DOI: 10.5061/dryad.h9w0vt4p4
title: MHC class II genes mediate susceptibility and resistance to coronavirus infections in bats

description: Understanding the immunogenetic basis of coronavirus (CoV) susceptibility
 in major pathogen reservoirs, such as bats, is central to infer their
 zoonotic potential. Members of the cryptic Hipposideros bat
 species complex differ in CoV susceptibility, but the underlying
 mechanisms remain unclear. The genes of the major histocompatibility
 complex (MHC) are the best understood genetic basis of pathogen
 resistance, and differences in MHC diversity are one possible reason for
 asymmetrical infection patterns among closely related species. Here, we
 aimed to link asymmetries in observed CoV (CoV-229E, CoV-2B, and
 CoV-2Bbasal) susceptibility to immunogenetic differences amongst four
 Hipposideros bat species. From the 2,072 bats assigned to their respective
 species using the mtDNA cytochrome b gene, members of the most numerous
 and ubiquitous species, Hipposideros caffer D, were most infected with
 CoV-229E and SARS-related CoV-2B. Using a subset of 569 bats we determined
 that much of the existent allelic and functional (i.e., supertype) MHC DRB
 class II diversity originated from common ancestry. One MHC supertype
 shared amongst all species, ST12, was consistently linked to
 susceptibility with CoV-229E, which is closely related to the common cold
 agent HCoV-229E, and infected bats with ST12 had a lower body condition.
 The same MHC supertype was connected to resistance to CoV-2B, and bats
 with ST12 were less likely be co-infected with CoV-229E and CoV-2B. Our
 work suggests a role of immunogenetics in determining CoV susceptibility
 in bats. We advocate for the preservation of functional genetic and
 species diversity in reservoirs as means of mitigating the risk of disease
 spillover.
description: Bats were live-trapped at five locations with one to three
 roosting sites in 12 two-month-long capture periods between September 2010
 and August 2012 in Central Ghana, West Africa (Figure 1A). If possible,
 all bats were classified to species level using morphological
 characteristics. Morphometric details such as forearm length or weight
 were described elsewhere (Baldwin et al., 2021). Two minimally invasive
 wing punches (3mm) were taken from each bat and stored in molecular-grade
 ethanol at -20°C for DNA extraction. Additionally, faecal samples were
 collected and stored in RNAlater at -80°C for virus and microbiome
 screening. DNA extraction was performed using wing
 punch tissue from 2,072 bats of the 6,654 bats assigned to the
 <em>H. caffer</em> complex or <em>H. abae</em>.
 The extraction followed an ammonium acetate protocol (Nicholls et al.,
 2000). Building on previous primer designs for the
 <em>Hipposideros</em> species complex (Vallo et al., 2008),
 the mtDNA cytochrome b gene (cytb) was amplified by polymerase chain
 reaction (PCR) using adapted primers suitable for high-throughput Illumina
 sequencing. After sequencing, the cytb gene was confirmed by homology
 analysis using the NCBI BLAST search. Subsequently, all sequences were
 analysed in Geneious 11.1.5  and assigned to the lineages B, C, or D of
 the <em>H. caffer</em> species complex (henceforth called
 species, Baldwin et al., 2014, 2021; Vallo et al., 2008) or the sympatric
 species <em>H. abae</em> using the MAFFT alignment tool (Katoh
 & Standley, 2013). RNA was purified from
 approximately 20 mg of faecal material suspended in 500 µl RNAlater
 stabilizing solution using the MagNA Pure 96 system (Roche, Penzberg,
 Germany) with elution volumes set at 100 µl. We used a real-time reverse
 transcription-PCR assay designed to detect several alpha- and beta-CoVs
 and genetically related bat CoVs using the SSIII RT-PCR kit (Life
 Technologies, Karlsruhe, Germany) and a cycling protocol in a LightCycler
 480 (Roche, Penzberg, Germany) as described previously (Corman et al.,
 2015; Drexler et al., 2009; Pfefferle et al., 2009). Bats were categorised
 as positive for a specific CoV if the CT-value was equal to or smaller
 than 38.0 (Corman et al., 2015). A 171 bp fragment
 within the MHC class II DRB exon 2 loci of 575 Hipposideros samples was
 amplified using primers modified from (Schad et al., 2011).
 <em>Hipposideros</em> samples were MHC-genotyped using an
 Illumina platform (see Supplementary Material). The samples collectively
 reached beyond the threshold sample size (&gt;200) suggested for wild
 populations (Gaigher et al., 2019). The MHC class II DRB exon 2 sequences
 were analysed using the genotyping pipeline ACACIA (Allele CAlling
 proCedure for Illumina Amplicon sequencing data; (Gillingham et al.,
 2021). The ACACIA workflow and post hoc elimination of singletons and
 alleles with low reliability, preserved reliable MHC allele information
 for a total of 569 bats. Allelic MHC diversity was then grouped into
 functional supertypes based on shared amino acid motifs at positively
 selected sites (PSSs), following the assumption that PSSs likely belong
 to, or are closely linked to, functionally important antigen-binding-sites
 (Roved et al., 2022; Schwensow et al., 2019; Sepil et al., 2013).
 Positively selected sites were identified for each species separately
 using CODEML integrated into the program PAML4 (Yang, 2007). Four PAML
 models were tested: M1a (nearly neutral), M2a (positive selection), M7
 (beta), and M8 (beta and ω). M2a and M8 performed equally well, evidencing
 selection acting on specific sites. For subsequent supertype assignment
 across species, a total of 14 ‘consensus’ PSS sites were selected based on
 being a) identified by both M2a and M8 with a posterior probability of at
 least 95% and b) selected in at least 3 of the 4 species. Antigen-binding
 specificity can be quantified by z-values describing the physio-chemical
 properties of amino acids encoded by the codon present at PSSs (Sandberg
 et al., 1998). A matrix containing the z values of each allele’s PSS amino
 acid was used in the functions find.clusters and dapc of the ‘adegenet’ R
 package (Jombart et al., 2010) to cluster alleles into groups (i.e. MHC
 supertypes or STs) with similar binding functionality. 
 This was the methodological backbone of the data uploaded here.
 For more information please refer to the open-access publication and its
 supplementary material.
description: We provide three datasets
 (Hip_MHC-CoV-data_Schmidetal2023MolEcol.csv;
 Hip_community-data_Schmidetal2023MolEcol.csv; and
 Hip_community-CoV-data_Schmidetal2023MolEcol.csv) as CSV format. They form
 the backbone of the analysis performed in the associated R Markdown
 (Schmid.Meyer_MolEcol23_Rscript.Rmd). All statistical
 data analyses were computed in the open-access platform R. 
 Additionally, we provide an Excel file
 (Hip_MHCII-DRB2_sequences_Schmidetal2023MolEcol) with four sheets listing
 the MHC class II DRB exon 2 allele sequences identified among the four
 hipposiderid species.