Identification and characterization of novel bat coronaviruses in Spain

Abstract

The zoonotic transmission of bat coronaviruses poses a threat to human health. However, the diversity of bat-borne coronaviruses remains poorly characterized in many geographical areas. Here, we recovered eight coronavirus genomes by performing a metagenomic analysis of fecal samples from hundreds of individual bats captured in Spain, a country with high bat diversity. Three of these genomes corresponded to potentially novel coronavirus species belonging to the alphacoronavirus genus. Phylogenetic analyses revealed that some of these viruses are closely related to coronaviruses previously described in bats from other countries, suggesting a shared viral reservoir worldwide. Using viral pseudotypes, we investigated the receptor usage of the identified viruses and found that one of them can use human ACE2, albeit with lower affinity than SARS-CoV-2. However, the receptor usage of the other viruses remains unknown. This study broadens our understanding of coronavirus diversity and identifies research priorities for the prevention of zoonotic viral outbreaks.

Fig 1. Collection of fecal samples from 23 bat species across Spain.

The number of samples collected from each bat species and the number of individuals captured in each area are indicated in parentheses. The locations and bat species in which coronaviruses were detected are shown in bold. The provinces where samples were collected are coloured in red (Murcia), yellow (Valencia), orange (Castellón), purple (Teruel), blue (Salamanca), pink (Lugo) and green (Cantabria). This map was created using R (https://www.R-project.org/) and the geospatial data of Spain obtained from GADM v4.1 (https://geodata.ucdavis.edu/gadm/gadm4.1/shp/gadm41_ESP_shp.zip). GADM data are freely available for academic use (https://gadm.org/license.html).

Fig 2. Phylogenetic positioning of the newly described viruses within the family Coronaviridae.

Maximum-likelihood (ML) trees were built using RdRP (A) and helicase (B) amino acid sequences. Sequences from representative ICTV-approved viral species were included. The Gamma- and Deltacoronavirus taxonomic groups are collapsed by genus. Microhyla letovirus 1 was used as an outgroup to root the trees. Viruses described in this study are shown in colors. Bootstrap values higher than 80 are indicated with red circles. The scale bar indicates the evolutionary distance in amino acid substitutions per site.

Fig 3. Spike sequences in the context of Sarbecovirus (A), Minunacovirus (B), Nyctacovirus (C) and Pedacovirus (D) phylogenies.

ML trees were built using the 20 BLASTp hits for each identified spike sequence and the ICTV-approved viral species for each subgenus. Viruses described in this study are shown in colors. Bootstrap values higher than 80 are indicated with red circles. The scale bar indicates the evolutionary distance in amino acid substitutions per site.

Fig 4. Receptor usage of the identified coronaviruses.

A. Western blot validation of spike incorporation into VSV pseudotypes and receptor expression. The spike was detected with an anti-His-Tag antibody and VSV-M was used as a loading control, whereas receptors were detected with an anti-Flag-Tag antibody and GAPDH was used as a loading control. B. Heat map showing the percentage of infected cells after transfection of the indicated bat or human orthologues and inoculation with pseudotyped viruses carrying the indicated spike proteins. For each combination, the average of three replicate (n = 3) assays are shown. The two APN alleles present in M. schreibersii were considered. A two-way ANOVA with Dunnett’s correction for multiple tests was performed to compare all the receptors to the empty. * P < 0.05, **** P < 0.0001. C. Infection inhibition assay by soluble hACE2 against RhBetaCoV-Murcia2022 and SARS-CoV-2 VSV pseudotypes. Each dot is the average of two technical replicates, lines correspond to a sigmoidal 2-parameter fit, and shaded areas correspond to 95% confidence intervals.