Serological Detection of SARS-CoV-2 Antibodies in Naturally-Infected Mink and Other Experimentally-Infected Animals

Abstract

The recent emergence of SARS-CoV-2 in humans from a yet unidentified animal reservoir and the capacity of the virus to naturally infect pets, farmed animals and potentially wild animals has highlighted the need for serological surveillance tools. In this study, the luciferase immunoprecipitation systems (LIPS), employing the spike (S) and nucleocapsid proteins (N) of SARS-CoV-2, was used to examine the suitability of the assay for antibody detection in different animal species. Sera from SARS-CoV-2 naturally-infected mink (n = 77), SARS-CoV-2 experimentally-infected ferrets, fruit bats and hamsters and a rabbit vaccinated with a purified spike protein were examined for antibodies using the SARS-CoV-2 N and/or S proteins. From comparison with the known neutralization status of the serum samples, statistical analyses including calculation of the Spearman rank-order-correlation coefficient and Cohen's kappa agreement were used to interpret the antibody results and diagnostic performance. The LIPS immunoassay robustly detected the presence of viral antibodies in naturally infected SARS-CoV-2 mink, experimentally infected ferrets, fruit bats and hamsters as well as in an immunized rabbit. For the SARS-CoV-2-LIPS-S assay, there was a good level of discrimination between the positive and negative samples for each of the five species tested with 100% agreement with the virus neutralization results. In contrast, the SARS-CoV-2-LIPS-N assay did not consistently differentiate between SARS-CoV-2 positive and negative sera. This study demonstrates the suitability of the SARS-CoV-2-LIPS-S assay for the sero-surveillance of SARS-CoV-2 infection in a range of animal species.

Keywords: SARS-CoV-2; animal; luciferase immunoprecipitation systems; mink; nucleocapsid; sera; spike.

Figure 1

Boxplot showing the differences between the LIPS-S negative and positive samples and the relative similar levels of antibodies found in the negative samples across species.

Figure 2

Distribution of the LIPS-S assay antibody values based of the sample’s known SARS-CoV-2 antibody neutralization status. The RLU determined by LIPS is shown on the Y-axis. Of note, all the negative samples are located below the blue threshold line of mean plus three standard deviations. A threshold line of mean plus five standard deviations (red dotted line) is also included for reference.

Figure 3

Distribution of the LIPS-N assay RLU values based of the sample’s SARS-CoV-2 antibody neutralization Note that all true negative samples are located below the blue threshold line of mean plus three standard deviations. A threshold line of mean plus five standard deviations (red dotted line) is also included for reference.

Figure 4

Distribution of RLU values determined by LIPS-S assay using sera from naturally-infected mink samples across the lower threshold line (blue) and according to the serum collection date. Note that, independent of the serum collection times, the LIPS-S assay correctly identified all samples according to their positive and negative status as determine by antibody neutralization activity.

Figure 5

Distribution of RLU values generated using the sera from naturally SARS-CoV-2-infected mink samples across the threshold line for the LIPS-N assay according to the serum collection date. Note that the RLU values generated from NT positive sera declined over time using LIPS-N and an increased number of NT positive sera fall below the threshold line.