Projects
MOLECULAR PATHOGENESIS AND ADAPTATION OF SARS-COV-2 IN WHITE-TAILED DEER AND IMPLICATIONS FOR SPILLOVER TO CATTLE AND PIGS
Topic: Coronaviruses
Summary
SARS-CoV-2, the virus responsible for COVID-19, has been found in various animal hosts, including white-tailed deer in the USA. These findings raise concerns about the potential transmission of the virus from deer to livestock, particularly cattle. To address this spillover risk, it's crucial to monitor how SARS-CoV-2 evolves in farmed deer and its potential infection of cattle. However, current methods for detecting and sequencing the virus are expensive, not tailored for animal samples, and unsuitable for field use. This project aims to develop cost-effective and adaptable molecular methods for monitoring SARS-CoV-2 in agricultural animals.The project will develop two methods: 1. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) with solid-state nanopore sensing and 2. an amplicon-based sequencing method rhAmpSeq.This project brings together a diverse team of experts in virology, molecular biology, biomedical engineering, clinical veterinary microbiology, and veterinary diagnostics to develop and rigorously validate these methods. These proposed methods hold great promise for cost-effective, flexible, and rapid detection and genetic characterization of SARS-CoV-2. This research aligns with the American Rescue Plan (ARP) Surveillance Program's goal of developing surveillance tools for rapidly detecting and characterizing infectious agents like SARS-CoV-2.?
Objectives & Deliverables
SARS-CoV-2 has accumulated dozens of mutations during its less than three-year circulation in humans, leading to multiple variants. Differing susceptibility to SARS-CoV-2 variant infection and patterns of innate immune response to variants in respiratory cells is associated with varied disease outcomes in humans, highlighting the significance of this initial interaction between virus and host in disease pathogenesis. SARS-CoV-2 has the ability to infect many animal species, and multiple natural spillover events of captive and free-living animals have been documented. We and other research teams have showed widespread natural SARS-CoV-2 infection including Delta and Omicron variants in white tailed-deer, the most abundant large mammal species in the USA. Unlike humans, pathogenicity and fitness of recent SARS-CoV-2 variants in white-tailed deer or other livestock and companion animals have not been explored. Multiple efforts to survey SARS-CoV-2 in white-tailed deer and other wildlife have been initiated. Limited experimental studies showed differential fitness of SARS-CoV-2 variants in large animals. For example, experimental infection of white-tailed deer revealed that the alpha variant of SARS-CoV-2 outcompeted the ancient B1 lineage. Most experimental and mechanistic studies investigating SARS-CoV-2 pathogenicity in animals were performed with the earlier lineages of SARS-CoV-2 that are not currently circulating.The most recent SARS-CoV-2 variants, Delta and Omicron, and the multiple offshoots of the Omicron variant have demonstrated increased transmissibility and the ability to circumvent host defenses. Currently, the role of animals in the virus ecology and evolution and risk to animal health remain unknown; including- Is there a difference in the fitness of Delta and Omicron variants in white-tailed deer? Can the SARS-CoV-2 adaptation in deer make it more efficient to infect cattle and pigs? Have the new SARS-CoV-2 variants spilled over into cattle and pigs in the USA?The scientific premise of our proposal is that the species and tissue tropism of SARS-CoV-2 are evolving, andthe replication and innate immune profiles in primary cells in-vitro could be predictors of the viral fitness in the host.Our proposed project uses well-established primary respiratory epithelial cell culture systems, species-specific serology assays, and cutting-edge molecular biology methods, including single-cell sequencing and antigen cartography, to characterize the fitness of recent and emerging SARS-CoV-2 variants to infect deer, cattle, and pigs.The goal of the proposed project is to evaluate the risk of SARS-CoV-2 transmission from deer to cattle and pigs. Our overarching hypothesis is that the continued circulation and evolution of SARS-CoV-2 will change its pathogenicity to deer, cattle, and pigs, with the following three Objectives:Objective 1. To understand the replication fitness of recent and emerging SARS-CoV-2 variants in cattle, pig, and white-tailed deerAim: Characterize the difference in susceptibility and replication kinetics of recent and emerging SARS-CoV-2 variants in cattle, pig, and white-tailed deer respiratory cells.Investigate the comparative replication of recent and emerging SARS-CoV-2 variants in deer, cattle, and pig tracheal cells.Investigate susceptibility of cattle and pig tracheal cells to deer-adapted SARS-CoV-2.Objective 2. To investigate the innate immune response modulation by SARS-CoV-2 variants in cattle, pig and white-tailed deer respiratory cellsAim: Characterize the differences in innate immune response of cattle, pig and white-tailed deer respiratory cells infected with different SARS-CoV-2 variantsInvestigate the innate immune gene expression profiles in deer, cattle, and pig tracheal cells infected with recent and emerging SARS-CoV-2 variants.Investigate the innate immune gene expression profiles in cattle and pig tracheal cells infected with deer-adapted SARS-CoV-2.Identify viral genetic features that correspond to differential host innate immune response to SARS-CoV-2 in deer, cattle and pig tracheal cells.Objective 3. To understand the seroprevalence of SARS-CoV-2 antibodies in cattle and pigsAim: Serosurveillance to monitor SARS-CoV-2 spillover infection of cattle and pigsMonitor cattle and pig seropositivity against SARS-CoV-2 using validated indirect ELISA (iELISA).Characterize the antibody landscapes and antigenic distance to SARS-CoV-2 spike variants in seropositive cattle and pigs.To address these critical questions, we propose to use advanced molecular approaches, including well-established primary airway epithelial cell models, single cell sequencing, species-specific indirect ELISA assay, and antigen cartography, to provide the following key deliverables.• Characterize the differences in the pattern of infection and host response of white-tailed deer cells to current and emerging SARS-CoV-2 variant infection• Characterize the differences in the fitness of human- and white-tailed deer-isolated SARS-CoV-2 variants in deer, cattle, and pig• Identification of crucial host response signatures that contribute to the pathogenesis and clinical outcome of SARS-CoV-2 infection in deer, cattle, and pig• Serological identification of spillover of SARS-CoV-2 into livestock, with data-driven predictions of the responsible viral variant.
Challenges
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.
Principle Investigator(s)
Planned Completion date: 14/06/2026
Effort: $650,000.00
Project Status
ACTIVE
Principal Investigator(s)
National Institute of Food and Agriculture
Researcher Organisations
UNIV OF PITTSBURGH
United Kingdom