Despite only a few human-to-animal transmissions of severe acute respiratory syndrome coronavirus 2 (SARS‑CoV‑2) that were initially reported, several animal species have been found to be susceptible to SARS-CoV-2. As a result, the World Organization for Animal Health has defined COVID-19 as an Emerging Disease in animals.
Study: Detailed epitope mapping of SARS-CoV-2 nucleoprotein reveals specific immunoresponse in cats and dogs housed with COVID-19 patients. Image Credit: Ermolaev Alexander / Shutterstock.com
Previously, a novel immunoassay was developed and based on paramagnetic beads coated with recombinant SARS-CoV-2 nucleoprotein (Np) and a flow cytometry-based system. This assay was used to screen both samples collected from both cats and dogs during both the pre-and current pandemic era to determine the susceptibility of these domesticated animals to SARS-CoV-2.
The tests with this assay demonstrated that both the SARS-CoV-2 spike (S) and nucleocapsid (N) proteins are equally sensitive, especially in the early phase of infection. Furthermore, the use of neutralization assays like the plaque reduction neutralization test (PRNT) could also function as reference standards to confirm positive results for both N- and S-based serological assays.
About the study
In a recent Research in Veterinary Science study, the researchers conducted a detailed epitope mapping of Np and developed an experimental double antigen-based enzyme-linked immunosorbent assay (ELISA) using horseradish peroxidase (HRP)-conjugate C-terminal subunit.
In this study, nucleotide sequences for the SARS-CoV-2 N protein and that isolated from other animal-associated coronaviruses, as well as SARS-CoV-1 and the Middle East Respiratory Syndrome Coronavirus (MERS-CoV), were obtained from GenBank for sequence alignments. In addition, human serum samples were collected from 23 patients with COVID-19, which consisted of 13 males and 10 females from March 2020 to November 2020.
Overall, 506 animal sera were collected during the same time period. Among these samples, 15 animals were positive for SARS-CoV-2. Notably, all animal sera were tested with double antigen ELISA and PRNT tests.
A set of pre-pandemic sera from 234 dogs and 35 cats was used to assess the specificity of the Np double antigen assay. Three sera from cats that were previously positive for Feline Coronavirus, three dog sera that were positive for Canine Respiratory Coronavirus, five dog sera that were positive for Canine Enteric Coronavirus, and eight bovine sera that were positive for Bovine Coronavirus, were also tested.
The researchers also tested a hyperimmune serum generated in a goat that was repeatedly immunized with the whole recombinant Np that was expressed in E. coli as their positive control.
Overall, nine subunits were expressed in the prokaryotic system. Serum panel from human patients was reactive mainly against the C-terminal half of Np with a lower degree of reactivity. This finding indicates that the immunodominant region of Np is located between D and E fragments, potentially overlapping with few residues upstream and downstream.
Sera from a dog hospitalized for interstitial pneumonia was compared to a set of sera from pet animals housed with COVID-19 patients. These samples were tested against the same nine subunits ELISA and were found to be highly reactive towards the same overlapping fragment (D + E) recognized by human sera. Once this immunodominant region of D+E was identified, the double antigen ELISA was developed to detect immunoglobulins (Igs) isolated from multiple species.
It was also noted that sera from the pre-pandemic era, as well as those obtained from animals that were naturally infected with different coronaviruses, were well below the cut-off value for the double antigen ELISA test. However, three dog sera out of 278 were found to be reactive by double antigen ELISA. Among these samples, one serum elicited a high reactivity against the Canine Respiratory Coronavirus (CRCoV) antigen, while the other was completely negative to CRCoV and Canine Enteric coronavirus antigens.
Taken together, the current study found that epitope mapping of SARS-CoV-2 Np led to the identification of the immunodominant region located in the C-terminal portion of the protein. A double antigen-based ELISA using this portion as HRP conjugate enhances serological testing of various animals and could therefore be a useful tool for multispecies monitoring of COVID-19 in susceptible animals.
- Colitti, B., Bonfante, F., Grazioli, S., et al. (2022). Detailed epitope mapping of SARS-CoV-2 nucleoprotein reveals specific immunoresponse in cats and dogs housed with COVID-19 patients. Research In Veterinary Science. doi:10.1016/j.rvsc.2021.12.020.