Middle East Respiratory Syndrome Coronavirus in Bats, Saudi Arabia

Ziad A. Memish; Nischay Mishra, Kevin J. Olival; Shamsudeen F. Fagbo; Vishal Kapoor; Jonathan H. Epstein; Rafat AlHakeem; Abdulkareem Durosinloun; Mushabab Al Asmari; Ariful Islam; Amit Kapoor; Thomas Briese; Peter Daszak; Abdullah A. Al Rabeeah; W. Ian Lipkin

Disclosures

Emerging Infectious Diseases. 2013;19(11):1819-1823. 

In This Article

The Study

In October 2012 and April 2013, three agencies collected samples from bats in regions where MERS cases had been identified (Figure 1). The agencies are the Ministry of Health of Saudi Arabia, the Center for Infection and Immunity of Columbia University, and EcoHealth Alliance.

Figure 1.

Bat sampling sites and locations of home and workplace of index case-patient with Middle East respiratory syndrome, Bisha, Saudi Arabia.

During the October investigation, the team interviewed the family of an index case-patient in Bisha and collected samples from bats <12 km from his home, in an abandoned date palm orchard, and <1 km from his place of employment, a hardware store that fronted a garden and date palm orchard. Although neither family members nor employees recalled seeing bats, the team observed roosting bats and guano in abandoned wells and ruins within 12 km of his home and insectivorous bats at dusk in the garden behind his store. Over 3 weeks, 96 bats representing 7 species (Rhinopoma hardwickii, Rhinopoma microphyllum, Taphozous perforatus, Pipistrellus kuhlii, Eptesicus bottae, Eidolon helvum, and Rosettus aegyptiacus) were captured in mist nets and harp traps, then released after visual speciation and collection of morphometric measurements; wing punch biopsy samples; blood; throat swab samples; and rectal swab samples or fecal pellets. Samples were collected into viral transport medium or lysis buffer.

During the 3-week April investigation, fecal samples were collected on tarps laid out at bat roosting sites in and around Bisha, Unaizah, and Riyadh. Representative animals at each roosting site were captured, identified morphologically, and released after wing punch biopsy samples were collected for speciation by DNA analysis. Samples were collected into cryovials.

All samples were stored in liquid nitrogen and conveyed to Riyadh for storage at −80°C before being transported to Columbia University in New York in dry nitrogen. The October 2012 shipment was inadvertently opened at customs in the United States and sat at room temperature for 48 hours before transfer to Columbia University; at arrival, all samples had thawed. The April 2013 samples arrived intact.

Total nucleic acid was extracted from samples by using the NucliSENS easyMAG system (bioMérieux, Durham, NC, USA) and subjected to 8 PCRs with primers and protocols designed to amplify regions within the helicase, RNA-dependent RNA polymerase (RdRp), and nucleocapsid or envelope proteins of CoVs.[6–9] Products were sequenced and analyzed for similarity to GenBank database entries by using the BLASTn and BLASTx programs (http://www.ncbi.nlm.nih.gov/blast/Blast.cgi). Primer sequences are shown in Table 1. The identity of bat species yielding specific viral products was determined by amplifying and sequencing a fragment of the cytochrome B gene.[10] All visual classifications of species were confirmed except for that of T. perforatus bats. There is no reference sequence for T. perforatus bats in GenBank. However, because the closest reference sequence was from T. nudiventris bats, at 84% identity we presume that the product represents bona fide T. perforatus bat cytochrome B gene sequence. Representative cytochrome B sequences have been uploaded to GenBank (accession nos. KF498635–KF498641).

Table 1 indicates the CoV genera identified by using individual primer sets. As anticipated, pan-CoV assays detected α- and β-CoVs. One assay specific for MERS CoV[9] also detected α-CoVs. This finding reinforces the need for sequence confirmation of PCR products. Table 2 indicates the CoV species identified with respect to location, sample type, and bat species. CoV sequences were amplified from rectal swab samples or fecal pellets and from roost feces but not from serum, throat swab samples, or urine. Alpha CoV sequences were amplified more frequently than β-CoV sequences (223 vs 4). Whereas α- and β-CoV sequences were amplified from CoVs from T. perforatus, E. helvum, and R. hardwickii bats, only alpha sequences were amplified from CoVs from P. kuhlii bat samples.

CoV sequences were amplified from 220 of 732 roost feces samples and 7 of 91 rectal swab samples or fecal pellets. A product obtained by PCR amplification of nucleic acid from a fecal pellet of a T. perforatus bat captured in October 2012 in Bisha showed 100% nt identity to the human β-CoV 2c EMC/2012 cloned from the index case-patient in Bisha. A phylogenetic analysis of CoVs obtained in this study is shown in Figure 2. CoV sequences have been uploaded in GenBank (accession nos. KF493884–KF493888).

Figure 2.

Phylogenetic tree showing genetic relatedness between coronaviruses identified in bat samples from Saudi Arabia (boldface), MERS coronaviruses, and other published coronavirus sequences available in GenBank. The maximum-likelihood tree of…

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