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Further Evidence for Bats as the Evolutionary Source of Middle East Respiratory Syndrome Coronavirus


Further Evidence for Bats as the Evolutionary Source of Middle East Respiratory Syndrome Coronavirus

S J Anthony et al. mBio.


The evolutionary origins of Middle East respiratory syndrome (MERS) coronavirus (MERS-CoV) are unknown. Current evidence suggests that insectivorous bats are likely to be the original source, as several 2c CoVs have been described from various species in the family Vespertilionidae Here, we describe a MERS-like CoV identified from a Pipistrellus cf. hesperidus bat sampled in Uganda (strain PREDICT/PDF-2180), further supporting the hypothesis that bats are the evolutionary source of MERS-CoV. Phylogenetic analysis showed that PREDICT/PDF-2180 is closely related to MERS-CoV across much of its genome, consistent with a common ancestry; however, the spike protein was highly divergent (46% amino acid identity), suggesting that the two viruses may have different receptor binding properties. Indeed, several amino acid substitutions were identified in key binding residues that were predicted to block PREDICT/PDF-2180 from attaching to the MERS-CoV DPP4 receptor. To experimentally test this hypothesis, an infectious MERS-CoV clone expressing the PREDICT/PDF-2180 spike protein was generated. Recombinant viruses derived from the clone were replication competent but unable to spread and establish new infections in Vero cells or primary human airway epithelial cells. Our findings suggest that PREDICT/PDF-2180 is unlikely to pose a zoonotic threat. Recombination in the S1 subunit of the spike gene was identified as the primary mechanism driving variation in the spike phenotype and was likely one of the critical steps in the evolution and emergence of MERS-CoV in humans.IMPORTANCE Global surveillance efforts for undiscovered viruses are an important component of pandemic prevention initiatives. These surveys can be useful for finding novel viruses and for gaining insights into the ecological and evolutionary factors driving viral diversity; however, finding a viral sequence is not sufficient to determine whether it can infect people (i.e., poses a zoonotic threat). Here, we investigated the specific zoonotic risk of a MERS-like coronavirus (PREDICT/PDF-2180) identified in a bat from Uganda and showed that, despite being closely related to MERS-CoV, it is unlikely to pose a threat to humans. We suggest that this approach constitutes an appropriate strategy for beginning to determine the zoonotic potential of wildlife viruses. By showing that PREDICT/PDF-2180 does not infect cells that express the functional receptor for MERS-CoV, we further show that recombination was likely to be the critical step that allowed MERS to emerge in humans.

Keywords: MERS coronavirus; Uganda; bat; spike; zoonoses.


Map showing the distribution of Pipistrellus hesperidus (based on International Union for Conservation of Nature [IUCN] data) and the location of the bat sampled for the study.
PREDICT/PDF-2180 and NeoCoV are ancestral to MERS-CoV. Maximum likelihood phylogenetic reconstructions of 2c coronaviruses (nucleotide) show that PREDICT/PDF-2180 and NeoCoV are consistently basal to, or form sister clades with, MERS-like CoV (human/camel strains), except in subunit 1 of the spike protein. Human OC43 is the outgroup. All genes were shown to be under purifying selection (ω).
The spike protein of PREDICT/PDF-2180 is highly divergent. (A) A nucleotide identity Simplot shows that PREDICT/PDF-2180 and NeoCoV are closely related to MERS-CoV across much of the genome but are highly divergent in subunit 1 of the spike protein, suggesting that they may have different receptor binding properties. (B) Variation in key amino acid binding residues (*) and modeling to human DPP4 both suggest that PREDICT/PDF-2180 is unable to bind to DPP4.
Uganda spike protein does not permit entry into Vero cells. (A) Genome organization of MERS-CoV encoding the Uganda spike glycoprotein. (Bi) Reverse transcription-PCR detection of leader-containing nested subgenomic mRNAs encoding the nucleocapsid transcript, E transcript, and ORF5 and ORF4a transcripts (p0, RNA-transfected cells; p1, passage 1; p2, passage 2). (Bii) Reverse transcription-PCR amplification of leader-containing mRNA 2 containing the Uganda S gene. Note the loss of the leader-containing transcripts in p1 and p2, demonstrating the loss of infectivity associated with insertion of the Uganda S gene. Ladder, 1 kb.
PDF-2180 spike unable to mediate infection of primary human airway cultures. (A) Primary human airway epithelial (HAE) cells grown on an air-liquid interface were infected with wild-type MERS-CoV (black bars) or passage 0 of PDF-2180/MERS chimeric CoV (red bars) and assayed by plaque assay on Vero cells. ND, none detected. (B) Reverse transcription-PCR detection of leader-containing nested subgenomic mRNAs encoding the nucleocapsid transcript, E transcript, and ORF5 and ORF4a transcripts following infection. Ladder, 1 kb; WT, wild type.

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