Coronavirus antibodies in African bat species

Abstract

Asian bats have been identified as potential reservoir hosts of coronaviruses associated with severe acute respiratory syndrome (SARS-CoV). We detected antibody reactive with SARS-CoV antigen in 47 (6.7%) of 705 bat serum specimens comprising 26 species collected in Africa; thus, African bats may harbor agents related to putative group 4 CoV.

Figure 1

Results of Western blot analysis with recombinant severe acute respiratory syndrome–associated coronavirus (SARS-CoV) nucleocapsid (N) and spike (S) protein. Shown are examples for SARS-CoV ELISA–positive (2, 17, 26, 31) and –negative (38, 321) bat serum specimens tested using full-length recombinant SARS-CoV N and a fragment of the S protein (amino acids 318–510). Serum specimens were diluted 1:2,500 (left strips) and 1:5,000 (right strips). Secondary detection was performed by incubating the nitrocellulose strips with horseradish peroxidase (HRP)–labeled goat-antibat immunoglobulin (Ig) (Bethyl, Montgomery, AL, USA) (1:10,000). For chemiluminescence, SuperSignal Dura substrate (Pierce, Rockford, IL, USA) was added and films exposed for 1 min. Serum 17* was used as a reference for comparing blots. For evaluation purposes, strips were also incubated with human SARS-CoV–positive (A, B) and –negative serum specimens C and D (HCoV-NL63 positive) at the same dilutions, using goat-antihuman Ig HRP (1:20,000) for secondary detection. Serum specimens that produced signals at a dilution of 1:5,000 were recorded as positive (+).

Figure 2

Results of indirect immunofluorescence (IF) test with Vero E6 cells infected with severe acute respiratory syndrome–associated coronavirus (SARS-CoV). The SARS-CoV diagnostic IIFT kit (EUROIMMUN AG, Lübeck, Germany) was used with minor modifications: bat and reference human serum specimens were diluted 1:100 (found to be the optimal dilution for bat sera) in sample buffer, and secondary detection was performed with goat-antibat immunoglobulin (Ig) (Bethyl, Montgomery, AL, USA) followed by fluorescein isothiocyanate (FITC)–labeled donkey-antigoat Ig (Dianova, Hamburg, Germany) (A–F) or FITC-labeled goat-antihuman Ig (G–I). Frames A–D, SARS-CoV ELISA–positive bat serum specimens 2, 17, 26, 31; E–F, ELISA-negative bat serum specimens 38 (showing unspecific signals) and 306; G–H, SARS-CoV–positive human control serum specimens A and B; I, negative human serum C. All photographs were taken at equivalent microscope settings. Scale bars represent 20 μm.