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Severe acute respiratory syndrome coronavirus (SARS-CoV) and the closely related SARS-CoV-2 are emergent highly pathogenic human respiratory viruses causing acute lethal disease associated with lung damage and dysregulated inflammatory responses. SARS-CoV envelope protein (E) is a virulence factor involved in the activation of various inflammatory pathways. Here, we study the contribution of host miRNAs to the virulence mediated by E protein. Small RNAseq analysis of infected mouse lungs identified miRNA-223 as a potential regulator of pulmonary inflammation, since it was significantly increased in SARS-CoV-WT virulent infection compared to the attenuated SARS-CoV-DE infection. In vivo inhibition of miRNA-223-3p increased mRNA levels of pro-inflammatory cytokines and NLRP3 inflammasome, suggesting that during lung infection, miRNA-223 might contribute to restrict an excessive inflammatory response. Interestingly, miRNA-223-3p inhibition also increased the levels of the CFTR transporter, which is involved in edema resolution and was significantly downregulated in the lungs of mice infected with the virulent SARS-CoV-WT virus. At the histopathological level, a decrease in the pulmonary edema was observed when miR-223-3p was inhibited, suggesting that miRNA-223-3p was involved in the regulation of the SARS-CoVinduced inflammatory pathology. These results indicate that miRNA-223 participates in the regulation of E protein-mediated inflammatory response during SARS-CoV infection by targeting different host mRNAs involved in the pulmonary inflammation, and identify miRNA-223 as a potential therapeutic target in SARS-CoV infection. IMPORTANCE The SARS-CoV-2 pandemic has emphasized the need to understand the mechanisms of severe lung inflammatory pathology caused by human deadly coronaviruses in order to design new antiviral therapies. Here, we identify miRNA-223-3p as a host miRNA involved in the regulation of lung inflammatory response mediated by envelope (E) protein during SARS-CoV infection. miRNAs downregulate the expression of cellular mRNAs and participate in complex networks of mRNA–miRNA interactions that regulate cellular processes. The inhibition of miRNA-223 in infected mice by intranasal administration of antisense RNAs led to changes in the expression of host factors involved in inflammation (cytokines, chemokines, and NLRP3 inflammasome) and in the resolution of lung edema ion transporter CFTR. These results confirmed the contribution of miRNA-223 to the regulation of SARS-CoV-induced pathogenic processes and support the therapeutic potential of inhibiting miRNAs during coronavirus infection using RNA interference approaches., This work was supported by grants from the Government of Spain (BIO2016-75549-R; PID2019-107001RBI00 AEI/FEDER, UE; SEV 2017-0712 and PIE_INTRAMURAL_LINEA 1-202020E079), CSIC (PIE_INTRAMURAL -202020E043), the European Zoonotic Anticipation and Preparedness Initiative (ZAPI) (IMI_JU_115760), the European Commission (H2020-SC1-2019, ISOLDA Project No. 848166-2), and the U.S. National Institutes of Health (NIH) (2P01AI060699). L.M. received a fellowship from the Ministry of Science an Innovation of Spain (BIO2010-16705) and a contract from ZAPI.

23 Pág. Centro de Investigación en Sanidad Animal (CISA), No vaccines or specific antiviral drugs are authorized against Middle East respiratory syndrome coronavirus (MERS-CoV) despite its high mortality rate and prevalence in dromedary camels. Since 2012, MERS-CoV has been causing sporadic zoonotic infections in humans, which poses a risk of genetic evolution to become a pandemic virus. MERS-CoV genome encodes five accessory proteins, 3, 4a, 4b, 5 and 8b for which limited information is available in the context of infection. This work describes 4b as a virulence factor in vivo, since the deletion mutant of a mouse-adapted MERS-CoV-Δ4b (MERS-CoV-MA-Δ4b) was completely attenuated in a humanized DPP4 knock-in mouse model, resulting in no mortality. Attenuation in the absence of 4b was associated with a significant reduction in lung pathology and chemokine expression levels at 4 and 6 days post-infection, suggesting that 4b contributed to the induction of lung inflammatory pathology. The accumulation of 4b in the nucleus in vivo was not relevant to virulence, since deletion of its nuclear localization signal led to 100% mortality. Interestingly, the presence of 4b protein was found to regulate autophagy in the lungs of mice, leading to upregulation of BECN1, ATG3 and LC3A mRNA. Further analysis in MRC-5 cell line showed that, in the context of infection, MERS-CoV-MA 4b inhibited autophagy, as confirmed by the increase of p62 and the decrease of ULK1 protein levels, either by direct or indirect mechanisms. Together, these results correlated autophagy activation in the absence of 4b with downregulation of a pathogenic inflammatory response, thus contributing to attenuation of MERS-CoV-MA-Δ4b., This work was supported by grants from the Government of Spain (BIO2016-75549-R; PID2019-107001RB-I00 AEI/FEDER, UE; SEV 2017-0712 and PIE_INTRAMURAL_LINEA 1-202020E079), CSIC (PIE_INTRAMURAL -202020E043), the European Zoonotic Anticipation and Preparedness Initiative (ZAPI) (IMI_JU_115760), the European Commission (H2020-SC1-2019, ISOLDA Project No. 848166-2), and the U.S. National Institutes of Health (NIH) (2P01AI060699 to I.S.). J.C. and J.H-T: received fellowships from the Ministry of Science an Innovation of Spain (BIO2013- 42869-R and PID2019-107001RB-I00 AEI/FEDER). M.B-P. received a contract from ISOLDA_848166 H2020-SC1-2019-Two-Stage-RTD.The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

9 páginas, 4 figuras, 1 tabla., Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19, has infected several animal species, including dogs, presumably via human-to-animal transmission. Most infected dogs reported were asymptomatic, with low viral loads. However, in this case we detected SARS-CoV-2 in a dog from the North African coastal Spanish city of Ceuta presenting hemorrhagic diarrhea, a disease also reported earlier on in an infected dog from the USA. Case presentation: In early January 2021, a West Highland Terrier pet dog from Ceuta (Spain) presented hemorrhagic diarrhea with negative tests for candidate microbial pathogens. Since the animal was in a household whose members suffered SARS-CoV-2 in December 2020, dog feces were analyzed for SARS-CoV-2, proving positive in a two-tube RT-PCR test, with confirmation by sequencing a 399-nucleotide region of the spike (S) gene. Furthermore, next-generation sequencing (NGS) covered > 90% SARS-CoV-2 genome sequence, allowing to classify it as variant B.1.177. Remarkably, the sequence revealed the Ile402Val substitution in the spike protein (S), of potential concern because it mapped in the receptor binding domain (RBD) that mediates virus interaction with the cell. NGS reads mapping to bacterial genomes showed that the dog fecal microbiome fitted best the characteristic microbiome of dog's acute hemorrhagic diarrhea. Conclusion: Our findings exemplify dog infection stemming from the human SARS-CoV-2 pandemic, providing nearly complete-genome sequencing of the virus, which is recognized as belonging to the B.1.177 variant, adding knowledge on variant circulation in a geographic region and period for which there was little viral variant characterization. A single amino acid substitution found in the S protein that could have been of concern is excluded to belong to this category given its rarity and intrinsic nature. The dog's pathology suggests that SARS-CoV-2 could affect the gastrointestinal tract of the dog., This research received external funding to CR-G and EM from “AgenciaValenciana de Innovación: COVID-19. Ayudas de concesión directa a soluciones científco-innovadoras directamente relacionadas con la lucha contra la COVID-19,” (Ref COVID-19-203); from the “Conselleria de Innovación Universidades, Ciencia y Sociedad Digital: Subvenciones a grupos de investigación emergentes” (Ref, GV/2021/163); and from a grant to VR (PID2020-120322RBC21) from the Spanish Agencia Estatal de Investigación (Plan Estatal de I+D+i)

The ongoing evolution of SARS-CoV-2 has resulted in the emergence of Omicron, which displays striking immune escape potential through mutations at key antigenic sites on the spike protein. Many of these mutations localize to the spike protein ACE2 receptor-binding domain, annulling the neutralizing activity of therapeutic antibodies that were effective against other Variants of Concern (VOCs) earlier in the pandemic. Here, we identified a receptor-blocking human monoclonal antibody, 87G7, that retained potent in vitro neutralizing activity against SARS-CoV-2 variants including the Alpha, Beta, Gamma, Delta and Omicron (BA.1/BA.2) VOCs. Using cryo-electron microscopy and site-directed mutagenesis experiments, we showed that 87G7 targets a patch of hydrophobic residues in the ACE2-binding site that are highly conserved in SARS-CoV-2 variants, explaining its broad neutralization capacity. 87G7 protected mice and/or hamsters prophylactically against challenge with all current SARS-CoV-2 VOCs, and showed therapeutic activity against SARS-CoV-2 challenge in both animal models. Our findings demonstrate that 87G7 holds promise as a prophylactic or therapeutic agent for COVID-19 that is more resilient to SARS-CoV-2 antigenic diversity., The MANCO project has received funding from the European Union's Horizon 2020 research and innovation program under grant agreement No 101003651). This work made use of the Dutch national e-infrastructure with the support of the SURF Cooperative using grant no. EINF-2453. This research was funded by the Deutsche Forschungsgemeinschaft (DFG; German Research Foundation) -398066876/GRK 2485/1; BMBF (Federal Ministry of Education and Research) project entitled RAPID (Risk assessment in re-pandemic respiratory infectious diseases), 01KI1723G, Ministry of Science and Culture of Lower Saxony in Germany (14 - 76103-184 CORONA-15/20)

Transgenic human monoclonal antibodies derived from humanized mice against different epitopes of the Middle East respiratory syndrome coronavirus (MERS-CoV), and chimeric llama-human bispecific heavy chain-only antibodies targeting the Rift Valley fever virus (RVFV), were produced using a CHO-based transient expression system. Two lead candidates were assessed for each model virus before selecting and progressing one lead molecule. MERS-7.7G6 was used as the model antibody to demonstrate batch-to-batch process consistency and, together with RVFV-107-104, were scaled up to 200 L. Consistent expression titers were obtained in different batches at a 5 L scale for MERS-7.7G6. Although lower expression levels were observed for MERS-7.7G6 and RVFV-107-104 during scale up to 200 L, product quality attributes were consistent at different scales and in different batches. In addition to this, peptide mapping data suggested no detectable sequence variants for any of these candidates. Functional assays demonstrated comparable neutralizing activity for MERS-7.7G6 and RVFV-107-104 generated at different production scales. Similarly, MERS-7.7G6 batches generated at different scales were shown to provide comparable protection in mouse models. Our study demonstrates that a CHO-based transient expression process is capable of generating consistent product quality at different production scales and thereby supports the potential of using transient gene expression to accelerate the manufacturing of early clinical material.

Historically part of the coronavirus (CoV) family, torovirus (ToV) was recently classified in the new family Tobaniviridae. While reverse genetics systems have been established for various CoVs, none exist for ToVs. Here, we developed a reverse genetics system using an infectious full-length cDNA clone of bovine ToV (BToV) in a bacterial artificial chromosome (BAC). Recombinant BToV harboring genetic markers had the same phenotype as wild-type (wt) BToV. To generate two types of recombinant virus, the hemagglutinin-esterase (HE) gene was edited, as cell-adapted wtBToV generally loses full-length HE (HEf), resulting in soluble HE (HEs). First, recombinant viruses with HEf and hemagglutinin (HA)-tagged HEf or HEs genes were rescued. These exhibited no significant differences in their effect on virus growth in HRT18 cells, suggesting that HE is not essential for viral replication in these cells. Thereafter, we generated a recombinant virus (rEGFP) wherein HE was replaced by the enhanced green fluorescent protein (EGFP) gene. rEGFP expressed EGFP in infected cells but showed significantly lower levels of viral growth than wtBToV. Moreover, rEGFP readily deleted the EGFP gene after one passage. Interestingly, rEGFP variants with two mutations (C1442F and I3562T) in nonstructural proteins (NSPs) that emerged during passage exhibited improved EGFP expression, EGFP gene retention, and viral replication. An rEGFP into which both mutations were introduced displayed a phenotype similar to that of these variants, suggesting that the mutations contributed to EGFP gene acceptance. The current findings provide new insights into BToV, and reverse genetics will help advance the current understanding of this neglected pathogen. IMPORTANCE ToVs are diarrhea-causing pathogens detected in various species, including humans. Through the development of a BAC-based BToV, we introduced the first reverse genetics system for Tobaniviridae. Utilizing this system, recombinant BToVs with a full-length HE gene were generated. Remarkably, although clinical BToVs generally lose the HE gene after a few passages, some recombinant viruses generated in the current study retained the HE gene for up to 20 passages while accumulating mutations in NSPs, which suggested that these mutations may be involved in HE gene retention. The EGFP gene of recombinant viruses was unstable, but rEGFP into which two NSP mutations were introduced exhibited improved EGFP expression, gene retention, and viral replication. These data suggested the existence of an NSP-based acceptance or retention mechanism for exogenous RNA or HE genes. Recombinant BToVs and reverse genetics are powerful tools for understanding fundamental viral processes, pathogenesis, and BToV vaccine development.

Coronavirus disease 2019 (COVID-19) is especially severe in aged populations1. Vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are highly effective, but vaccine efficacy is partly compromised by the emergence of SARS-CoV-2 variants with enhanced transmissibility2. The emergence of these variants emphasizes the need for further development of anti-SARS-CoV-2 therapies, especially for aged populations. Here we describe the isolation of highly virulent mouse-adapted viruses and use them to test a new therapeutic drug in infected aged animals. Many of the alterations observed in SARS-CoV-2 during mouse adaptation (positions 417, 484, 493, 498 and 501 of the spike protein) also arise in humans in variants of concern2. Their appearance during mouse adaptation indicates that immune pressure is not required for selection. For murine SARS, for which severity is also age dependent, elevated levels of an eicosanoid (prostaglandin D2 (PGD2)) and a phospholipase (phospholipase A2 group 2D (PLA2G2D)) contributed to poor outcomes in aged mice3,4. mRNA expression of PLA2G2D and prostaglandin D2 receptor (PTGDR), and production of PGD2 also increase with ageing and after SARS-CoV-2 infection in dendritic cells derived from human peripheral blood mononuclear cells. Using our mouse-adapted SARS-CoV-2, we show that middle-aged mice lacking expression of PTGDR or PLA2G2D are protected from severe disease. Furthermore, treatment with a PTGDR antagonist, asapiprant, protected aged mice from lethal infection. PTGDR antagonism is one of the first interventions in SARS-CoV-2-infected animals that specifically protects aged animals, suggesting that the PLA2G2D–PGD2/PTGDR pathway is a useful target for therapeutic interventions., This work is supported in part by grants from the National Institutes of Health USA (NIH; P01 AI060699 (S.P. and P.B.M.) and R01 AI129269 (S.P.)) and BIOAGE Labs (S.P.). The Pathology Core is partially supported by the Center for Gene Therapy for Cystic Fibrosis (NIH grant P30 DK-54759) and the Cystic Fibrosis Foundation. P.B.M. is supported by the Roy J. Carver Charitable Trust. L.-Y.R.W. is supported by Mechanism of Parasitism Training Grant (T32 AI007511). We thank M. Gelb (University of Washington) for Pla2g2d−/− mice.

The coronavirus spike glycoprotein, located on the virion surface, is the key mediator of cell entry and the focus for development of protective antibodies and vaccines. Structural studies show exposed sites on the spike trimer that might be targeted by antibodies with cross-species specificity. Here we isolated two human monoclonal antibodies from immunized humanized mice that display a remarkable cross-reactivity against distinct spike proteins of betacoronaviruses including SARS-CoV, SARS-CoV-2, MERS-CoV and the endemic human coronavirus HCoV-OC43. Both cross-reactive antibodies target the stem helix in the spike S2 fusion subunit which, in the prefusion conformation of trimeric spike, forms a surface exposed membrane-proximal helical bundle. Both antibodies block MERS-CoV infection in cells and provide protection to mice from lethal MERS-CoV challenge in prophylactic and/or therapeutic models. Our work highlights an immunogenic and vulnerable site on the betacoronavirus spike protein enabling elicitation of antibodies with unusual binding breadth., Here, the authors report the isolation and characterization of two human monoclonal antibodies (mAbs) from immunized mice with trimeric spike ectodomains of three human betacoronaviruses HCoV-OC43, SARS-CoV and MERSCoV, and show that while exhibiting cross-reactivity, the mAbs only neutralize MERS-CoV but not SARS-CoV nor SARS-CoV-2, likely due to the subtle epitope differences in the spike S2 fusion subunit.

Self-amplifying RNA replicons are promising platforms for vaccine generation. Their defects in one or more essential functions for viral replication, particle assembly, or dissemination make them highly safe as vaccines. We previously showed that the deletion of the envelope (E) gene from the Middle East respiratory syndrome coronavirus (MERS-CoV) produces a replication-competent propagation- defective RNA replicon (MERS-CoV-ΔE). Evaluation of this replicon in mice expressing human dipeptidyl peptidase 4, the virus receptor, showed that the single deletion of the E gene generated an attenuated mutant. The combined deletion of the E gene with accessory open reading frames (ORFs) 3, 4a, 4b, and 5 resulted in a highly attenuated propagation-defective RNA replicon (MERS-CoV-Δ[3,4a,4b,5,E]). This RNA replicon induced sterilizing immunity in mice after challenge with a lethal dose of a virulent MERS-CoV, as no histopathological damage or infectious virus was detected in the lungs of challenged mice. The four mutants lacking the E gene were genetically stable, did not recombine with the E gene provided in trans during their passage in cell culture, and showed a propagation-defective phenotype in vivo. In addition, immunization with MERS-CoV-Δ[3,4a,4b,5,E] induced significant levels of neutralizing antibodies, indicating that MERS-CoV RNA replicons are highly safe and promising vaccine candidates, Government of Spain and European Union co-financed grant BIO2016-75549-R; by Government of Spain grants PID2019-107001RB-I00, Proyecto Intramural Especial (PIE) 202020E079, and PIE–Consejo Superior de Investigaciones Científicas (CSIC) 202020E043; by European Commission grants ZAPI_IMI_JU_115760, ISOLDA_848166, and MANCO_101003651; and by IH grant 2P01AI060699

Middle East respiratory syndrome coronavirus (MERS-CoV) causes a highly lethal pneumonia that emerged in 2012. There is limited information on MERS-CoV pathogenesis, as data from patients are scarce and the generation of animal models reproducing MERS clinical manifestations has been challenging. Human dipeptidyl peptidase 4 knock-in (hDPP4-KI) mice and a mouse-adapted MERS-CoV strain (MERSMA-6-1-2) were recently described. hDPP4-KI mice infected with MERSMA-6-1-2 show pathological signs of respiratory disease, high viral titers in the lung, and death. In this work, a mouse-adapted MERS-CoV infectious cDNA was engineered by introducing nonsynonymous mutations contained in the MERSMA-6-1-2 genome into a MERS-CoV infectious cDNA, leading to a recombinant mouse-adapted virus (rMERS-MA) that was virulent in hDDP4-KI mice. MERS-CoV adaptation to cell culture or mouse lungs led to mutations and deletions in genus-specific gene 5 that prevented full-length protein expression. In contrast, analysis of 476 MERS-CoV field isolates showed that gene 5 is highly stable in vivo in both humans and camels. To study the role of protein 5, two additional viruses were engineered expressing a full-length gene 5 (rMERS-MA-5FL) or containing a complete gene 5 deletion (rMERS-MA-Δ5). rMERS-MA-5FL virus was unstable, as deletions appeared during passage in different tissue culture cells, highlighting MERS-CoV instability. The virulence of rMERS-MA-Δ5 was analyzed in a sublethal hDPP4-KI mouse model. Unexpectedly, all mice died after infection with rMERS-MA-Δ5, in contrast to those infected with the parental virus, which contains a 17-nucleotide (nt) deletion and a stop codon in protein 5 at position 108. Expression of interferon and proinflammatory cytokines was delayed and dysregulated in the lungs of rMERS-MA-Δ5-infected mice. Overall, these data indicated that the rMERS-MA-Δ5 virus was more virulent than the parental one and suggest that the residual gene 5 sequence present in the mouse-adapted parental virus had a function in ameliorating severe MERS-CoV pathogenesis.IMPORTANCE Middle East respiratory syndrome coronavirus (MERS-CoV) is a zoonotic virus causing human infections with high mortality rate (∼35%). Animal models together with reverse-genetics systems are essential to understand MERS-CoV pathogenesis. We developed a reverse-genetics system for a mouse-adapted MERS-CoV that reproduces the virus behavior observed in humans. This system is highly useful to investigate the role of specific viral genes in pathogenesis. In addition, we described a virus lacking gene 5 expression that is more virulent than the parental one. The data provide novel functions in IFN modulation for gene 5 in the context of viral infection and will help to develop novel antiviral strategies.

Summary Middle East Respiratory Syndrome Coronavirus (MERS-CoV) likely originated in bats and passed to humans through dromedary camels; however, the genetic mechanisms underlying cross-species adaptation remain poorly understood. Variation in the host receptor, dipeptidyl peptidase 4 (DPP4), can block the interaction with the MERS-CoV spike protein and form a species barrier to infection. To better understand the species adaptability of MERS-CoV, we identified a suboptimal species-derived variant of DPP4 to study viral adaption. Passaging virus on cells expressing this DPP4 variant led to accumulation of mutations in the viral spike which increased replication. Parallel passages revealed distinct paths of viral adaptation to the same DPP4 variant. Structural analysis and functional assays showed that these mutations enhanced viral entry with suboptimal DPP4 by altering the surface charge of spike. These findings demonstrate that MERS-CoV spike can utilize multiple paths to rapidly adapt to novel species variation in DPP4., Graphical Abstract, Highlights • MERS-CoV infected cells expressing DPP4 from 16 bat species • MERS-CoV spike rapidly adapted to species variation in DPP4 • Viral adaptations modified the surface charge of viral spike • Different routes of spike adaptation enhanced entry with the same DPP4 variant, MERS-CoV is a zoonotic pathogen capable of infecting numerous species. However, our understanding of how this virus adapts to new species remains unclear. Letko et al. experimentally observe several different routes in the stepwise, adaptive evolution of MERS-CoV to a unique host-species variant of the viral receptor.

Porcine enteric coronaviruses (CoVs) cause severe disease in the porcine herds worldwide, leading to important economic losses. Despite the knowledge of these viruses since the 1970s, vaccination strategies have not been implemented, leading to continuous re-emergence of novel virulent strains. Live attenuated vaccines historically have been the most efficient. We consider that the new trend is the development of recombinant vaccines by using reverse genetics systems to engineer attenuated viruses, which could be used as effective and safe modified live vaccine candidates. To this end, host cell signaling pathways influencing porcine CoV virulence should be identified. Similarly, the identity of viral proteins involved in the modulation of host cell pathways influencing CoV pathogenesis should be analyzed. With this information, and using reverse genetics systems, it is possible to design viruses with modifications in the viral proteins acting as virulence factors, which may lead to attenuated viruses and, therefore, vaccine candidates. In addition, novel antiviral drugs may be developed once the host cell pathways and the molecular mechanism affecting porcine CoV replication and virulence are known. This review is focused in the host cell responses to enteric porcine CoV infection and the viral proteins involved in pathogenesis., This work was supported by grants from the Government of Spain (BIO2013-42869-R), a U.S. National Institutes of Health (NIH) project (2P01AI060699-06A1), and financial support of IMI and European Commission and in-kind contributions from EFPIA partners (ZAPI project, IMI Grant Agreement n°115760). SZ received a contract from NIH. We thank Marga González for her technical assistance.

Severe acute respiratory syndrome coronavirus (SARS-CoV) is capable of inducing a storm of proinflammatory cytokines. In this study, we show that the SARS-CoV open reading frame 3a (ORF3a) accessory protein activates the NLRP3 inflammasome by promoting TNF receptor-associated factor 3 (TRAF3)-mediated ubiquitination of apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC). SARS-CoV and its ORF3a protein were found to be potent activators of pro-IL-1beta gene transcription and protein maturation, the 2 signals required for activation of the NLRP3 inflammasome., This work was supported by the Hong Kong Health and Medical Research Fund (13121032, 14130822, and HKM-15- M01), Hong Kong Research Grants Council (T11-707/15-R, C7011-15R, and 17124415), Government of Spain (BIO2013- 42869-R), U.S. National Institutes of Health (NIH) National Institute of Allergy and Infectious Diseases (2P01AI060699- 06A1), and European Commission (Innovative Medicines Initiative Grant Agreement 115760). C.C.-R. received a fellowship from Fundacion La Caixa.

Virus vaccines have to be immunogenic, sufficiently stable, safe, and suitable to induce long-lasting immunity. To meet these requirements, vaccine studies need to provide a comprehensive understanding of (i) the protective roles of antiviral B and T-cell-mediated immune responses, (ii) the complexity and plasticity of major viral antigens, and (iii) virus molecular biology and pathogenesis. There are many types of vaccines including subunit vaccines, whole-inactivated virus, vectored, and live-attenuated virus vaccines, each of which featuring specific advantages and limitations. While nonliving virus vaccines have clear advantages in being safe and stable, they may cause side effects and be less efficacious compared to live-attenuated virus vaccines. In most cases, the latter induce long-lasting immunity but they may require special safety measures to prevent reversion to highly virulent viruses following vaccination. The chapter summarizes the recent progress in the development of coronavirus (CoV) vaccines, focusing on two zoonotic CoVs, the severe acute respiratory syndrome CoV (SARS-CoV), and the Middle East respiratory syndrome CoV, both of which cause deadly disease and epidemics in humans. The development of attenuated virus vaccines to combat infections caused by highly pathogenic CoVs was largely based on the identification and characterization of viral virulence proteins that, for example, interfere with the innate and adaptive immune response or are involved in interactions with specific cell types, such as macrophages, dendritic and epithelial cells, and T lymphocytes, thereby modulating antiviral host responses and viral pathogenesis and potentially resulting in deleterious side effects following vaccination.

Replication of the coronavirus genome requires continuous RNA synthesis, whereas transcription is a discontinuous process unique among RNA viruses. Transcription includes a template switch during the synthesis of subgenomic negative-strand RNAs to add a copy of the leader sequence. Coronavirus transcription is regulated by multiple factors, including the extent of base-pairing between transcription-regulating sequences of positive and negative polarity, viral and cell protein-RNA binding, and high-order RNA-RNA interactions. Coronavirus RNA synthesis is performed by a replication-transcription complex that includes viral and cell proteins that recognize cis-acting RNA elements mainly located in the highly structured 5' and 3' untranslated regions. In addition to many viral nonstructural proteins, the presence of cell nuclear proteins and the viral nucleocapsid protein increases virus amplification efficacy. Coronavirus RNA synthesis is connected with the formation of double-membrane vesicles and convoluted membranes. Coronaviruses encode proofreading machinery, unique in the RNA virus world, to ensure the maintenance of their large genome size., This work was supported by grants from the Ministry of Science and Innovation of Spain (MINECO project BIO2013-42869-R), the European Community's Seventh Framework Programme (FP7/2007–2013; project EMPERIE EC grant agreement number 223498), and the US National Institutes of Health (NIH) (projects 2P01AI060699-06A1 and CRIP-HHSN266200700010C). S.Z. received a contract from NIH project 2P01AI060699-06A1.

Base on the sequence of S genes, which encode spike proteins, we previously identified three different types (North American, S INDEL, and S large-DEL types) of porcine epidemic diarrhea virus (PEDV) that have re-emerged in Japan since 2013. Based on experimental infections with the North American and S large-DEL types, we also hypothesized that PEDV virulence may be linked to the S1 subunit of the S protein. To test this hypothesis, we have now assayed in gnotobiotic piglets various recombinant PEDVs generated by reverse genetics. Piglets inoculated with CV777 maintained in National Institute of Animal Health, along with piglets infected with a recombinant form of the same virus, developed subclinical to mild diarrhea. In contrast, severe watery diarrhea, dehydration, weight loss, astasia, and high mortality were observed in piglets inoculated with recombinant strains in which the S gene was partially or fully replaced with corresponding sequences from the highly virulent Japanese PEDV isolate OKN-1/JPN/2013. Indeed, symptoms resembled those in piglets inoculated with the OKN-1/JPN/2013, and were especially pronounced in younger piglets. Collectively, the data demonstrate that the S1 subunit of the S protein is an important determinant of PEDV virulence, and advance development of new vaccine candidate., T.S. was partially supported by a grant from National Agricultural Research Organization. W.K. was supported by grants from The Ito Foundation and The Mishima Kaiun Memorial Foundation. Y.T. was supported by a grant from The Futaba Research Grant Program of the Futaba Foundation.

Dromedary camel heavy chain–only antibodies may provide novel intervention strategies against MERS coronavirus., Middle East respiratory syndrome coronavirus (MERS-CoV) continues to cause outbreaks in humans as a result of spillover events from dromedaries. In contrast to humans, MERS-CoV–exposed dromedaries develop only very mild infections and exceptionally potent virus-neutralizing antibody responses. These strong antibody responses may be caused by affinity maturation as a result of repeated exposure to the virus or by the fact that dromedaries—apart from conventional antibodies—have relatively unique, heavy chain–only antibodies (HCAbs). These HCAbs are devoid of light chains and have long complementarity-determining regions with unique epitope binding properties, allowing them to recognize and bind with high affinity to epitopes not recognized by conventional antibodies. Through direct cloning and expression of the variable heavy chains (VHHs) of HCAbs from the bone marrow of MERS-CoV–infected dromedaries, we identified several MERS-CoV–specific VHHs or nanobodies. In vitro, these VHHs efficiently blocked virus entry at picomolar concentrations. The selected VHHs bind with exceptionally high affinity to the receptor binding domain of the viral spike protein. Furthermore, camel/human chimeric HCAbs—composed of the camel VHH linked to a human Fc domain lacking the CH1 exon—had an extended half-life in the serum and protected mice against a lethal MERS-CoV challenge. HCAbs represent a promising alternative strategy to develop novel interventions not only for MERS-CoV but also for other emerging pathogens.

Viroporins are viral proteins with ion channel (IC) activity that play an important role in several processes, including virus replication and pathogenesis. While many coronaviruses (CoVs) encode two viroporins, severe acute respiratory syndrome CoV (SARS-CoV) encodes three: proteins 3a, E, and 8a. Additionally, proteins 3a and E have a PDZ-binding motif (PBM), which can potentially bind over 400 cellular proteins which contain a PDZ domain, making them potentially important for the control of cell function. In the present work, a comparative study of the functional motifs included within the SARS-CoV viroporins was performed, mostly focusing on the roles of the IC and PBM of E and 3a proteins. Our results showed that the full-length E and 3a proteins were required for maximal SARS-CoV replication and virulence, whereas viroporin 8a had only a minor impact on these activities. A virus missing both the E and 3a proteins was not viable, whereas the presence of either protein with a functional PBM restored virus viability. E protein IC activity and the presence of its PBM were necessary for virulence in mice. In contrast, the presence or absence of the homologous motifs in protein 3a did not influence virus pathogenicity. Therefore, dominance of the IC and PBM of protein E over those of protein 3a was demonstrated in the induction of pathogenesis in mice., IMPORTANCE Collectively, these results demonstrate key roles for the ion channel and PBM domains in optimal virus replication and pathogenesis and suggest that the viral viroporins and PBMs are suitable targets for antiviral therapy and for mutation in attenuated SARS-CoV vaccines.

Middle East respiratory syndrome coronavirus (MERS-CoV) is a novel human coronavirus that emerged in 2012, causing severe pneumonia and acute respiratory distress syndrome (ARDS), with a case fatality rate of ~36%. When expressed in isolation, CoV accessory proteins have been shown to interfere with innate antiviral signaling pathways. However, there is limited information on the specific contribution of MERS-CoV accessory protein 4b to the repression of the innate antiviral response in the context of infection. We found that MERS-CoV 4b was required to prevent a robust NF-κB dependent response during infection. In wild-type virus infected cells, 4b localized to the nucleus, while NF-κB was retained in the cytoplasm. In contrast, in the absence of 4b or in the presence of cytoplasmic 4b mutants lacking a nuclear localization signal (NLS), NF-κB was translocated to the nucleus leading to the expression of pro-inflammatory cytokines. This indicates that NF-κB repression required the nuclear import of 4b mediated by a specific NLS. Interestingly, we also found that both in isolation and during infection, 4b interacted with α-karyopherin proteins in an NLS-dependent manner. In particular, 4b had a strong preference for binding karyopherin-α4 (KPNA4), which is known to translocate the NF-κB protein complex into the nucleus. Binding of 4b to KPNA4 during infection inhibited its interaction with NF-κB-p65 subunit. Thereby we propose a model where 4b outcompetes NF-κB for KPNA4 binding and translocation into the nucleus as a mechanism of interference with the NF-κB-mediated innate immune response., This work was supported by grants from the Government of Spain (BIO2013-42869-R and BIO2016-75549-R AEI/FEDER, UE), the European Zoonotic anticipation and preparedness initiative ZAPI (IMI_115760), and U.S. National Institutes of Health (NIH) (contract #0258-3413/ HHSN266200700010C (LE), 2P01 AI060699 (LE and SP), and R01 AI129269 (SP). JC received a contract from the Government of Spain (BIO2013- 42869-R). ARF was supported by institutional NRSA training grant T32-AI007260 and an individual NIH NRSA grant (F32-AI113973).

Infection of polarized intestinal epithelial cells by porcine epidemic diarrhea virus (PEDV) was characterized. Indirect immunofluorescence assay, real-time PCR, and transmission electron microscopy confirmed PEDV can be successfully propagated in immortalized swine small intestine epithelial cells (IECs). Infection involved porcine aminpeptidase N (pAPN), a reported cellular receptor for PEDV, transient expression of pAPN and siRNA targeted pAPN increased and decreased the infectivity of PEDV in IECs, respectively. Subsequently, polarized entry into and release from both Vero E6 and IECs was analyzed. PEDV entry into polarized cells and pAPN grown on membrane inserts occurs via apical membrane. The progeny virus released into the medium was also quantified which demonstrated that PEDV is preferentially released from the apical membrane. Collectively, our data demonstrate that pAPN, the cellular receptor for PEDV, mediates polarized PEDV infection. These results imply the possibility that PEDV infection may proceed by lateral spread of virus in intestinal epithelial cells., Highlights • PEDV infection of polarized intestinal epithelial cells (IECs) was characterized. • Porcine aminpeptidase N (pAPN) facilitated PEDV infection in IECs. • PEDV entry into and release from polarized cell via its apical membrane. • PEDV infection may proceed by lateral spread of virus in IECs.

Cell surface aminopeptidase N (APN) is a membrane-bound ectoenzyme that hydrolyzes proteins and peptides and regulates numerous cell functions. APN participates in tumor cell expansion and motility, and is a target for cancer therapies. Small drugs that bind to the APN active site inhibit catalysis and suppress tumor growth. APN is also a major cell entry receptor for coronavirus, which binds to a region distant from the active site. Three crystal structures that we determined of human and pig APN ectodomains defined the dynamic conformation of the protein. These structures offered snapshots of closed, intermediate and open APN, which represent distinct functional states. Coronavirus envelope proteins specifically recognized the open APN form, prevented ectodomain progression to the closed form and substrate hydrolysis. In addition, drugs that bind the active site inhibited both coronavirus binding to cell surface APN and infection; the drugs probably hindered APN transition to the virus-specific open form. We conclude that allosteric inhibition of APN functions occurs by ligand suppression of ectodomain motions necessary for catalysis and virus cell entry, as validated by locking APN with disulfides. Blocking APN dynamics can thus be a valuable approach to development of drugs that target this ectoenzyme., GM was a recipient of a La Caixa fellowship. JR was supported by the Juan de la Cierva program and RR by NIH grant 2P01AI054456-06A1. The work was supported by grants from the Spanish Ministry of Science (BFU2011-23940 and BIO2014-52683-R to JMC).

Trabajo presentado en el XIV Congreso Nacional de Virología, celebrado en Cádiz (España), del 11 al 14 de junio de 2017, Severe acute respiratory syndrome coronavirus (SARS-CoV) causes lethal disease in humans, which is characterized by exacerbated inflammatory response and extensive lung pathology. In addition to protein components, viral genomes can also encode non-coding RNAs (ncRNAs) differing in size, biogenesis, and function, which are not necessarily comparable to those produced by host cells. To address the relevance of small non-coding RNAs in SARS-CoV pathology, we deep sequenced RNAs from the lungs of infected mice, which reproduce the pulmonary pathology observed in humans. Bioinformatics analysis of RNAs sequencesrevealed small RNAs generated from the SARS-CoV genome (svRNAs).Three abundant 18-22 ntsvRNAs derived from the nsp3 (svRNA-nsp3.1 and nsp3.2) and N (svRNA-N) genomic regions of SARS-CoV, which represented 18% outof thetotalsmallviral sequences, were selected for further study. The generation of these svRNAs was confirmed by RTqPCRin different cell types infectedeither by WT or E genemutant SARS-CoV.Production of SARS-CoV svRNAs was cell type and host species independent, but it was dependent on the extent of viral replication. Biogenesis of svRNAs was evaluated in cells defective in Drosha and Dicer enzymes, which are responsible for miRNA canonical processing.svRNAgeneration resulted to be independent of these host RNase IIInucleases,suggesting that svRNA synthesis was mainly dependent on alternative mechanisms involving viral proteins or cellular factors induced during infection. The production of svRNAs during cell culture infection had a positive, although modest effect, on SARSCoV growth. svRNAs specifically repressed the expression of reporter mRNA targets with complementary 3’ UTR sequences, suggesting a post-transcriptional silencing effect of similar character to cellular miRNAs. To assess the contributionof svRNAs in pulmonary pathogenesis, svRNA-N antisense oligonucleotides or antagomirs were intranasally inoculated prior to SARS-CoV infection in mice. Inhibition of svRNA-N in vivo significantly reduced inflammatory cell infiltration and edema observed in the lung of SARS-CoV infected mice in the absence of inhibitors. Accordingly, a significant decrease in the expression of pro-inflammatory cytokines CCL2, IL-6 and CXCL10, associated with SARSCoV pathology, was also observed. Viral titers in the lungs were not affected by svRNA-N Inhibition, indicating that the reduced inflammation should not be attributed to decreased viral growth, but to the contribution of svRNAs to the inflammatory lung pathology induced by SARS-CoV. Together, these results indicate that svRNAs generated from SARS-CoV genome contribute to pathogenesis and highlight the potential of svRNA-N antagomirsin antiviral therapy.

Severe acute respiratory syndrome coronavirus (SARS-CoV) is the etiological agent of a respiratory disease that has a 10% mortality rate. We previously showed that SARS-CoV lacking the E gene (SARS-CoV-¿E) is attenuated in several animal model systems. Here, we show that absence of the E protein resulted in reduced expression of proinflammatory cytokines, decreased numbers of neutrophils in lung infiltrates, diminished lung pathology, and increased mouse survival, suggesting that lung inflammation contributed to SARS-CoV virulence. Further, infection with SARS-CoV-¿E resulted in decreased activation of NF-¿B compared to levels for the wild-type virus. Most important, treatment with drugs that inhibited NF-¿B activation led to a reduction in inflammation and lung pathology in both SARS-CoV-infected cultured cells and mice and significantly increased mouse survival after SARS-CoV infection. These data indicated that activation of the NF-¿B signaling pathway represents a major contribution to the inflammation induced after SARS-CoV infection and that NF-¿B inhibitors are promising antivirals in infections caused by SARS-CoV and potentially other pathogenic human coronaviruses., This work was supported by grants from the Ministry of Science and Innovation of Spain (BIO2010-16705), the Seventh Framework Programme (FP7/2007-2013) of the European Commission (EC) under the project EMPERIE (EC grant agreement number 223498), and the U.S. National Institutes of Health (2P01AI060699-06A1 and CRIP-HHSN266200700010C). M.L.D. received a contract from the project EMPERIE (EC grant agreement number 223498), and J.A.R.-N. and C.C.-R. received a contract from Fundación La Caixa.

Coronaviruses (CoVs) are positive-stranded RNA viruses with potential as immunization vectors, expressing high levels of heterologous genes and eliciting both secretory and systemic immune responses. Nevertheless, its high recombination rate may result in the loss of the full-length foreign gene, limiting their use as vectors. Transmissible gastroenteritis virus (TGEV) was engineered to express porcine reproductive and respiratory syndrome virus (PRRSV) small protein domains, as a strategy to improve heterologous gene stability. After serial passage in tissue cultures, stable expression of small PRRSV protein antigenic domains was achieved. Therefore, size reduction of the heterologous genes inserted in CoV-derived vectors led to the stable expression of antigenic domains. Immunization of piglets with these TGEV vectors led to partial protection against a challenge with a virulent PRRSV strain, as immunized animals showed reduced clinical signs and lung damage. Further improvement of TGEV-derived vectors will require the engineering of vectors with decreased recombination rate., Highlights • Foreign gene stability in TGEV-derived vectors was achieved by decreasing insert size. • A set of PRRSV antigens was stably expressed by TGEV-derived vectors. • PRRSV M protein was used as a scaffold for PRRSV antigenic domains expression. • Piglet immunization with rTGEVs encoding PRRSV antigens led to partial protection.

Porcine epidemic diarrhea virus (PEDV) is an enteric coronavirus causing high morbidity and mortality in porcine herds worldwide. Although both inactivated and live attenuated vaccines have been extensively used, the emergence of highly virulent strains and the recurrent outbreaks even in vaccinated farms highlight the need of effective vaccines. Engineering of genetically defined live attenuated vaccines is a rational approach for novel vaccine development. In this line, we engineered an attenuated virus based on the transmissible gastroenteritis virus (TGEV) genome, expressing a chimeric spike protein from a virulent United States (US) PEDV strain. This virus (rTGEV-RS-SPEDV) was attenuated in highly-sensitive five-day-old piglets, as infected animals did not lose weight and none of them died. In addition, the virus caused very minor tissue damage compared with a virulent virus. The rTGEV-RS-SPEDV vaccine candidate was also attenuated in three-week-old animals that were used to evaluate the protection conferred by this virus, compared with the protection induced by infection with a virulent PEDV US strain (PEDV-NVSL). The rTGEV-RS-SPEDV virus protected against challenge with a virulent PEDV strain, reducing challenge virus titers in jejunum and leading to undetectable challenge virus RNA levels in feces. The rTGEV-RS-SPEDV virus induced a humoral immune response specific for PEDV, including neutralizing antibodies. Altogether, the data indicated that rTGEV-RS-SPEDV is a promising vaccine candidate against virulent PEDV infection, This research was funded by grants from the Government of Spain (BIO2013-42869-R and BIO2016-75549-R AEI/FEDER, UE), Ceva Animal Health, the European Zoonotic Anticipation and Preparedness Initiative (ZAPI) (IMI_JU_115760), and the U.S. National Institutes of Health (NIH) (2P01AI060699 awarded to L.E. and I.S.).

Infection of conventional mice with a mouse adapted (MA15) severe acute respiratory syndrome (SARS) coronavirus (CoV) reproduces many aspects of human SARS such as pathological changes in lung, viremia, neutrophilia, and lethality. However, established mouse cell lines highly susceptible to mouse-adapted SARS-CoV infection are not available. In this work, efficiently transfectable mouse cell lines stably expressing the murine SARS-CoV receptor angiotensin converting enzyme 2 (ACE2) have been generated. These cells yielded high SARS-CoV-MA15 titers and also served as excellent tools for plaque assays. In addition, in these cell lines, SARS-CoV-MA15 induced the expression of proinflammatory cytokines and IFN-beta, mimicking what has been observed in experimental animal models infected with SARS-CoV and SARS patients. These cell lines are valuable tools to perform in vitro studies in a mouse cell system that reflects the species used for in vivo studies of SARS-CoV-MA15 pathogenesis, This work was supported by grants from the Ministry of Science and Innovation of Spain (BIO2010-16705), the European Community's Seventh Framework Programme (FP7/2007–2013) under the project “EMPERIE” EC Grant Agreement number 223498, and U.S. National Institutes of Health (NIH) (2P01AI060699-06A1, W000306844). JAR received a fellowship from the Fundacion La Caixa

Coronavirus (CoV) transcription requires a high-frequency recombination process that links newly synthesized minus-strand subgenomic RNA copies to the leader region, which is present only once, at the 5′ end of the genome. This discontinuous RNA synthesis step is based on the complementarity between the transcription-regulating sequences (TRSs) at the leader region and those preceding each gene in the nascent minus-strand RNA. Furthermore, the template switch requires the physical proximity of RNA genome domains located between 20,000 and 30,000 nucleotides apart. In this report, it is shown that the efficacy of this recombination step is promoted by novel additional long-distance RNA-RNA interactions between RNA motifs located close to the TRSs controlling the expression of each gene and their complementary sequences mapping close to the 5′ end of the genome. These interactions would bring together the motifs involved in the recombination process. This finding indicates that the formation of high-order RNA structures in the CoV genome is necessary to control the expression of at least the viral N gene. The requirement of these long-distance interactions for transcription was shown by the engineering of CoV replicons in which the complementarity between the newly identified sequences was disrupted. Furthermore, disruption of complementarity in mutant viruses led to mutations that restored complementarity, wild-type transcription levels, and viral titers by passage in cell cultures. The relevance of these high-order structures for virus transcription is reinforced by the phylogenetic conservation of the involved RNA motifs in CoVs., This work was supported by grants from the Ministry of Science and Innovation of Spain (BIO2010-16707 and PET2008-0310) and the European Community’s Seventh Framework Programme (FP7/2007-2013), under the projects EMPERIE (EC grant agreement 223498) and PoRRSCon (EC grant agreement 245141). P.A.M.-G. received a fellowship from the Ministry of Science and Innovation of Spain (BES-2008- 001932).

Innate immune response is the first line of antiviral defense resulting, in most cases, in pathogen clearance with minimal clinical consequences. Viruses have developed diverse strategies to subvert host defense mechanisms and increase their survival. In the transmissible gastroenteritis virus (TGEV) as a model, we previously reported that accessory gene 7 counteracts the host antiviral response by associating with the catalytic subunit of protein phosphatase 1 (PP1c). In the present work, the effect of the absence of gene 7 on the host cell, during infection, was further analyzed by transcriptomic analysis. The pattern of gene expression of cells infected with a recombinant mutant TGEV, lacking gene 7 expression (rTGEV-Δ7), was compared to that of cells infected with the parental virus (rTGEV-wt). Genes involved in the immune response, the interferon response, and inflammation were upregulated during TGEV infection in the absence of gene 7. An exacerbated innate immune response during infection with rTGEV-Δ7 virus was observed both in vitro and in vivo . An increase in macrophage recruitment and activation in lung tissues infected with rTGEV-Δ7 virus was observed compared to cells infected with the parental virus. In summary, the absence of protein 7 both in vitro and in vivo led to increased proinflammatory responses and acute tissue damage after infection. In a porcine animal model, which is immunologically similar to humans, we present a novel example of how viral proteins counteract host antiviral pathways to determine the infection outcome and pathogenesis.

Zoonotic coronaviruses, including the one that caused severe acute respiratory syndrome (SARS), cause significant morbidityand mortality in humans. No specific therapy for any human coronavirus is available, making vaccine development critical forprotection against these viruses. We previously showed that recombinant SARS coronavirus (SARS-CoV) (Urbani strain based)lacking envelope (E) protein expression (rU-ΔE) provided good but not perfect protection in young mice against challenge withvirulent mouse-adapted SARS-CoV (MA15). To improve vaccine efficacy, we developed a second set of E-deleted vaccine candidateson an MA15 background (rMA15-E). rMA15-ΔE is safe, causing no disease in 6-week-, 12-month-, or 18-month-old BALB/c mice. Immunization with this virus completely protected mice of three ages from lethal disease and effected more-rapidvirus clearance. Compared to rU-ΔE, rMA15-ΔE immunization resulted in significantly greater neutralizing antibody and SARSCoV-specific CD4 and CD8 T cell responses. After challenge, inflammatory cell infiltration, edema, and lung destruction weredecreased in the lungs of rMA15-ΔE-immunized mice compared to those in rU-ΔE-immunized 12-month-old mice. Collectively,these results show that immunization with a species-adapted attenuated coronavirus lacking E protein expression is safe andprovides optimal immunogenicity and long-term protection against challenge with lethal virus. This approach will be generallyuseful for development of vaccines protective against human coronaviruses as well as against coronaviruses that cause disease indomestic and companion animals. © 2013, American Society for Microbiology., This work was supported by grants from the NIH (PO1 AI060699) and from the Ministry of Science and Innovation of Spain (BIO2010-16705) and the European Community project EMPERIE (GA223498). J.A.R.-N. was supported by a fellowship from the Fundacion La Caixa

Advances in transient expression technologies have allowed the production of milligram quantities of proteins within a matter of days using only small amounts (tens of grams) of plant tissue. Among the proteins that have been produced using this approach are the structural proteins of viruses which are capable of forming virus-like particles (VLPs). As such particulate structures are potent stimulators of the immune system, they are excellent vaccine candidates both in their own right and as carriers of additional immunogenic sequences. VLPs of varying complexity derived from a variety of animal viruses have been successfully transiently expressed in plants and their immunological properties assessed. Generally, the plant-produced VLPs were found to have the expected antigenicity and immunogenicity. In several cases, including an M2e-based influenza vaccine candidate, the plant-expressed VLPs have been shown to be capable of stimulating protective immunity. These findings raise the prospect that low-cost plant-produced vaccines could be developed for both veterinary and human use.

Middle East respiratory syndrome (MERS) remains a serious international public health threat. With the goal of accelerating the development of countermeasures against MERS coronavirus (MERS-CoV), funding agencies, nongovernmental organizations, and researchers across the world assembled in Riyadh, Saudi Arabia, on November 14¿15, 2015, to discuss vaccine development challenges. The meeting was spearheaded by the Saudi Ministry of Health and co-organized by the International Vaccine Institute, South Korea. Accelerating the development of a preventive vaccine requires a better understanding of MERS epidemiology, transmission, and pathogenesis in humans and animals. A combination of rodent and nonhuman primate models should be considered in evaluating and developing preventive and therapeutic vaccine candidates. Dromedary camels should be considered for the development of veterinary vaccines. Several vaccine technology platforms targeting the MERS-CoV spike protein were discussed. Mechanisms to maximize investment, provide robust data, and affect public health are urgently neede, V.S.M. is supported by the Intramural Research Program of the National Institute of Allergy and Infectious Diseases, US National Institutes of Health.

The full-length genome of the highly lethal feline infectious peritonitis virus (FIPV) strain DF-2 was sequenced and cloned into a bacterial artificial chromosome (BAC) to study the role of ORF3abc in the FIPV-feline enteric coronavirus (FECV) transition. The reverse genetic system allowed the replacement of the truncated ORF3abc of the original FIPV DF-2 genome with the intact ORF3abc of the canine coronavirus (CCoV) reference strain Elmo/02. The in vitro replication kinetics of these two viruses was studied in CrFK and FCWF-4 cell lines, as well as in feline peripheral blood monocytes. Both viruses showed similar replication kinetics in established cell lines. However, the strain with a full-length ORF3 showed markedly lower replication of more than 2 log10 titers in feline peripheral blood monocytes. Our results suggest that the truncated ORF3abc plays an important role in the efficient macrophage/monocyte tropism of type II FIPV. © 2012, American Society for Microbiology., This work was supported by the Award of Excellence from the Swedish University of Agricultural Sciences, research grants from the AGRIA Animal Insurance Company (Agria Djurförsäkring), OTKA, and NKTH (Mobilitás 08-C OTKA 81187), the János Bolyai Fellowship from the Hungarian Academy of Sciences (BO/00414/10), and the Ministry of Science and Innovation of Spain (MCINN) (BIO2010-16705).

STAT1-deficient mice are more susceptible to infection with severe acute respiratory syndrome coronavirus (SARS-CoV) than type I interferon (IFN) receptor-deficient mice. We used mice lacking functional receptors for both type I and type III IFN (double knockout, dKO) to evaluate the possibility that type III IFN plays a decisive role in SARS-CoV protection.Wefound that viral peak titres in lungs of dKO and STAT1-deficient mice were similar, but significantly higher than in wild-type mice. The kinetics of viral clearance from the lung were also comparable in dKO and STAT1-deficient mice. Surprisingly, however, infected dKO mice remained healthy, whereas infected STAT1-deficient mice developed liver pathology and eventually succumbed to neurological disease. Our data suggest that the failure of STAT1-deficient mice to control initial SARS-CoV replication efficiently in the lung is due to impaired type I and type III IFN signalling, whereas the failure to control subsequent systemic viral spread is due to unrelated defects in STAT1-deficient mice., C. D. was supported by the German Ministry of Education (project code SARS II 01KI1005A) and grants from the European Commission (FP7-ANTIGONE number 278976 and FP7-EMPERIE number 223498). L. E. was supported by a grant from the Ministry of Science and Innovation of Spain (BIO-2010-60978). P. S. was supported by a grant from the German Science Foundation (SFB620).

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.

Severe acute respiratory syndrome (SARS) coronavirus (CoV) envelope (E) protein is a transmembrane protein. Several subcellular locations and topological conformations of E protein have been proposed. To identify the correct ones, polyclonal and monoclonal antibodies specific for the amino or the carboxy terminus of E protein, respectively, were generated. E protein was mainly found in the endoplasmic reticulum-Golgi intermediate compartment (ERGIC) of cells transfected with a plasmid encoding E protein or infected with SARS-CoV. No evidence of E protein presence in the plasma membrane was found by using immunofluorescence, immunoelectron microscopy and cell surface protein labeling. In addition, measurement of plasma membrane voltage gated ion channel activity by whole-cell patch clamp suggested that E protein was not present in the plasma membrane. A topological conformation in which SARS-CoV E protein amino terminus is oriented towards the lumen of intracellular membranes and carboxy terminus faces cell cytoplasm is proposed. © 2011 Elsevier Inc., This work was supported by grants from the Ministry of Science and Innovation of Spain (BIO2007-60978 and BIO2010-16705), the European Community’s Seventh Framework Programme (FP7/2007-2013) under the project “EMPERIE” EC Grant Agreement number 223498, and U.S. National Institutes of Health (ARRA-W000151845). JLN received a fellowship from the Consejo Superior de Investigaciones Cientificas (CSIC) of Spain

The N-terminal S1 region of the transmissible gastroenteritis virus (TGEV) spike (S) glycoprotein contains four antigenic sites (C, B, D and A, from the N- to the C-terminal end) and is engaged in host-cell receptor recognition. The most N-terminal portion of the S1 region, which comprises antigenic sites C and B, is needed for the enteric tropism of TGEV, whereas the major antigenic site A at the C-terminal moiety is required for both respiratory and enteric cell tropism, and is engaged in recognition of the aminopeptidase N (APN) receptor. This study determined the kinetics for binding of a soluble S1 protein to the APN protein. Moreover, the S1 region of the TGEV S protein was dissected, with the aim of identifying discrete modules displaying unique antigenic sites and receptor-binding functions. Following protease treatments and mammalian cell expression methods, four modules or domains (D1–D4) were defined at the S1 region. Papain treatment identified an N-terminal domain (D1) resistant to proteolysis, whereas receptor binding defined a soluble and functional APN receptor-binding domain (D3). This domain was recognized by neutralizing antibodies belonging to the antigenic site A and therefore could be used as an immunogen for the prevention of viral infection. The organization of the four modules in the S1 region of the TGEV S glycoprotein is discussed.

Cholesterol is a major constituent of detergent-resistant membrane microdomains (DRMs). We localized transmissible gastroenteritis virus (TGEV) spike (S) protein in DRMs in the viral envelope. Though S protein was not solubilized by cold non-ionic detergents, this behavior was unchanged when cholesterol was depleted from viral membrane by methyl-β-cyclodextrin (MβCD) and the protein did not comigrate with cellular DRM marker proteins in flotation analyses. Therefore, the S protein is not anchored in the viral membrane DRMs as they are known to occur in the plasma membrane. Cholesterol depletion from viral membrane may not affect the adsorption process as neither the sialic acid binding activity nor the binding to aminopeptidase N was reduced post-MβCD treatment. Reduced infectivity of cholesterol-depleted TGEV was observed only when the adsorption process occurred at 37 °C but not when the virus was applied at 4 °C. Cholesterol is important for a post-adsorption step, allowing membrane rearrangements that facilitate virus entry. © 2010 Elsevier B.V., National Natural Science Foundation of China (30700591) to J.Y. National Natural Science Foundation of China (30700590; 30972195), Funding supported by Program for New Century Excellent Talents in Heilongjiang Provincial University (1155--NCET--005) and a visiting scholarship from Deutscher Akademischer Austauschdienst (DAAD) to X.R. are acknowledged. G.H. was supported by Deutsche Forschungsgemeinschaft (SFB621) and by the German Ministry of Education and Research (BMBF: FKZ 01KI0702)

PRRSV is the causative agent of the most important infectious disease affecting swine herds worldwide, producing great economic losses. Commercially available vaccines are only partially effective in protection against PRRSV. Moreover, modified live vaccines may allow virus shedding, and could revert generating virulent phenotypes. Therefore, new efficient vaccines are required. Vaccines based on recombinant virus genomes (virus vectored vaccines) against PRRSV could represent a safe alternative for the generation of modified live vaccines. In this paper, current vectored vaccines to protect against PRRSV are revised, including those based on pseudorabies virus, poxvirus, adenovirus, and virus replicons. Special attention has been provided to the use of transmissible gastroenteritis virus (TGEV) as vector for the expression of PRRSV antigens. This vector has the capability of expressing high levels of heterologous genes, is a potent interferon-α inducer, and presents antigens in mucosal surfaces, eliciting both secretory and systemic immunity. A TGEV derived vector (rTGEV) was generated, expressing PRRSV wild type or modified GP5 and M proteins, described as the main inducers of neutralizing antibodies and cellular immune response, respectively. Protection experiments showed that vaccinated animals developed a faster and stronger humoral immune response than the non-vaccinated ones. Partial protection in challenged animals was observed, as vaccinated pigs showed decreased lung damage when compared with the non-vaccinated ones. Nevertheless, the level of neutralizing antibodies was low, what may explain the limited protection observed. Several strategies are proposed to improve current rTGEV vectors expressing PRRSV antigens.

A strain of transmissible gastroenteritis virus (TGEV), designated H16, was isolated in PK-15 cells and passaged serially to level 165. Vaccines based on passages 155–165 in cell cultures are available commercially as vaccines for the prevention and control of infections with TGEV in China. Nucleoprotein (N) sequences of the virus at passages 155 and 165 were aligned and compared using a computer software program. The suitability of restriction fragment length polymorphism (RFLP) analysis for differentiation of the vaccine strain from the other TGEVs was investigated. The RFLP analysis identified a change in the cleavage sites of AclI at passages 155 and 165. This RFLP pattern of the N gene differentiated the Chinese vaccine strain from its parental strain, the 11 TGEVs studied and the other reported TGEVs in the GenBank. Using phylogenetic analysis, the Chinese TGEVs were divided into three groups (G1, G2, and G3). The G3 Chinese TGEVs possessed several specific nucleotides and amino acids that were not found in the G1 and G2 Chinese TGEVs or the other reference TGEVs. Analysis of the phylogenetic trees revealed that the G3 TGEVs represent a separate group that is distinct from the non-Chinese TGEVs and from Chinese TGEVs isolated previously. These findings suggest that Chinese strains of TGEV are evolving continuously.

Transmissible gastroenteritis virus (TGEV), the etiological agent of transmissible gastroenteritis (TGE), is the major cause of viral enteritis and fetal diarrhea in swine neonates, resulting in significant economic losses to the swine industry. The Chinese vaccine strain H165 of TGEV was derived from a virulent field strain H16 by serial passage in vitro. Strain H165 has been proven to be safe in piglets and pregnant sows and displays efficacy against TGEV infection. In this study, we report the complete genome sequences of strains H165 and H16, obtained by sequencing several overlapping fragments amplified from viral RNA and our findings from sequence and phylogenetic analyses. The genomes were 28,569 nucleotides in length, including the poly (A) tail. No deletions or insertions were detected in the H16 genome sequence after continuous passage in vitro; however, we found 27 nucleotide mutations in strain H165 compared with strain H16, resulting in 16 amino acid changes distributed among the genes 1, S, 3, and sM. An A to G nucleotide mutation was found in the intergenic region between the 3a and 3b genes. Furthermore, six unique nucleotides identified in the genome sequence of H165 could be used as makers to differentiate the H165 vaccine strain from wild-type TGEV strains. Our findings from phylogenetic analysis may enhance our understanding of the evolution of TGEV, as well as the other coronaviruses.

The severe acute respiratory syndrome coronavirus (SARS-CoV) caused substantial morbidity and mortality in 2002-2003. Deletion of the envelope (E) protein modestly diminished virus growth in tissue culture but abrogated virulence in animals. Here, we show that immunization with rSARS-CoV-ΔE or SARS-CoV-Δ[E,6-9b] (deleted in accessory proteins (6, 7a, 7b, 8a, 8b, 9b) in addition to E) nearly completely protected BALB/c mice from fatal respiratory disease caused by mouse-adapted SARS-CoV and partly protected hACE2 Tg mice from lethal disease. hACE2 Tg mice, which express the human SARS-CoV receptor, are extremely susceptible to infection. We also show that rSARS-CoV-ΔE and rSARS-CoV-Δ[E,6-9b] induced anti-virus T cell and antibody responses. Further, the E-deleted viruses were stable after 16 blind passages through tissue culture cells, with only a single mutation in the surface glycoprotein detected. The passaged virus remained avirulent in mice. These results suggest that rSARS-CoV-ΔE is an efficacious vaccine candidate that might be useful if SARS recurred. © 2009 Elsevier Inc. All rights reserved., This work was supported by grants from the National Institutes of Health (US) (PO1 AI060699-01) (SP); RO1 AI079424-01A1 (SP and LE), the Ministry of Education and Science of Spain (BIO2007-60978) (LE), and the European Commission (EMPERIE PROJECT, Ref. No. 223498) (LE). J.N. was supported by an NIH training grant (T32 AI007533).

Purified nucleocapsid protein (N protein) from transmissible gastroenteritis virus (TGEV) enhanced hammerhead ribozyme self-cleavage and favored nucleic acid annealing, properties that define RNA chaperones, as previously reported. Several TGEV N-protein deletion mutants were expressed in Escherichia coli and purified, and their RNA binding ability and RNA chaperone activity were evaluated. The smallest N-protein domain analyzed with RNA chaperone activity, facilitating DNA and RNA annealing, contained the central unstructured region (amino acids 117 to 268). Interestingly, N protein and its deletion mutants with RNA chaperone activity enhanced template switching in a retrovirus-derived heterologous system, reinforcing the concept that TGEV N protein is an RNA chaperone that could be involved in template switching. This result is in agreement with the observation that in vivo , N protein is not necessary for TGEV replication, but it is required for efficient transcription.

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.

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.