Severe Acute Respiratory Syndrome Coronavirus Envelope Protein Regulates Cell Stress Response and Apoptosis

Severe acute respiratory syndrome virus (SARS-CoV) that lacks the envelope (E) gene (rSARS-CoV-ΔE) is attenuated in vivo. To identify factors that contribute to rSARS-CoV-ΔE attenuation, gene expression in cells infected by SARS-CoV with or without E gene was compared. Twenty-five stress response genes were preferentially upregulated during infection in the absence of the E gene. In addition, genes involved in signal transduction, transcription, cell metabolism, immunoregulation, inflammation, apoptosis and cell cycle and differentiation were differentially regulated in cells infected with rSARS-CoV with or without the E gene. Administration of E protein in trans reduced the stress response in cells infected with rSARS-CoV-ΔE or with respiratory syncytial virus, or treated with drugs, such as tunicamycin and thapsigargin that elicit cell stress by different mechanisms. In addition, SARS-CoV E protein down-regulated the signaling pathway inositol-requiring enzyme 1 (IRE-1) of the unfolded protein response, but not the PKR-like ER kinase (PERK) or activating transcription factor 6 (ATF-6) pathways, and reduced cell apoptosis. Overall, the activation of the IRE-1 pathway was not able to restore cell homeostasis, and apoptosis was induced probably as a measure to protect the host by limiting virus production and dissemination. The expression of proinflammatory cytokines was reduced in rSARS-CoV-ΔE-infected cells compared to rSARS-CoV-infected cells, suggesting that the increase in stress responses and the reduction of inflammation in the absence of the E gene contributed to the attenuation of rSARS-CoV-ΔE

SARS-CoV-2 and Dengue Virus Coinfection in a Mexican Pediatric Patient: A Case Report from Early Molecular Diagnosis

Mexico is an endemic region for dengue virus (DENV). The increase in this disease coincides with outbreaks of COVID-19, both of which are single-stranded positive RNA viruses. These characteristics make it difficult to distinguish each disease because they share clinical and laboratory features, which can consequently result in misdiagnoses. This is why the use of precision confirmatory tests (qRT-PCR) are crucial for early diagnosis. We herein report a pediatric patient who presented a coinfection for DENV and COVID-19, “SARS-CoV-2/Dengue”. This patient initially presented a fever, cough, and headache and, three days later, developed generalized pain and epistaxis. Blood studies revealed thrombocytopenia and leukopenia, and the patient was admitted to the hospital for a probable DENV infection. Within 48 h, qRT-PCR tests specific for SARS-CoV-2 and DENV were performed and resulted as positive. The patient immediately received pharmacological treatment with azithromycin, oseltamivir, and metamizole. During hospitalization (9 days), the patient had no signs of respiratory distress and maintained normal body temperature and normal blood oxygen saturation. This case warns of the need for early diagnosis and adequate clinical and pharmacological management in the face of a “SARS-CoV-2/Dengue” coinfection. Early molecular detection of both viruses and timely treatment helped the patient to achieve a favorable recovery

The PDZ-Binding Motif of Severe Acute Respiratory Syndrome Coronavirus Envelope Protein Is a Determinant of Viral Pathogenesis

<div><p>A recombinant severe acute respiratory syndrome coronavirus (SARS-CoV) lacking the envelope (E) protein is attenuated <i>in vivo</i>. 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.</p></div

Primers used for the generation of recombinant SARS-CoV-E-PBM mutants.

<p>Primers used for the generation of recombinant SARS-CoV-E-PBM mutants.</p

Virulence and viral growth of SARS-CoV-E-PBM-infected mice.

<p>16-week-old BALB/c mice were intranasally inoculated with 100,000 pfu of wt, ΔE, ΔPBM, mutPBM and potPBM viruses. Weight loss (A) and survival (B) were monitored for 10 days. Data represent two independent experiments with 5 mice per group. Differences in weight loss between attenuated and virulent viruses were statistically significant (<i>P</i><0.01). (C) Viral titer in lungs was determined at 2 and 4 days post infection (n = 3, each day). Error bars represent standard deviations. Statistically significant data are indicated with one (<i>P</i><0.05) asterisk.</p

Effect of SARS-CoV E protein PBM on host gene expression.

<p>(A) Comparison of gene expression in lungs of infected mice using microarrays: wt versus mock-infected, mutPBM versus mock-infected and mutPBM versus wt-infected mice. Red spots indicate upregulated gene transcripts (fold change, >2) and green spots indicate downregulated gene transcripts (fold change, <−2). Only genes with a FDR of <0.01 were considered as candidate genes. (B) Candidate genes that were downregulated in mutPBM infected mice compared wt infected ones, were grouped on Gene Ontology terms. Numbers on the <i>x</i> axis indicate DAVID FDR values. (C) Selection of differentially expressed genes found in at least one functional group using DAVID software. The numbers indicate the fold change for each gene in mutPBM versus wt-infected mice. (D) Expression of inflammatory cytokines evaluated by RT-qPCR. Three independent experiments were analyzed with similar results in all cases. Error bars represent standard deviations of the mean of results from three experiments.</p

Effect of p38 MAPK inhibitor in rSARS-CoV-MA15-infected mice.

<p>16-week-old BALB/c mice were mock-infected or inoculated intranasally with 100,000 pfu of wt virus. At 4 hpi and every 12 h from days 1 to 8, mock pfu of wt virus. At 4 hpi and every 12 h from days 1 to 8, mock hpi and every 12 h from days 1 to 8, mock h from days 1 to 8, mock-infected and wt-infected mice were intraperitoneally injected with SB203580 (6 mg mg/kg of body weight/day). (A) Animals were monitored daily for mortality. Data represent three independent experiments with 5 mice per group. (B) The active phosphorylated (p-HSP27) and total HSP27 in lungs of three infected mice per condition were evaluated by Western blot analysis. (C) Phospho and total HSP27 amounts were quantified by densitometric analysis. The graph shows the phosphorylated HSP27/total HSP27 ratio at 2 dpi in lungs of mock dpi in lungs of mock-infected mice or mice infected with the parental virus, treated or not with SB203580. Error bars represent the means of three mice analyzed for each condition. Statistically significant data are indicated with one (<i>P</i><0.05) asterisk.</p

Identification of the Mechanisms Causing Reversion to Virulence in an Attenuated SARS-CoV for the Design of a Genetically Stable Vaccine

<div><p>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 <i>in vivo</i>, 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 <i>in vitro</i> 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 <i>in vitro</i> and <i>in vivo</i>. 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.</p></div