MERS-CoV nucleic acid and antibody prevalence
The nasal swab of one sheep (1.8 %) repeatedly tested positive for MERS-CoV nucleic acid by RT-qPCR (Ct values 32.5-34.8; Table 1), and the nasal swab of one goat tested borderline positive (Ct values 38.1-39.4; Table 1). The positive sheep was a two-year-old female, presumably locally raised Nuaimi breed. It was kept in a pen at the market used for sheep only, which was the second closest pen to the camel area (approximately 25 m away; Fig. 1, Supplementary Table 1). The borderline positive goat, a one-year-old locally raised male Omani goat, was kept in one of the pens directly adjacent to the camel pens, with only approximately 20 m between them (Fig. 1, Supplementary Table 1).
The RT-qPCR-positive sheep sample was confirmed by two different RT-PCRs. The obtained sequences [lengths without primer sequences:N – 293 bp (GenBank acc. number: MZ558082), ORF1b – 341 bp (GenBank acc. number: MZ558083)] were >99 % identical to many MERS-CoV sequences belonging to clade B, lineage 5 [according to the classification by , including human and camel strains from the UAE, Saudi Arabia, South Korea, Jordan, Qatar, and the United Kingdom. Unfortunately, we were not able to uncover the complete MERS-CoV genomes using NGS; however we succeeded to establish the complete N gene sequences of the virus-positive sheep and five selected MERS-CoV-positive dromedaries (Figure 2). The sheep-derivedN gene sequences obtained by NGS and Sanger sequencing were identical. By NGS, the average sequencing coverage of the N gene from the sheep (S90_N) and five camel samples (C53_N, C5_N, C6_N, C28_N, and C50_N) was >6X across all samples (Figure 2A). The percentage of sequence identity of the N gene of the same samples along with three reference MERS-CoV sequences (GenBank acc. numbers KF192507, MG757604 and MK462253) revealed that the sheep sample showed high nucleotide identity (>99 %) with all other MERS-CoV sequences (Figure 2B). In detail, multiple sequence alignment revealed five positions of nucleotide variations between the sheep-derived MERS-CoV sequence and the camel-derived sequences and/or reference sequences (representative positions 533 and 637 of theN gene are shown in Figure 2C). The GenBank accession numbers of the complete MERS-CoV N gene sequences are: sheep-derived (S90_N): MZ558076; dromedary-derived: C53_N - MZ558080; C5_N - MZ558077; C6_N - MZ558078; C28_N - MZ558079; and C50_N - MZ558081. Unfortunately, the borderline-positive result of the goat sample could not be confirmed by either RT-PCR or NGS.
The serological survey revealed that, although in the initial screening with 1:40 dilutions 15 serum samples (eight sheep, four goats, three cattle) appeared questionable, the presence of antibodies could not be confirmed in any sample by retesting using a dilution series of 1:10 – 1:320 (Supplementary Tables 1 and 2).