodiffusion) fully compatible with the outcomes of selectivity experiments. Third, by measuring channel conductance in mixtures of neutral diphytanoyl phosphatidylcholine (DPhPC) lipids and negatively charged DPhPS lipids in low and high salt concentrations we conclude that the protein-lipid conformation in the channel is likely the same in charged and neutral lipids. Overall, the whole set of experiments supports the proteolipidic structure of SARS-CoV E channels and explains the large difference in channel conductance observed between neutral and charged membranes. © 2013 Elsevier B.V., This work was supported by grants from the Ministry of Science and Innovation of Spain (MICINN. FIS2010-19810, BIO2007-60978 and BIO2010-16705), Fundació Caixa Castelló-Bancaixa (P1-1B2012-03), Generalitat Valenciana (Prometeu/2012/069), the European Community's Seventh Framework Programme (FP7/2007–2013) under the project “EMPERIE” EC grant agreement number 223498, and the U.S. National Institutes of Health (NIH) (2P01AI060699-06A1, W000306844). CVB received a fellowship from UJI. JLN received a fellowship from CSIC.

Here we report the rescue of a recombinant porcine reproductive and respiratory syndrome virus (PRRSV) carrying an enhanced green fluorescent protein (EGFP) reporter gene as a separate transcription unit. A copy of the transcription regulatory sequence for ORF6 (TRS6) was inserted between the N protein and 3′-UTR to drive the transcription of the EGFP gene and yield a general purpose expression vector. Successful recovery of PRRSV was obtained using an RNA polymerase II promoter to drive transcription of the full-length virus genome, which was assembled in a bacterial artificial chromosome (BAC). The recombinant virus showed growth replication characteristics similar to those of the wild-type virus in the infected cells. In addition, the recombinant virus stably expressed EGFP for at least 10 passages. EGFP expression was detected at approximately 10 h post infection by live-cell imaging to follow the virus spread in real time and the infection of neighbouring cells occurred predominantly through cell-to-cell-contact. Finally, the recombinant virus generated was found to be an excellent tool for neutralising antibodies and antiviral compound screening. The newly established reverse genetics system for PRRSV could be a useful tool not only to monitor virus spread and screen for neutralising antibodies and antiviral compounds, but also for fundamental research on the biology of the virus., This study was funded by grants from the National Natural Science Foundation of China (U0931003/L01) and the National High-Tech R&D Program of China (2011AA10A208) to EMZ, the National Natural Science Foundation of China (31302103) to WCB, the European Community’s Seventh Frame-work Programme (PoRRSCon, FP7-KBBE-2009-3-245141) and the Ministry of Science and Innovation of Spain (MCINN) (BIO2010-16075) to FA and LE.

Following infection of haplotype defined NIH-miniswine with virulent transmissible gastroenteritis coronavirus (TGEV), isolated mesenteric lymph node CD4+ T-cells mounted a specific proliferative response against infectious or inactivated purified virus in secondary in vitro stimulation. A specific, dose-dependent response to the three major recombinant viral proteins: spike (S), membrane (M), and nucleoprotein (N), purified by affinity chromatography, was characterized. Induction of in vitro antibody synthesis was analyzed. The purified recombinant viral proteins induced the in vitro synthesis of neutralizing TGEV-specific antibodies when porcine TGEV-immune cells were stimulated with each of the combinations made with two of the major structural proteins: S + N, S + M, and to a minor extent with M + N, but not by the individual proteins. S-protein was dissociated from purified virus using NP-40 detergent and then micellar S-protein oligomers (S-rosettes) were formed by removing the detergent. These occurred preferentially by the association of more than 10 S-protein trimmers. These S-rosettes in collaboration with either N or M-proteins elicited TGEV-specific antibodies with titers up to 84 and 60%, respectively, of those induced by the whole virus. N-protein could be partially substituted by a 15-mer peptide that represents a T helper epitope previously identified in N-protein (Antón et al. (1995)). These results indicate that the induction of high levels of TGEV-specific antibodies requires stimulation by at least two viral proteins, and that optimum responses are induced by a combination of S-rosettes and the nucleoprotein.

Antigenic site D from the spike protein of transmissible gastroenteritis virus (TGEV), which is a continuous epitope critical in neutralization, has been expressed as a fusion protein with E. coli heat-labile toxin B subunit (LT-B) in attenuated S. typhimurium. Synthetic peptides containing the sequence of site D induced TGEV neutralizing antibodies when inoculated subcutaneously in both rabbits and swine. A synthetic oligonucleotide encoding residues 373-398 of TGEV S protein, including antigenic site D, was cloned in frame with the 3' end of LT-B gene, into a plasmid used to transform S. typhimurium delta asd chi 3730. A collection of 6 recombinant plasmids designated pYALTB-D I-VI encoding LTB-site D fusions with a variable number of site D sequences were selected. Four of the 6 LTB-site D fusion products expressed in S. typhimurium chi 3730 formed oligomers (pentamers) that dissociated at70 degrees. S. typhimurium chi 3730 (pYALTB-D) V and VI expressed the oligomer forming products with higher antigenicity. Partially purified LTB-site D fusion product expressed from S. typhimurium chi 3730 (pYALTB-D) V induced anti-TGEV neutralizing antibodies in rabbits. Recombinant vaccine strain S. typhimurium delta cya delta crp delta asd chi 3987 transformed with plasmid pYALTB-D V expressed constitutively products that formed oligomers presumably containing 20 copies of site D, and showed a high stability in vitro. This recombinant strain was orally inoculated in rabbits and induced TGEV specific antibodies in both serum and intestinal secretion.

Three transmissible gastroenteritis virus (TGEV) defective RNAs were selected by serial undiluted passage of the PUR46 strain in ST cells. These RNAs of 22, 10.6, and 9.7 kb (DI-A, DI-B, and DI-C, respectively) were detected at passage 30, remained stable upon further passage in cell culture, and significantly interfered with helper mRNA synthesis. RNA analysis from purified virions showed that the three defective RNAs were efficiently packaged. Virions of different densities containing either full-length or defective RNAs were sorted in sucrose gradients, indicating that defective and full-length genomes were independently encapsidated. DI-B and DI-C RNAs were amplified by the reverse transcription-polymerase chain reaction, cloned, and sequenced. DI-B and DI-C genomes are formed by three and four discontinuous regions of the wild-type genome, respectively. DI-C contains 2144 nucleotides (nt) from the 5'-end of the genome, two fragments of 4540 and 2531 nt mostly from gene 1b, and 493 nt from the 3' end of the genome. DI-B and DI-C RNAs include sequences with the pseudoknot motif and encoding the polymerase, metal ion binding, and helicase motifs. DI-B RNA has a structure closely related to DI-C RNA with two main differences: it maintains the entire ORF 1b and shows heterogeneity in the size of the 3' end deletion. This heterogeneity maps at the beginning of the S gene, where other natural TGEV recombination events have been observed, suggesting that either a process of template switching occurs with high frequency at this point or that the derived genomes have a selective advantage.