Middle East respiratory syndrome coronavirus quasispecies that include homologues of human isolates revealed through whole-genome analysis and virus cultured from dromedary camels in Saudi Arabia

Abstract

ABSTRACT Complete Middle East respiratory syndrome coronavirus (MERS-CoV) genome sequences were obtained from nasal swabs of dromedary camels sampled in the Kingdom of Saudi Arabia through direct analysis of nucleic acid extracts or following virus isolation in cell culture. Consensus dromedary MERS-CoV genome sequences were the same with either template source and identical to published human MERS-CoV sequences. However, in contrast to individual human cases, where only clonal genomic sequences are reported, detailed population analyses revealed the presence of more than one genomic variant in individual dromedaries. If humans are truly infected only with clonal virus populations, we must entertain a model for interspecies transmission of MERS-CoV wherein only specific genotypes are capable of passing bottleneck selection. IMPORTANCE In most cases of Middle East respiratory syndrome (MERS), the route for human infection with the causative agent, MERS coronavirus (MERS-CoV), is unknown. Antibodies to and viral nucleic acids of MERS-CoV have been found in dromedaries, suggesting the possibility that they may serve as a reservoir or vector for human infection. However, neither whole viral genomic sequence nor infectious virus has been isolated from dromedaries or other animals in Saudi Arabia. Here, we report recovery of MERS-CoV from nasal swabs of dromedaries, demonstrate that MERS-CoV whole-genome consensus sequences from dromedaries and humans are indistinguishable, and show that dromedaries can be simultaneously infected with more than one MERS-CoV. Together with data indicating widespread dromedary infection in the Kingdom of Saudi Arabia, these findings support the plausibility of a role for dromedaries in human infection.

FIG 1

(A) Real-time PCR analysis of cell culture supernatant after inoculation of Vero cells with nasal swab samples KSA-363 and KSA-378. (B) Phylogenetic analysis of MERS-CoV sequences from dromedary camels in Saudi Arabia and other genome-length MERS-CoV sequences available on 7 April 2014. GenBank accession numbers are given in parentheses for each sequence (England2 sequence is available at http://www.hpa.org.uk/Topics/InfectiousDiseases/InfectionsAZ/MERSCoV/respPartialgeneticsequenceofnovelcoronavirus/); bootstrap values of >60% indicate statistical support for the respective nodes; the scale bar indicates the number of substitutions/site. (C) (i) Clippings from the multiple MERS-CoV sequence alignment indicating sequence variation among human MERS-CoV sequences and potential variation of sequences within individual DC samples (indicated by x). (ii) Sequences obtained by direct sequencing of PCR products from the same region. (iii) Sequence analysis of individual clones generated from the PCR amplification products. Two PCRs were performed, including nt 24190 to 24300 and 24510 to 2530.

FIG 1

(A) Real-time PCR analysis of cell culture supernatant after inoculation of Vero cells with nasal swab samples KSA-363 and KSA-378. (B) Phylogenetic analysis of MERS-CoV sequences from dromedary camels in Saudi Arabia and other genome-length MERS-CoV sequences available on 7 April 2014. GenBank accession numbers are given in parentheses for each sequence (England2 sequence is available at http://www.hpa.org.uk/Topics/InfectiousDiseases/InfectionsAZ/MERSCoV/respPartialgeneticsequenceofnovelcoronavirus/); bootstrap values of >60% indicate statistical support for the respective nodes; the scale bar indicates the number of substitutions/site. (C) (i) Clippings from the multiple MERS-CoV sequence alignment indicating sequence variation among human MERS-CoV sequences and potential variation of sequences within individual DC samples (indicated by x). (ii) Sequences obtained by direct sequencing of PCR products from the same region. (iii) Sequence analysis of individual clones generated from the PCR amplification products. Two PCRs were performed, including nt 24190 to 24300 and 24510 to 2530.