MERS-CoV nucleic acid screening
Nasal swabs of sheep (n=55) and goats (n= 45), as well as serum samples
of all animals (n=152) were screened for MERS-CoV nucleic acid by
reverse transcription real-time (RT-q)PCR. For this, 200 µl of each
sample were subjected to automated nucleic acid extraction employing
QIAamp Viral RNA Mini Kit on QIAcube HT (both Qiagen, Hilden, Germany).
MERS-CoV-specific RT-qPCR was performed using qScript XLT One Step
RT-qPCR ToughMix (QuantaBio, MA, USA) and previously described primers
and probe in the open reading frame (ORF ) 1a gene .
RT-qPCR-positive samples were additionally tested by conventional
RT-PCRs (OneStep RT-PCR Kit, Qiagen, Hilden, Germany) employing two
previously published primer pairs within the nucleocapsid (N ) andORF1b genomic regions of MERS-CoV . Amplicons from the RT-PCRs
were subjected to Sanger sequencing (Eurofins Genomics, Ebersberg,
Germany). In addition, next generation sequencing (NGS) was performed on
putative positive samples and five selected MERS-CoV-positive samples
from dromedaries in nearby pens. For this, adapter sequences were
trimmed using bcl2fastq2 conversion software v2.20.0, and high-quality
sequencing reads were aligned to three reference MERS-CoV genome
sequences (GenBank acc. numbers KF192507, MG757604 and MK462253) using
BWA v0.7.17. Consensus sequences were generated for the N gene
using GATK v3.8–1–0 and BCFtools v1.3.1. Multiple sequence alignment
of the N gene of MERS-CoV (1.2 kb) derived from one sheep and
five dromedary camels was performed using MAFFT v7.455.