MOLECULAR PATHOGENESIS AND ADAPTATION OF SARS-COV-2 IN WHITE-TAILED DEER AND IMPLICATIONS FOR SPILLOVER TO CATTLE AND PIGS

<b>Project Methods</b><br> Comparative Replication in cell culture:Viral whole-genome sequencing will be performed using our defined ARTIC-based workflow for targeted amplicon sequencing for each virus stock. To generate deer adapted SARS-CoV-2, the strain of interest will be passaged three times in deer PTEC cells to produce stock. Stock viruses will be used to inoculate the established primary tracheal epithelial cells with SARS-CoV-2 at a multiplicity of infection (MOI) of 1 for two hours on ice to synchronize the cell cycle. Virus will then be removed from cells, cells will be washed and provided with fresh media, and virus-infected cells will be grown at 37?/5% CO2. Cell supernatants will be harvested at 2, 6, 12, 24, 48, and 72 hours post inoculation (hpi) for the quantification of viral genome copy equivalents by real-time RT-PCR using a standard curve. The supernatants will also be used to determine infectious titers expressed as TCID50.Following removal of the supernatant, cells will be fixed with 4% paraformaldehyde and stained to detect N protein using rabbit anti-N antibody (Absolute Antibody, Ab01691-23.0) and double-stranded RNA (representing replicating SARS-CoV-2 RNA) using mouse J2 monoclonal antibody (Abcam 288755).The experiments will be conducted in triplicates and will be repeated thrice. All experiments will be conducted in our animal biosafety level 3 (ABSL-3) facility with appropriate respiratory protection and barrier clothing procedures for personnel.Statistical analysis:Statistical analyses will be performed using R software. One-way ANOVA with multiple comparisons (Tukey test) will be used for testing differences in mean viral RNA and TCID50titers between virus inoculum sets. The area of dsRNA per cell across all FOV from all wells of each virus inoculum will be averaged and evaluated against the means of other viral inocula by one-way ANOVA with multiple comparisons (Tukey test).Cell Infection for innate immune response:Stock viruses will be used to inoculate the cattle, pig and deer tracheal epithelial cells as described above. Mock-infected cells will be inoculated with an equivalent volume of culture medium and washed, incubated and grown along with the virus-infected cells. Cell supernatants will be harvested at 2, 6, 12, 24, 48, and 72 hours post inoculation (hpi). One aliquot will subsequently be used to quantify the infectious virus titer by TCID50. Following the supernatant removal, virus- and mock-infected cell lysates will be collected in RNA lysis buffer (Buffer RLT Plus – QIAGEN). Total RNA will be isolated from lysates using RNeasy Plus Kit (Qiagen). The RNA will be converted to cDNA using qScript cDNA Supermix (Quanta Bio). The resulting cDNA will be used to conduct quantitative gene expression analysis.Infection experiments to evaluate innate immune gene expression induced by deer-adapted viruses will be conducted in bovine and porcine PTEC.Innate immune profiling:The cDNA will be used to conduct quantitative gene expression analysis of intracellular viral E gene, negative-sense viral RNA, the genes identified in our preliminary studies as differentially regulated in SARS-CoV-2-variant infected deer RPLNf (IFNβ, BST2, OAS1, Mx1), and genes identified by others in human cells to be differentially expressed in Alpha versus ancestral B.1 infection (Ly6E, IFIT3, CXCL10, ISG15, and RSAD2). Gene expression will be normalized to the expression of one or more optimal reference genes. Genes 18S, TBP, GAPDH, H3F3A, ACTB, PPIA and YWHAZ will be evaluated as reference genes. The experiments will be conducted in triplicates and will be repeated thrice.Statistical Analysis:Statistical analyses will be performed using R software. Dunnett's multiple comparison test (ANOVA) will be used to compare mean measurements of gene expression and TCID50titers from ancestral B.1 lineage infection with those of variant infections at each timepoint.Single-cell RNAseq (scRNAseq):The two most interesting variants will be compared, with two wells of cells per variant used for scRNAseq library preparation of approximately 4,000 cells per well. At the selected timepoint, infected cells will be harvested using trypsin with EDTA to generate single-cell suspensions. Cells will be counted and then fixed to inactivate virus. Following removal from the ABSL-3 laboratory, samples will be processed at the Genomics Core Facility with a Next GEM Single Cell 3' kit using a Chromium X droplet-based processor. Sequencing libraries will be constructed using the Nextera XT DNA kit and processed on an Illumina NextSeq 2000.Indirect ELISA (iELISA):Our validated livestock iELISAs against Spike RBD (described in Gontuet al.) will be applied to the cattle and pig serum samples. Each assay will include hyperimmune serum produced in these species as a positive control. Negative controls will consist of pre-pandemic sera and commercially acquired serum from pathogen-free cattle and pigs. Sera will be tested at 1:50 dilution, and absorbance will be measured at 450 nm using a Synergy Neo2 multimode microplate reader. To evaluate potential cross-reactivity of the animal sera against coronaviruses common in that species, we will screen all SARS-CoV-2-specific cattle sera against bovine coronavirus and pig sera against porcine coronaviruses using live virus neutralization assays in appropriate cell lines (MDBK, ST, PK-15).Statistical analysis:iELISA absorbance values above the pre-determined cut-off (established with pre-pandemic serum samples) will be interpreted as positive, and those at or below as negative.Pseudotyped Virus Neutralization Test (pVNT):SARS-CoV-2 spike pseudoviruses will be produced using the third-generation lentiviral packaging plasmids as described. pVNTs will be performed on all positive sera by incubating ~10000 RLUs of each pseudovirus with 2-fold serial dilutions of sera for one hour at 37°C. Pseudovirus/sera mixtures will then be used to inoculate 96-well plates seeded with 1.3 x 104293T ACE2 cells per well. Pseudovirus infectivity will be determined at 72 hpi by quantifying luciferase activity with BrightGlow luciferase assay read with a Synergy Neo2 multi-mode microplate reader. Each serum will run in duplicate in two independent experiments against each pseudovirus.Statistical Analysis:Percentage neutralization will be calculated based on the luciferase activity of cells infected with the virus only, representing no neutralization. For each serum, the measured percent neutralization will be plotted against corresponding serum dilution, and non-linear regression curves will be produced using R software to determine the 50% neutralization titer (NT50).Antigenic cartography:To generate antibody landscapes, an antigenic map will first be generated from pVNT titer measurements from our collection of reference sera. Data of each serum specimen and its titer against each tested variant will be tabulated. A 2-dimensional antigenic map will be computed from the table using the Racmacs package in R software with 1000 optimizations. Reference sera will consist of hyperimmune sera previously collected in our laboratory from cattle and pigs experimentally injected with SARS-CoV-2 B.1 spike protein and additional deer SARS-CoV-2-specific sera already in our laboratory. The quality of the map will be verified by assessing the root mean squared errors of maps created from the data in 1, 2, 3, 4 and 5 dimensions.