Source: PENNSYLVANIA STATE UNIVERSITY submitted to
SUSCEPTIBILITY AND THE POTENTIAL ADAPTATION OF SARS-COV-2 IN LIVESTOCK
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
EXTENDED
Funding Source
Reporting Frequency
Annual
Accession No.
1023601
Grant No.
2020-67015-32175
Project No.
PENW-2020-05957
Proposal No.
2020-05957
Multistate No.
(N/A)
Program Code
A1711
Project Start Date
Jul 15, 2020
Project End Date
Jul 14, 2023
Grant Year
2020
Project Director
Kuchipudi, S. V.
Recipient Organization
PENNSYLVANIA STATE UNIVERSITY
408 Old Main
UNIVERSITY PARK,PA 16802-1505
Performing Department
Veterinary and Biomedical Scie
Non Technical Summary
The COVID-19 pandemic has dispersed a new strain of coronavirus (called SARS-CoV-2) throughout the world. During the rapid global spread, the new virus continues to mutate and there are now numerous genetic versions of SARS-CoV-2. The U.S. livestock sector plays an indispensable role in the safe and reliable food supply, employment, and economic development, so it is critical to understand if SARS-CoV-2 viruses can pose a threat to livestock. The goals of this project are to investigate the susceptibility of livestock to SARS-CoV-2 and to determine if the virus may adapt and efficiently spread among livestock. The research will use a combination of experimental infection studies using cell cultures and animals along with computer models to assess the chance for the virus to efficiently infect livestock species. Additionally, the project will develop diagnostic tests and use them to monitor the presence of antibodies to SARS-CoV-2 in livestock animals. Chickens, cattle, and pigs are major animal agriculture species in the U.S., and through this project, a better understanding of how SARS-CoV-2 may affect them will be gained.
Animal Health Component
100%
Research Effort Categories
Basic
25%
Applied
75%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
3114030110150%
3114030104030%
3114030117020%
Knowledge Area
311 - Animal Diseases;

Subject Of Investigation
4030 - Viruses;

Field Of Science
1040 - Molecular biology; 1101 - Virology; 1170 - Epidemiology;
Goals / Objectives
1). To determine the susceptibility of livestock to SARS-CoV-2 in a BSL3 environment.2). To investigate the potential of transmission of SARS-CoV-2 to livestock and the likely adaptation in animals.
Project Methods
All experiments with live infectious SARS-CoV-2 will be conducted in our animal biosafety level 3 (ABSL-3) facility with appropriate respiratory protection and barrier clothing procedures for personnel.Aim 1: Determine the susceptibility of livestock to SARS-CoV-21a: Investigate the comparative replication of Asian, European and North American SARS-CoV-2 isolates in livestock primary respiratory cells.:We will initially determine the comparative replication ability of three different isolates of SARS-CoV-2 from Asia, Europe, and North America (strains Hong Kong/VM20001061/2020, Italy-INMI1, and USA-WA1/2020). To simulate the real-life settings, we will initially grow the SARS-CoV-2 isolates in human respiratory cells before infecting animal cells. At 6, 12 and 24 hpi, we will fix the cells and immunostain for SARS-CoV-2 viral protein expression. We will also determine the replication kinetics of SARS-CoV-2 in avian, porcine, and bovine cells compared with human cells. Cells will be infected with an MOI of 0.1 or 1.0, and virus production at 12, 24, 48, and 72 hpi will be evaluated by fifty-percent tissue-culture infective dose (TCID50) and RT-PCR. The amount of SARS-CoV-2 protein and virus production will be compared to determine the comparative ability of different strains of SARS-CoV-2 to replicate in livestock cells.1b: Susceptibility of chicken to experimental SARS-CoV-2 infection:We will carry out infection studies in chickens with a SARS-CoV2 isolate that we find to better replicate in chicken cells (Aim 1a). We will investigate susceptibility to experimental SARS-CoV2 infection in two age groups of SPF chicken. We will document the pattern of clinical disease, virus shedding, and seroconversion.Aim 2: To investigate the potential of transmission of SARS-CoV-2 to livestock and the likely adaptation in animals2a: Prevalence of SARS-CoV-2 in poultry, cattle and pigs: As part of this aim, we will raise SARS-CoV-2 specific antisera in chickens, pigs and cattle that will serve as positive control reagents for the indirect ELISA and Luciferase Immunoprecipitation System (LIPS) serological assays. We will monitor the presence of SARS-CoV-2 specific antibody in poultry, cattle and pigs using these two assays using test serum samples submitted for routine diagnosis and surveillance programs (for example Avian Influenza, PA Bull Test sales, etc.) to the Pennsylvania Animal Diagnostic Lab System (PADLS) and networked laboratories.2b. Establishing the mutational landscape in SARS-CoV-2 quasispecies from chicken, pig andbovine cells:We will passage the SARS-CoV-2 in chicken, pig, and cattle primary cells three times, and cell culture supernatants collected from the three passages will be used to analyze the viral quasispecies by Next Generation Sequencing (NGS).2c: Investigate the potential for SARS-CoV-2 adaptation in livestock:? Expanding on our preliminary binding energy analysis, we will use molecular modeling to perform in silico mutation analysis of the SARS-CoV-2 S protein RBD and the resulting effect on Rosetta binding energy. Each residue will be computationally mutated to all possible alternatives to identify the maximum improvement in the binding energy of ACE-2 with SARS-CoV-2 S protein upon a single residue change. Next, the consequences of consecutive mutations (second residue, third residue, etc.) will be explored. Binding energy analysis will also be applied to the mutations observed in quasispecies identified through sequencing in Aim 2b. All calculations will be performed using resources at the Penn State Institute for Cyberscience. Finally, the RBD configurations determined to computationally be best adapted to each species will be experimentally tested. The ideal RBD and spike protein sequence candidates will be expressed as proteins and used to perform binding assays on respiratory and gastrointestinal tissues from chicken, pig, and cattle.

Progress 07/15/20 to 07/14/21

Outputs
Target Audience:Computation model for prediction of the impact of amino acid changes in SARS-CoV-2 spike protein on the infectivity of the virus has been published in the Proceedings of the National Academy of Sciences (PNS) journal to disseminate the results tothe wider scientific community. A new article was also published through Penn State News to disseminate the information to the broader general public. https://www.pnas.org/content/118/42/e2106480118 https://news.psu.edu/story/670936/2021/09/29/research/new-tool-predicts-changes-may-make-covid-variants-more-infectious Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Three graduate students, two Postdoctoral Scholars a research associate, and four undergraduate researchers worked closely with the project under the direct supervision of the PI and Co-PIs. As part of the training, all the graduate students and a postdoc got proficient working in a BSL-3 facility. Undergraduate students were trained on developing and validating serological assays. Sequencing, protein prediction, and bioinformatics workflows were developed and standardized. How have the results been disseminated to communities of interest?The sero-surveillnace results will be presented at the 2021 annual meeting of the Conference of Research Workers in Animal Diseases (CRWAD): "Development and validation of serological assays to detect the spillover of SARS-CoV-2 in livestock". CRWAD is one of the oldest and largest international research conferences featuring cutting-edge research on animal health and disease, population health, and translational medicine to veterinarians and animal health scientists. Manuscript published in PNAS:Chen, Chen, et al. "Computational prediction of the effect of amino acid changes on the binding affinity between SARS-CoV-2 spike RBD and human ACE2." Proceedings of the National Academy of Sciences 118.42 (2021). Results are shared with extension veterinarians in the team who connects regularly with the livestock farmers in Pennsylvania. What do you plan to do during the next reporting period to accomplish the goals? Currently, we are working on developing a computational model that can predict potential SARS-CoV-2 variants that increase the infection susceptibility of livestock animals like pigs, cattle, chickens, and deer. The novel computational tool (CNN_seq) is based on a convolutional neural network and takes only the sequence of the complex formed by ACE2 and RBD as the input, which considerably accelerates training and prediction processes. This enhancement in efficiency allows the use of a much-expanded dataset covering 8440 variants with single and multiple AA changes on either ACE2 or RBD proteins, leading to an accuracy of 83.28% and a correlation coefficient r of 0.85 for the obtained model. The CNN_seq model can be transferred to the animal case by combining the sequence of animal ACE2 with RBD and scanning on possible combinations of up to 4 AA changes on RBD to identify the variants that would potentially increase the ACE2-RBD binding affinity for these livestock animals. We plan to investigate the reason for replication inhibition of SARS-CoV-2 virus in cattle, pig and chicken primary epithelial cells through strand-specific RT-PCR of cellular contents of infected cells and transcriptomics (RNASeq) to identify host and/or viral factors that limit virus multiplication in these cells. We will further explore our predicted binding activities of SARS-CoV-2 variants in experimental settings by expressing bovine, porcine, or chicken ACE2 in BHK-21 cells. Infection studies in chickens with SARS-CoV2 will be attempted as we gain a deeper understanding of the molecular basics of transmission dynamics of SARS-CoV-2 isolates in chicken primary epithelial cells. Development and validation of serological assays for understanding the spillover events in several wildlife and livestock species are currently underway. A computational model that can predict potential SARS-CoV-2 variants that increase the infection susceptibility of livestock animals like pigs, cattle, chickens and deer will be developed. Two manuscripts are under preparation. They will be completed and submitted to peer-reviewed journals of high impact in the field of infectious diseases.

Impacts
What was accomplished under these goals? 1. Determine the susceptibility of livestock to SARS-CoV-2 1a: Investigate the comparative replication of SARS-CoV-2 isolates in livestock primary respiratory cells Culture conditions for successfully growing primary cattle, pig, and chicken cells were established. Multiple attempts were made to culture SARS-CoV-2 isolates on human primary tracheal/bronchial epithelial cells. While all isolates could be cultured, the growth of all isolates in this cell type was low. To obtain high titers of human-adapted SARS-CoV-2 for subsequent experiments, a human lung epithelial cell line overexpressing human cell receptor Angiotensin-converting enzyme 2 (A549-hACE2) was used instead of the primary human cells. Each isolate was passaged three times on A549-hACE2 cells. Samples from each passage were sequenced to monitor changes to the virus as it adapted to the human cells. Bovine and porcine primary tracheal epithelial cells and chicken primary lung cells were inoculated at a multiplicity of infection (MOI) 0.1 with the indicated human-cell-adapted isolates of SARS-CoV-2 and grown for up to 144 hours. A negligible increase in viral RNA was observed in the supernatant, and human A549-hACE2 cells infected simultaneously produced a minimum of 20-fold RNA increase over the same period. In addition, all samples collected from the bovine, porcine, and chicken cells demonstrated no competent viral production, with Tissue Culture Infectious Dose 50 (TCID50) results indicating viral levels ≤ 10 TCID50/mL. Overall, none of the animal cells were susceptible to the human-adapted SARS-CoV-2. In addition, embryonated chicken eggs also failed to produce any viable SARS-CoV-2 or increased viral RNA. The three SARS-CoV-2 isolates (Hong Kong/VM20001061/2020, Italy-INMI1, and USA-WA1/2020) used in these experiments were wild-type (Wuhan) lineage. Since 2020, several distinct genetic lineages have been described, with each variant type possessing unique mutations in spike protein and other viral proteins. Our computational predictions suggested that some of these spike variants may improve or reduce the binding affinity of SARS-CoV-2 to bovine, porcine, and chicken ACE2. We decided to explore if altered binding affinity might change the ability of SARS-CoV-2 to grow in these animal cells, starting with primary bovine tracheal epithelial cells. Representative isolates of wild-type, alpha, beta, and gamma lineages were used to infect cells at MOI 1. In this case, viruses were not the first human-cell-adapted. As observed before with the human-adapted viruses, little to no viral RNA was released into the supernatant of infected cells. Concurrently, no, or negligible increase in live virus as measured by TCID50 was observed following infection with any of the variants, and the live virus was near or below the limit of detection by 72 hours post-infection. Taken together, it has been observed that bovine - possibly chicken and porcine - primary respiratory cells are not susceptible to SARS-CoV-2. We plan to investigate the reason for this finding through strand-specific RT-PCR of cellular contents of infected cells and transcriptomics (RNASeq) to identify host and/or viral factors that limit SARS-CoV-2 in these cells. We will further explore our predicted binding activities of SARS-CoV-2 variants in experimental settings by expressing bovine, porcine or chicken ACE2 in BHK-21 cells. 1b: Susceptibility of chicken to experimental SARS-CoV-2 infection. The infection studies in chickens with a SARS-CoV2 described under objective 2a is currently underway as we are attempting to understand the colonization and transmission dynamics of SARS-CoV-2 isolates in chicken primary epithelial cells. 2.a. Seroprevalence of SARS-CoV-2 antibodies in domestic livestock In this aim, we developed and validated serological assays, including an indirect ELISA and Luciferase Immunoprecipitation System (LIPS) Assay for screening cattle, pigs, and chickens. Serum samples collected before December 2019 (pre-pandemic) and from 01/2020 through 10/2021(post-pandemic) from the PADLS and our network of animal diagnostic laboratories across the US were screened for the presence of anti-SARS-CoV-2 antibodies. These specimens included samples from unhealthy animals that are being investigated for disease and samples from healthy animals as part of regulatory or other surveillance efforts by state and local entities. Hyperimmune serum comprising polyclonal anti- SARS-CoV-2 antibodies were generated in chicken, pigs, and cattle as reference controls for validation and implementation of ELISA and LIPS assays. 2b. Establishing the mutational landscape in SARS-CoV-2 quasispecies from chicken, pig, andbovine cells. We passaged three different isolates of SARS-CoV-2 from Asia, Europe, and North America (strains Hong Kong/VM20001061/2020, Italy-INMI1, and USA-WA1/2020) in pig three times, and cell culture supernatants collected from the three passages will be used to analyze the viral genome mutations by Next Generation Sequencing (NGS). Although we observed several single nucleotide polymorphisms (SNPs) throughout the genomes, significant ones favoring or worsening binding of the spike protein to the host cell surface receptor or the downstream colonization was not observed using the computational prediction models. 2c. Investigate the potential for SARS-CoV-2 adaptation in livestock Using Monte Carlo simulations and biophysical energetic analyses of the SARS-CoV-1 spike protein and SARS-CoV-2 spike protein with human ACE2 proteins, we deciphered the interactions that govern the higher infectivity of SARS-CoV-2 compared to SARS-CoV-1.1 We further used molecular dynamics simulations to delineate the plausible interaction mechanism of human ACE2 protein with its membrane-bound partner protein ATR1. The association of the receptor-binding domain (RBD) of SARS-CoV-2 viral spike with a human angiotensin-converting enzyme (hACE2) represents the first required step for viral entry. Amino acid changes in the RBD have been implicated with increased infectivity and the potential for immune evasion. Hence, reliably predicting the effect of amino acid changes in the ability of the RBD to interact more strongly with the hACE2 receptor can help assess the public health implications and the potential for spillover and adaptation into other animals. We developed a two-step framework that first relies on 48 independent 4-ns molecular dynamics (MD) trajectories of RBD-hACE2 variants to collect binding energy terms.2 The second step implements a neural network to classify and quantitatively predict binding affinity using the decomposed energy terms as descriptors. We achieved an accuracy of 82.2% in terms of correctly classifying single amino-acid substitution variants of the RBD as worsening or improving binding affinity for hACE2 and a correlation coefficient r of 0.69 between predicted and experimentally calculated binding affinities. Both metrics are calculated using a 5-fold cross-validation test. Our method thus sets up a framework for effectively screening binding affinity change with unknown single and multiple amino-acid changes. This can be a valuable tool for predicting host adaptation and zoonotic spillover of current and future SARS-CoV-2 variants.

Publications

  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Computational prediction of the effect of amino acid changes on the binding affinity between SARS-CoV-2 spike RBD and human ACE2 Chen Chen, Veda Sheersh Boorla, Deepro Banerjee, Ratul Chowdhury, Victoria S. Cavener, Ruth H. Nissly, Abhinay Gontu, Nina R. Boyle, Kurt Vandegrift, Meera Surendran Nair, Suresh V. Kuchipudi, Costas D. Maranas Proceedings of the National Academy of Sciences Oct 2021, 118 (42) e2106480118; DOI: 10.1073/pnas.2106480118