Project description:This experiment aims to profile polyclonal antibody binding profiles in serum from vaccinated animals relative to antibody function in a virus neutralization assay. Rabbits received three vaccinations with a DNA vaccine encoding the spike protein of the SARS-CoV-2 index strain. Serum samples were selected based on a three-tier (low, intermediate, and high) capacity to cross-neutralize SARS-CoV-2 strains with known neutralization resistance. Following normalization of total anti-spike IgG levels, serum of each animal (n=3) were evaluated for antibody binding to 10mer cyclic constrained peptides spanning the entire spike protein and regions with known SARS-CoV-2 variant of concern spike mutations.
Project description:For the assessment of host response dynamics to SARS-CoV and SARS-CoV-2 infections in human airway epithelial cells at ambient temperature corresponding to the upper or lower respiratory tract. We performed a temporal transcriptome analysis on human airway epithelial cell (hAEC) cultures infected with SARS-CoV and SARS-CoV-2, as well as uninfected hAEC cultures, incubated either at 33°C or 37°C. hAEC cultures were harvested at 24, 48 72, 96 hpi and processed for Bulk RNA Barcoding and sequencing (BRB-seq), which allows a rapid and sensitive genome-wide transcriptomic analysis in a highly multiplexed manner. Transcriptome data was obtained from a total of 7 biological donors for pairwise comparisons of SARS-CoV or SARS-CoV-2 virus-infected to unexposed hAEC cultures at respective time points and temperatures.
Project description:To further investigate the underlying mechanisms of severe acute respiratory syndrome (SARS) pathogenesis and evaluate the therapeutic efficacy of potential drugs and vaccines it is necessary to use an animal model that is highly representative of the human condition in terms of respiratory anatomy, physiology and clinical sequelae. The ferret, Mustela putorius furo, supports SARS-CoV replication and displays many of the symptoms and pathological features seen in SARS-CoV-infected humans. We have recently established a SARS-CoV infection-challenge ferret platform for use in evaluating potential therapeutics to treat SARS. The main objective of the current study was to extend our previous results and identify early host immune responses upon infection and determine immune correlates of protection upon challenge with SARS-CoV in ferrets. Keywords: time course This study is a simple time course (58 day) examination of host responses in 35 SARS-CoV (TOR2) infected ferrets with the addition of a challenge inoculation of SARS CoV (TOR2) at day 29 post infection. Three mock-infected ferrets are included as negative controls. Due to the unavailability of ferret microarrays, Affymetrix Canine 2.0 oligonucleotide arrays were chosen following sequence analysis of our ferret cDNA library (~5000 clones) and demonstration of high levels of homology (>80%) between dog and ferret.
Project description:The purpose of this experiment was to investigate the transcriptional differences between mice infected with icSARS CoV, SARS MA15 wild type or SARS BatSRBD viruses. Overview of Experiment: Groups of 20 week old C57BL6 mice were infected with icSARS CoV, Wild Type SARS MA15 or SARS BatSRBD mutant viruses. Infections were done at 10^4 PFU or 10^5 PFU or time-matched mock infected. Time points were 1, 2, 4 and 7 d.p.i. There were 3-5 animals/dose/time point. Lung samples were collected for virus load, transcriptional and proteomics analysis. Weight loss and animal survival were also monitored.
Project description:HAE cultures were infected with SARS-CoV, SARS-dORF6 or SARS-BatSRBD and were directly compared to A/CA/04/2009 H1N1 influenza-infected cultures. Cell samples were collected at various hours post-infection for analysis. Time Points = 0, 12, 24, 36, 48, 60, 72, 84 and 96 hrs post-infection for SARS-CoV, SARS-dORF6 and SARS-BatSRBD. Time Points = 0, 6, 12, 18, 24, 36 and 48 hrs post-infection for H1N1. Done in triplicate for RNA Triplicates are defined as 3 different wells, plated at the same time and using the same cell stock for all replicates. Time matched mocks done in triplicate from same cell stock as rest of samples. Culture medium (the same as what the virus stock is in) will be used for the mock infections. Infection was done at an MOI of 2 for SARS viruses and an MOI of 1 for H1N1.
Project description:HAE cultures were infected with SARS-CoV, SARS-dORF6 or SARS-BatSRBD and were directly compared to A/CA/04/2009 H1N1 influenza-infected cultures. Cell samples were collected at various hours post-infection for analysis. Time Points = 0, 12, 24, 36, 48, 60, 72, 84 and 96 hrs post-infection for SARS-CoV, SARS-dORF6 and SARS-BatSRBD. Time Points = 0, 6, 12, 18, 24, 36 and 48 hrs post-infection for H1N1. Done in triplicate or quadruplicate for RNA Triplicates/quadruplicates are defined as 3/4 different wells, plated at the same time and using the same cell stock for all replicates. Time matched mocks done in triplicate from same cell stock as rest of samples. Culture medium (the same as what the virus stock is in) will be used for the mock infections. Infection was done at an MOI of 2.
Project description:We are studying the role of human sirtuin SIRT5 during viral infection with SARS-CoV-2. We performed RNA-sequencing of WT and SIRT5-KO human lung adenocarinoma A549 cells overexpressing ACE2 (A549-ACE2), in infected and mock-infected conditions. . Our analysis revealed that SARS-CoV-2 replication is attenuated in SIRT5-KO cells. In addition, SIRT5-KO cells expressed higher basal levels of innate immunity markers and mounted a stronger antiviral response. Our results indicate that SIRT5 is a proviral factor necessary for efficient viral replication.
Project description:Seasonal infection rates of individual viruses are influenced by synergistic or inhibitory interactions between coincident viruses. Endemic patterns of SARS-CoV-2 and influenza infection overlap seasonally in the Northern hemisphere and may be similarly influenced. We explored the immunopathologic basis of SARS-CoV-2 and influenza A (H1N1) interactions in Syrian hamsters. H1N1 given 48 hours prior to SARS-CoV-2 profoundly mitigated weight loss and lung pathology compared to SARS-CoV-2 infection alone. This was accompanied by normalization of granulocyte dynamics and accelerated antigen presenting populations in bronchoalveolar lavage and blood. Using nasal transcriptomics, we identified rapid upregulation of innate and antiviral pathways induced by H1N1 by the time of SARS-CoV-2 inoculation in 48 hour dual infected animals. Dual infected animals also experienced significant transient downregulation of mitochondrial and viral replication pathways. By quantitative RT-PCR, we confirmed reduced SARS-CoV-2 viral load and lower cytokine levels throughout disease course in lung of dual infected animals. Our data confirm that H1N1 infection induces rapid and transient gene expression that is associated with mitigation of SARS-CoV-2 pulmonary disease. These protective responses are likely to begin in the upper respiratory tract shortly after infection. On a population level, interaction between these two viruses may influence their relative seasonal infection rates.
Project description:We conducted a high-throughput drug repositioning screen using the LOPAC®1280 and the ReFRAME drug libraries to identify existing drugs that harbor antiviral activity against SARS-CoV-2, in a Vero E6 cell-based assay. We additionally performed RNA sequencing on control and SARS-CoV-2 infected Vero E6 cells to study the biological changes after SARS-CoV-2 infection and to elucidate the potential mechanisms underlying the positive hits identified from our high-throughput screen. Vero E6 cells were either mock-infected or infected with SARS-CoV-2 USA-WA1/2020 (MOI = 0.3) with three replicates. Cells were harvested 24 hours after infection, and total RNA was extracted using the Qiagen® RNeasy® Plus Mini Kit. The quality of the extracted RNA was assessed with the Agilent® 2100 Bioanalyzer. Libraries were prepared from total RNA following ribosome RNA depletion using standard protocol according to Illumina®. Total RNA sequencing was then performed on the Illumina® NextSeq system; 150bp paired-end runs were performed and 100 million raw reads per sample were generated.