Picornaviridae and Caliciviridae diversity in Madagascar fruit bats is driven by cross-continental genetic exchange

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Ranaivoson , View ORCID Profile Angelo Andrianianina , View ORCID Profile Santino Andry , Amy R. Henry , Rachel L. Davis , Farida Laboune , View ORCID Profile Elizabeth R. Longtine , Sucheta Godbole , View ORCID Profile Sophia Horigan , View ORCID Profile Emily Cornelius Ruhs , Vololoniaina Raharinosy , View ORCID Profile Tsiry Hasina Randriambolamanantsoa , View ORCID Profile Vincent Lacoste , View ORCID Profile Jean-Michel Heraud , View ORCID Profile Philippe Dussart , Daniel C. Douek , Cara E. Brook doi: https://doi.org/10.1101/2024.12.31.630946 Gwenddolen Kettenburg 1 Department of Ecology and Evolution, University of Chicago , IL, United States Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Gwenddolen Kettenburg For correspondence: gkettenburg{at}uchicago.edu Hafaliana C. Ranaivoson 1 Department of Ecology and Evolution, University of Chicago , IL, United States 2 Association Ekipa Fanihy , Antananarivo, Madagascar 3 Department of Zoology and Animal Biodiversity, University of Antananarivo , Antananarivo, Madagascar Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Hafaliana C. Ranaivoson Angelo Andrianianina 2 Association Ekipa Fanihy , Antananarivo, Madagascar 3 Department of Zoology and Animal Biodiversity, University of Antananarivo , Antananarivo, Madagascar Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Angelo Andrianianina Santino Andry 2 Association Ekipa Fanihy , Antananarivo, Madagascar 4 Department of Entomology, University of Antananarivo , Antananarivo, Madagascar Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Santino Andry Amy R. Henry 5 Human Immunology Section, Vaccine Research Center, NIAID, NIH , Bethesda, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Rachel L. Davis 5 Human Immunology Section, Vaccine Research Center, NIAID, NIH , Bethesda, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Farida Laboune 5 Human Immunology Section, Vaccine Research Center, NIAID, NIH , Bethesda, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Elizabeth R. Longtine 5 Human Immunology Section, Vaccine Research Center, NIAID, NIH , Bethesda, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Elizabeth R. Longtine Sucheta Godbole 6 PREMISE, Vaccine Research Center, NIAID, NIH , Bethesda, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Sophia Horigan 1 Department of Ecology and Evolution, University of Chicago , IL, United States Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Sophia Horigan Emily Cornelius Ruhs 1 Department of Ecology and Evolution, University of Chicago , IL, United States 7 Grainger Center for Bioinformatics, Field Museum of Natural History , Chicago, IL, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Emily Cornelius Ruhs Vololoniaina Raharinosy 8 Virology Unit, Institut Pasteur de Madagascar , Antananarivo, Madagascar , Find this author on Google Scholar Find this author on PubMed Search for this author on this site Tsiry Hasina Randriambolamanantsoa 8 Virology Unit, Institut Pasteur de Madagascar , Antananarivo, Madagascar , Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Tsiry Hasina Randriambolamanantsoa Vincent Lacoste 8 Virology Unit, Institut Pasteur de Madagascar , Antananarivo, Madagascar , Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Vincent Lacoste Jean-Michel Heraud 8 Virology Unit, Institut Pasteur de Madagascar , Antananarivo, Madagascar , Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Jean-Michel Heraud Philippe Dussart 8 Virology Unit, Institut Pasteur de Madagascar , Antananarivo, Madagascar , Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Philippe Dussart Daniel C. Douek 5 Human Immunology Section, Vaccine Research Center, NIAID, NIH , Bethesda, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Cara E. Brook 1 Department of Ecology and Evolution, University of Chicago , IL, United States 2 Association Ekipa Fanihy , Antananarivo, Madagascar Find this author on Google Scholar Find this author on PubMed Search for this author on this site Abstract Full Text Info/History Metrics Supplementary material Preview PDF ABSTRACT Bats are reservoir hosts for numerous well-known zoonotic viruses, but their broader virus-hosting capacities remain understudied. Picornavirales are an order of enteric viruses known to cause disease across a wide range of mammalian hosts, including Hepatitis A in humans and foot-and-mouth disease in ungulates. Host-switching and recombination drive the diversification of Picornavirales worldwide. Divergent Caliciviridae and Picornaviridae (families within the Picornavirales ) have been described in bats across mainland Africa, but surveillance for these viruses has been rare in the Southwest Indian Ocean Islands. Bats live in close proximity to and are consumed widely as a food source by humans in Madagascar, providing opportunities for zoonotic transmission. Prior work in Madagascar has described numerous evolutionarily divergent bat viruses, some with zoonotic potential. Using metagenomic Next Generation Sequencing of urine and fecal samples obtained from three species of endemic Malagasy fruit bats ( Eidolon dupreanum , Pteropus rufus , and Rousettus madagascariensis ), we recovered 13 full-length and 37 partial-length genomic sequences within the order Picornavirales (36 Picornaviridae and 14 Caliciviridae sequences), which we identify and describe here. We find evidence that genetic exchange between mainland African bat and Madagascar bat Picornavirales likely shaped the diversification patterns of these novel sequences through recombination events between closely related Picornavirales ; thus far, high host fidelity appears to have limited these viruses from spilling over into other species. INTRODUCTION Picornavirales is a viral order associated with a large taxonomic variety of hosts, spanning animals (both vertebrates and invertebrates), plants and insects. Viruses in this order are characterized by a single-stranded, positive-sense RNA genome that forms non-enveloped icosahedral virions 1 . These viruses can encode either one or two polyproteins 1 . Picornavirales families Picornaviridae and Caliciviridae are known to cause clinical disease in both human and other mammalian hosts. Hepatitis A, caused by hepatovirus A in the Picornaviridae family, is a human disease with about 1.5 million reported cases each year, characterized by symptoms ranging from nausea and vomiting to potentially fatal fulminant hepatitis 2 . Poliovirus, an enterovirus in the family Picornaviridae , has caused epidemics of poliomyelitis, sometimes resulting in irreversible paralysis, throughout the 19 th and first half of the 20 th century before the introduction of the Salk and Sabin vaccines in the 1950s 3 . Although poliovirus disease met the target of 99% global eradication in 2000, poliovirus outbreaks still occur in areas of low vaccine uptake 4 . Norovirus and Sapovirus , in the family Caliciviridae , cause gastroenteritis in young children and more severe complications in immunocompromised individuals 5 , 6 . Notably, viruses in the family Picornaviridae and Caliciviridae can infect some of the most common zoonotic hosts (bats, rodents, shrews) 7 , 8 , in addition to agriculturally significant hosts such as swine and cattle – both of which come into direct contact with humans 9 – 14 . Foot- and-mouth disease, caused by foot-and-mouth disease virus in the Picornaviridae family, affects cloven-hoofed animals, is highly contagious, and is characterized by clinical manifestations of vesicles in the oral cavity and feet 15 . Some Picornaviridae and Caliciviridae viruses display close genetic similarity between those hosted by humans and animals, but evidence of zoonoses is limited 14 , 16 – 19 . For example, some bat calicivirus virus-like particles (VLPs) experimentally generated in vitro have similar antigenic epitopes, which elicit histo-blood group binding, as do human and other mammalian noroviruses, but no natural observations of zoonoses for these viruses is known 14 . This diverse host range of Picornaviridae and Caliciviridae diversity can partially be equated to recombination and host-switching events that drive the evolution of these viruses 20 – 25 . Bats have garnered interest for their unique ability to host viruses known to be highly pathogenic in other mammals, including humans, without experiencing significant disease 26 – 31 . While bats are known to host viruses in the Picornavirales order, particularly in the Picornaviridae and Caliciviridae 14 , 32 – 39 families, these viruses are generally understudied as compared with a few more well-known bat virus clades such as coronaviruses, filoviruses, lyssaviruses and paramyxoviruses. Previous work from Cameroon used metagenomic Next Generation Sequencing (mNGS) to describe diverse Picornavirales in both Eidolon helvum and Epomorphus gambianus fruit bats, including novel kunsagiviruses and sapeloviruses that were divergent enough in amino acid composition to represent new species 36 . Divergent sapoviruses were also identified from the same bats 35 ; in other cases, animal-derived sapoviruses have been shown to cluster closely with human-infecting genotypes 40 – 43 . In a separate study focusing on Algerian bats, evidence of past recombination events was detected within the genome of a novel mischivirus 34 . Host-virus coevolutionary analysis indicated that host-switching likely drove the diversification of this novel virus, in concordance with previously described patterns for the Picornaviridae family at large 34 , 44 . To date, the majority of bat Picornavirales work has taken place in mainland Africa, with limited prior studies in the Southwest Indian Ocean Islands (SWIO: Madagascar, Seychelles, Mauritius, Réunion, and Comoros) 13 . Of the SWIO, Madagascar is particularly unique. An island country 400km off the coast of southeastern Africa, Madagascar boasts high levels of endemism and extraordinary evolutionary divergence among its flora and fauna due its isolation from mainland Africa and Asia for the past 80 million years 45 . Several evolutionarily distinct viruses have been previously identified in endemic Malagasy bats 46 – 52 , matching expectations that the isolated evolutionary landscape leads not only to diverse hosts but also diverse viruses. However, research regarding bat-hosted Picornavirales in Madagascar has been limited to only two prior studies. One of these studies described a unique hepatovirus in liver tissue collected from the Malagasy insectivorous bat, Miniopterus cf. manavi ; sequence analysis of small mammal-hosted hepatoviruses broadly suggests some degree of host-virus co-evolution in their evolutionary history, with a hypothesized ancestral origin in bats and shrews 13 . In the other study, one full-length kobuvirus sequence was detected via mNGS of fecal samples collected from an Eidolon dupreanum fruit bat 52 . Within Madagascar, host-switching, likely fostered by co-roosting, is the dominant evolutionary mechanism driving diversification of morbilli-related bat paramyxoviruses 49 . We aimed to elucidate the role of host-switching mechanisms in driving the diversification of bat-borne Picornaviridae and Caliciviridae in Madagascar. MATERIALS AND METHODS Sample collection Longitudinal monthly sampling of three endemic Malagasy fruit bats ( E. dupreanum, P. rufus, and Rousettus madagascariensis ) was carried out from 2013-2019 at species-specific roost sites across Madagascar in part with an ongoing effort to investigate seasonal viral dynamics, as described previously 46 , 47 , 51 – 53 . Over this period, 2156 bats were captured and processed under manual restraint as previously described 46 , 47 , 51 , 53 . Upon capture, bats were identified by species, age class, and sex, and individual fecal, throat and urine swabs were collected. All excreta samples were collected in viral transport medium (VTM) and frozen in liquid nitrogen until samples could be processed. A subset of 810 (271 fecal/539 urine) samples collected between 2013-2019 at the following roost sites was used for the molecular analyses outlined here: Angavobe cave (-18.944S, 47.949 E, E. dupreanum) ; Angavokely cave (-18.933 S, 47.758 E, E. dupreanum) ; Ambakoana roost (-18.513 S, 48.167 E, P. rufus) ; Maromizaha cave (-18.9623 S, 48.4525 E, R. madagascariensis) ; Andrafiabe cave (-12.9435 S, 49.0555 E, E. dupreanum and R. madagascariensis ); Cathedral cave (-12.952901 S, 49.046885 E, E. dupreanum ); Antsiroandoha cave (-12.959336 S, 49.123698 E, E. dupreanum ). This study was carried out in strict accordance with research permits obtained from the Madagascar Ministry of Forest and the Environment (permit numbers: 251/13, 166/14, 75/15, 92/16, 259/16, 019/18, 170/18, and 007/19,) and under guidelines posted by the American Veterinary Medical Association. All field protocols employed were pre-approved by the UC Berkeley Animal Care and Use Committee (IACUC Protocol # AUP-2017-10-10393), and every effort was made to minimize discomfort to animals. Sample processing Samples were frozen at the site of capture in liquid nitrogen and stored at -80°C at the Virology Unit at the Institut Pasteur de Madagascar. Subsequently, RNA extraction of the samples was completed using the Zymo Quick DNA/RNA Microprep Plus kit (Zymo Research, Irvine, CA, USA), according to the manufacturer’s instructions and including the step for DNAse digestion. Extracted RNA was then shipped to either the Chan Zuckerberg Biohub (CZB; sample date range 2018-2019) (San Francisco, CA, USA) or the Vaccine Research Center (VRC), National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH; sample date range 2013-2019) (Bethesda, USA) for mNGS. Briefly, RNA underwent library preparation using the NEBNext Ultra II RNA Library Prep Kit (New England Biolabs, Beverly, MA, USA) with the following modifications for CZB samples (date range 2018-2019): 25pg of External RNA Controls Consortium Spike-in mix (ERCCS, Thermo-Fisher) was added to each sample prior to RNA fragmentation; the input RNA mixture was fragmented for 8min at 94°C prior to reverse transcription; and a total of 14 cycles of PCR with dual-indexed TruSeq adapters was applied to amplify the resulting individual libraries. Quality was assessed by electrophoresis before performing large-scale paired-end sequencing (2 x 146bp) on the Illumina NovaSeq (Illumina, San Diego, CA, USA). The following modifications were made for VRC samples (date range 2018-2019): input RNA was fragmented for 7min at 94°C prior to reverse transcription; and a total of 12 cycles of PCR. Quality was assessed by electrophoresis (Bioanalyzer 2100, Agilent) before paired-end sequencing (2 x 150bp) on the Illumina NovaSeq 6000 (Illumina, San Diego, CA, USA). The CZID pipeline to parse output from individual libraries into FASTQ format is available on GitHub ( https://github.com/czbiohub/utilities ). Virus detection Raw reads were host-filtered, quality-filtered, and assembled on the Chan Zuckerberg Infectious Diseases (CZID) bioinformatics platform (v3.10 NR/NT 2019-12-01) 54 . A background profile named “bat” was created using all publicly available full-length bat genomes in GenBank at the time of sequencing (July 2019 for CZID samples, December 2023 for VRC samples) and used in host filtering. Samples were investigated using a command line BLAST pipeline, as described previously in novel coronavirus, henipavirus, kobuvirus, and astrovirus detection 46 , 47 , 51 , 52 . The following criteria were used to call “positive” samples: (1) at least two contigs with an average read depth of >2 reads/nucleotide were assembled and (2) showed significant nucleotide or protein BLAST alignment(s) (alignment length >100nt/aa and E-value 100 for protein BLAST) to Picornavirales present in NCBI NR/NT database (v12-01-2019). We used NCBI Virus taxids 12058/478825 for Picornaviridae /unclassified Picornaviridae and taxids 11974/179239 for Caliciviridae /unclassified Caliciviridae to develop nucleotide and protein libraries, against which we queried our novel sequences in command line BLASTn 55 and BLASTx 55 searches. Detailed instructions on our methods for parsing “positive” samples is available on our open-access GitHub repository (see Data Availability ). Genome annotation Once contigs were identified as Picornaviridae / Caliciviridae sequences, we sorted genomes into full and partial-length sequences. We downloaded annotated reference Picornaviridae / Caliciviridae sequences from NCBI and aligned to our novel sequences using MAFFT 56 (v7.450) with default settings in Geneious Prime (v08-18-2022). We annotated the polyprotein and peptide sequences using available Picornaviridae / Caliciviridae reference sequences that corresponded to the top BLAST hit. Reference sequences were also used to identify potential cleavage sites and conserved motifs within RNA-dependent RNA polymerase (RdRp), polyprotein major proteases, and NTPase-helicase. Phylogenetic analysis We constructed ten maximum-likelihood (ML) nucleotide phylogenetic trees in RAxML 57 , representing all genera in which we identified at least one novel sequence >2000bp in length. All trees were rooted using an outgroup of Sindbis virus (accession NC_001547 ). We first constructed (A) a phylogeny encompassing the conserved polymerase (3D peptide) region and included global background sequences from viral genera corresponding to those clades represented by all novel Madagascar sequences (unclassified bat picornavirus, Cardiovirus , Hepatovirus , Kobuvirus , Kunsagivirus , Mischivirus , Sapelovirus , Sapovirus , and Teschovirus ). Then, we constructed nine additional ML trees using full-length sequences from the following viral genera as references: (B) Cardiovirus , (C) Hepatovirus , (D) Kobuvirus , (E) Kunsagivirus , (F) Mischivirus , (G) Shanbavirus /unclassified bat picornavirus, (H) Sapelovirus , (I) Teschovirus , and (J) Sapovirus . Reference sequences and novel sequences were aligned using MAFFT 56 , and we used ModelTest-NG 58 (v0.1.7) to determine the best nucleotide substitution model for each alignment prior to building the ML-trees. We further detail NCBI virus taxid information, ModelTest output, alignment names, and size of overlap region per phylogeny in Supplemental table 1 . Sequence alignments and more detailed tree building instructions are available in our GitHub repository (see Data Availability ). Similarity analysis We conducted similarity analyses, using PySimPlot 59 , on all nucleotide and translated amino acid Picornaviridae and Caliciviridae full-length sequences recovered from CZID. PySimPlot commands were formulated using our novel Madagascar sequences as the query sequences; in cases where multiple novel sequences were identified within a single genus, we input one of those sequences as the query and included the others within the reference sequences. Otherwise, reference sequences for input to PySimPlot 59 were comprised of the top three full-genome BLASTx hits for the query. In all cases, sequences were aligned in MAFFT 56 , and both similarity analyses were carried out using a window size of 100aa and a step size of 20aa for amino acid comparisons and a window size of 100bp and a step size of 1bp for nucleotide comparisons. A full list of query sequences and reference sequences per alignment is available in our GitHub repository (see Data Availability ). RDP4 recombination analysis Recombination Detection Program 4 (RDP4) 60 was used to evaluate signals of recombination in sets of pairwise alignments; RDP4 identifies the potential recombinant sequence, as well as the potential major parental sequence (from which most of the genome is reputed to be sourced) and the minor parental sequence (from which some of the genome is reputed to be sourced) for this lineage. From each pairwise alignment, RDP4 identifies potentially recombinant sequences, in addition to specifying the region of each putatively recombinant genome most likely to have undergone recombination and the parent genome (either major or minor) most likely to have contributed to that region. We ran seven separate analyses within RDP4 (RDP, GENECONV, Bootscan, MaxChi, Chimaera, and 3Seq) to estimate likely recombination events and the location of the recombination event within all full-length (13) sequences identified in this study. We considered recombination events to be reliable if at least four analyses were significant using default settings with a cutoff P -value of 0.05. A full list of query sequences and reference sequences per alignment is available in our GitHub repository (see Data Availability ). RESULTS Sampling and demographic patterns We sequenced RNA extracted from 810 (271 fecal and 539 urine) samples from 803 individual bats (373 E. dupreanum , 146 P. rufus , and 284 R. madagascariensis ). In total, 24/803 bats were Picornaviridae positive (2.98%), and 7/803 bats were Caliciviridae positive (0.87%) ( Table 1 ). Of the 24 Picornaviridae positive bats, 16 were E. dupreanum , 1 was P. rufus , and 7 were R. madagascariensis . The 7 Caliciviridae positive bats were evenly split between E. dupreanum (4) and R. madagascariensis (4) with no infections identified in P. rufus . No bats were positive at the Ankarana caves (N=151). Picornaviridae and Caliciviridae prevalence was highest in E. dupreanum at the Angavokely/Angavobe roosts (16/281 bats Picornaviridae and 4/281 bats Caliciviridae ) with a similar prevalence reported in R. madagascariensis at the Maromizaha roost (7/225 bats Picornaviridae and 4/225 bats Caliciviridae ) ( Fig. 1A and Table 1 ). In P. rufus , only one bat out of 146 individuals was found to be positive for Picornaviridae ( Fig. 1A and Table 1 ). Two individual bats were coinfected: one was an E. dupreanum from Angavokely/Angavobe cave, from which we identified a partial genome for a Hepatovirus and a full genome for the only Kunsagivirus described in this study. The second individual was a R. madagascariensis from Maromizaha, which hosted a partial genome for a Sapovirus and a full genome for an unclassified bat picornavirus. Download figure Open in new tab Figure 1: (A) Map of sampling sites for Eidolon dupreanum, Pteropus rufus , and Rousettus madagascariensis at sites in Districts of Ambilobe ( E. dupreanum and R. madagascariensis : Ankarana caves), Moramanga ( P. rufus: Ambakoana roost and R. madagascariensis : Maromizaha cave), and Manjakandriana ( E. dupreanum : Angavobe/Angavokely caves), Madagascar. Pie charts show Picornaviridae (yellow) and Caliciviridae (orange) positive bats by site. Pie chart corresponds to sample size on a log10 scale. (B) Summary bar plot of diversity of viral genera (facets) and viral species (colors) identified from bats caught within the same sampling date at Angavokely/Angavobe caves ( E. dupreanum ) and Maromizaha cave ( R. madagascariensis ). Ambakoana roost (P . rufus ) was excluded due to only having one identified novel virus in a single bat. View this table: View inline View popup Download powerpoint Table 1: Sampling efforts by roost site and species, in addition to breakdown by Picornaviridae and Caliciviridae positives. Genome characterization and roost-site diversity We recovered 13 full-length (12 Picornaviridae and 1 Caliciviridae ) and 37 partial-length (24 Picornaviridae and 13 Caliciviridae ) sequences primarily from E. dupreanum (25 Picornaviridae from 15 individuals and 5 Caliciviridae from 4 individuals) and R. madagascariensis (10 Picornaviridae from 7 individuals and 9 Caliciviridae from 4 individuals) ( Fig. 1B ). Only one viral Picornaviridae sequence (full-genome) was recovered from an individual P. rufus ( Fig. 1B ). Within Picornaviridae , we identified sequences corresponding to the following viral genera: Cardiovirus (1 sequence from 1 viral species), Hepatovirus (4 sequences from 1 viral species), Kobuvirus (8 sequences from 2 viral species), Kunsagivirus (1 sequence from 1 viral species), Mischivirus (1 sequence from 1 viral species), Sapelovirus (11 sequences from 3 viral species, Teschovirus (3 sequences from 3 viral species), and unclassified “bat picornavirus” (7 sequences from 3 viral species). Caliciviridae was only represented by a single genus: Sapovirus (14 sequences from 7 viral species) ( Fig. 1B , Supplemental tables 2 and 3 ). Some Picornaviridae genera were only found in E. dupreanum ( Cardiovirus , Hepatovirus , Kobuvirus , and Kunsagivirus ) ( Fig. 1B , Supplemental table 2 ). In general, Picornaviridae sequences from E. dupreanum had highest identity to Picornaviridae identified from its sister species E. helvum ( Supplemental table 2 ), which is widely distributed across the African mainland continent but is absent from Madagascar 61 . Exceptions to this pattern included the Cardiovirus sequence which had the highest identity to a divergent encephalomyocarditis virus isolated from an orangutan from Singapore (NCBI accession QMI58083.1) 62 and a Teschovirus sequence with highest identity to a Ugandan Rousettus aegypticus bat teschovirus (accession XBH24020) 63 . Cardiovirus has been detected in bats before, namely East Asian Miniopterus fuliginosus bats 64 . It is possible that our novel Malagasy bat Cardiovirus would be more closely related to this Chiropteran -hosted Cardiovirus; however, the lack of publicly-available sequences from this previous study impedes these comparisons. One partial Teschovirus sequence from a Saudi Arabian E. helvum bat has also been described (accession KX420938 ) 65 , but is still not the highest identity to our novel E. dupreanum -derived Teschovirus . E. dupreanum -hosted Hepatovirus , Kobuvirus , Kunsagivirus, and Sapelovirus had highest identity to Ghanaian E. helvum -hosted hepatovirus H2 (accession YP_009179216.1) 13 , Malagasy E. dupreanum -hosted kobuvirus (accession WBP49885.1) 52 , Cameroonian E. helvum -hosted kunsagivirus B (accession YP_009345896.1) 66 , and Cameroonian E. helvum -hosted sapelovirus (accession YP_009345901.1) 66 respectively ( Supplemental table 2 ). One Picornaviridae genus, Mischivirus , was found only in P. rufus, in one individual ( Fig. 1B ). This novel sequence showed very low identity (46% over 81% genome coverage) to its closest match (accession YP_009121743.1, mischivirus C1 from a Hipposideros gigas bat from the Democratic Republic of the Congo (DRC)) ( Supplemental table 2 ). Among Picornaviridae genera exclusively hosted by R. madagascariensis (unclassified bat picornavirus), and those hosted by both E. dupreanum and R. madagascariensis ( Sapelovirus and Teschovirus ), we identified numerous viruses with high identity to viruses hosted by sister species R. aegypticus 67 sampled in Uganda and Kenya. The unclassified bat picornaviruses were most closely related to other previously-described “bat picornaviruses” 12 , 63 , 68 , 69 but, within this clade, form what appears to be a largely divergent Picornaviridae genus with an average BLASTx identity of ∼80% with ∼90% genome coverage to the closest match ( Supplemental table 2 ). R. madagascariensis -hosted Sapelovirus and Teschovirus had highest identity to East African (Uganda and Kenya) R. aegypticus -hosted Sapelovirus and Teschovirus sequences 63 ( Supplemental table 2 ). The representative Caliciviridae genus, Sapovirus , was found in both E. dupreanum and R. madagascariensis ( Fig. 1 , Supplemental table 3 ). Sapovirus sequences from E. dupreanum showed highest identity to a Cameroonian E. helvum -hosted Sapovirus (accession KX759623.1 ) 35 ( Supplemental table 3 ) and R. madagascariensis -hosted Sapovirus sequences had highest identity to East African R. aegypticus -hosted sapoviruses, mirroring the host patterns seen in the novel Picornaviridae sequences, as well ( Supplemental table 3 ). We identified as many as 4 different viral species sourced from the same group of bats captured within a single sampling session ( Fig. 1B ). Overall, the Angavokely roost site (with E. dupreanum bats) was represented the most frequently in the samples analyzed for this study and demonstrated the most unique viruses overall ( Fig. 1B and Supplemental tables 2 and 3 ), but the Maromizaha roost site (with R. madagascariensis bats) also demonstrated high virus diversity – with multiple repeats of the same virus genotype recovered from different individuals – within fewer viral genera. Genome annotation Picornaviridae We successfully annotated a single ORF-encoding polyprotein in Picornaviridae which includes the P1 region of structural polypeptides and the P2/P3 regions of replication-associated nonstructural polypeptides) and further identified cleavage sites within the polyprotein between each peptide of the P1 region (L, VP4, VP2, VP3, VP1), the P2 region (2A, 2B, 2C) and the P3 region (3A, 3B, 3C, and the RNA-dependent-RNA-polymerase [RdRp] 3D) ( Supplemental table 4 ). Following previous work 33 , 36 , 68 , we identified the conserved motifs, helicase GxxGxGKS, 2A protease GxCG, 3C protease GxCG, and RdRp motifs KDELR, YGDD, and FLKR. All novel Picornaviridae identified in our study had conserved RdRp motifs with no substitutions ( Table 2 ). The only genera with the 2A protease motif were the R. madagascariensis picornaviruses and all sapeloviruses. E. dupreanum cardiovirus had the same 2C GDAGQGKS helicase as the previously mentioned divergent simian encephalomyocarditis virus 62 ( Table 2 ). E. dupreanum hepatoviruses all had the same 2C motif GNRGGGKS as the comparable Cameroonian E. helvum hepatovirus H2 13 and a treeshrew hepatovirus (accession NC_028981 ) ( Table 2 ). E. dupreanum kobuvirus sequences were generally almost identical to a previously described E. dupreanum kobuvirus 52 with slight genotypic variation; the 2C helicase GPPGTGKS and 3C protease GLCG motifs were perfectly identical. We characterized a partial genome of a second kobuvirus species, E. dupreanum kobuvirus 2, but the segment unfortunately did not cover any of the conserved regions for further comparison. These motifs seem to be well conserved in kobuviruses across multiple host types such as bovine, human, canine, and other bats 70 – 73 ( Table 2 ). E. dupreanum kunsagivirus has the same 2C helicase GEPGTGKS and 3C protease GMCG as other African bat and rodent kunsagiviruses 66 , 74 . View this table: View inline View popup Download powerpoint Table 2: Conserved motifs in novel Picornaviridae sequences from Madagascar fruit bats. Bold denotes full-length sequences. Dashes indicate that the sequence recovered does not include that motif due to length. Absent motifs are otherwise noted. The divergent P. rufus mischivirus had a 2C helicase motif GKPGAGKS and 3C protease motif GYCG as seen in Chinese Miniopterus pusillus -hosted mischiviruses (accession OR867092 ), and an Algerian Miniopterus spp.-hosted mischivirus (accession MG888045 ). While the closest match for P. rufus mischivirus is a mischivirus C1 sequence from H. gigas bat from the DRC, it did not share the 2C helicase of GRPGAGKS or the 3C protease of GFCG ( Table 2 . Also divergent, the group of R. madagascariensis -hosted bat picornaviruses share the same 3C helicase motif GSPGCGKS, excepting one partial sequence of R. madagascariensis picornavirus 3 (accession PP766472 ) which instead had GQPGSGKS, a motif previously seen in bat picornavirus BtSY4 sequences from Chinese Rhinolophus bats (accessions PP746000 and OP963617 ) 69 . Otherwise, the GSPGCGKS motif is shared with East African E. helvum and R. aegypticus picornaviruses (sample accessions PP711943 and PP711928 ) 63 , as is the 2A protease GVCG motif ( Table 2 ). Teschoviruses from E. dupreanum and R. madagascariensis had the 2C helicase motif of GKPGQGKS, as seen in Chinese R. leschenaultii bats (accessions OR951333 and OR951334 ), differing from the 2C helicase GAPGQGKS seen in East African R. aegypticus bats (accession PP711934 ) and porcine teschoviruses (sample accession OM105029 ). Teschoviruses lack a 2A protease motif but have two 3C protease motifs that are generally conserved across bat and porcine hosts (GYCG and KICG), excepting R. madagascariensis teschovirus 2, which had a novel substitution of GFCG in the first 3C protease motif ( Table 2 ). Finally, the E. dupreanum sapeloviruses had a 2C helicase motif of GSPGTGKS and a 2A protease motif of GFCG, whereas R. madagascariensis sapelovirus 1 has a 2C helicase motif of GTPGTGKS and a 2A protease motif of GYCG ( Table 2 ). East African E. helvum -hosted sapeloviruses have the same motifs as E. dupreanum sapeloviruses (accessions PP711921 and PP711943 ) 63 , and East African R. aegypticus -hosted sapeloviruses have the same motifs as R. madagascariensis sapelovirus 1 (accession PP711911 ) 63 , indicating bat species-specific viral differences that are conserved between sister species. Caliciviridae In all Caliciviridae sequences we identified and annotated the ORF1-encoding polyprotein in addition to ORF2. Within ORF1 polyprotein we further identified cleavage sites between peptides NS1/NS2, Helicase, NS4, Vpg, Pro-Pol (RdRp), and VP1 in addition to annotating the small structural protein VP2 encoded by ORF2 ( Supplemental table 5 ). Within Caliciviridae , specifically sapoviruses, again following prior work 35 , we identified conserved helicase GAPGIGKT, Vpg KGKTK and DDEYDE, protease GxCG, RdRp conserved WKGL, KDELR, DYSKWDST, GLPSG, and YGDD, and finally VP1 PPG and GWS motifs. RdRp and VP1 motifs were generally conserved among E. dupreanum sapoviruses and R. madagascariensis sapoviruses, in addition to all comparison sequences ( Table 3 ). Slight variation existed among the Malagasy bat sapoviruses, notably a missing VpG KGKTK motif and a protease GSCG (other novel sequences are GDCG) in R. madagascariensis sapovirus 3: accession OQ818348 ( Table 3 ). R. madagascariensis sapovirus 2: accession OQ818347 had a RdRp motif of DFSKWDST (other novel sequences have DYSKWDST), also seen in East African R. aegypticus sapoviruses (accessions PP712001 and PP712004 ). NTPase GPPGIGKT was well conserved amongst the novel sapoviruses, also appearing in African bat sapoviruses (example accessions KX759619 and PP712001 ) , but also in a human sapovirus (accession MH922772 ) and a pig sapovirus (accession OM105025 ) ( Table 3 ). View this table: View inline View popup Download powerpoint Table 3: Conserved motifs in novel Caliciviridae sequences from Madagascar fruit bats. Bold denotes full-length sequences. Dashes indicate that the sequence recovered does not include that motif due to length. Absent motifs are otherwise noted. Phylogenetic analysis Summary phylogeny of Picornaviridae and Caliciviridae After identification of viral genera and species through BLAST ( Supplemental tables 2 and 3 ), we constructed an RdRp phylogeny which showed phylogenetic clustering of the novel Malagasy bat Picornaviridae and Caliciviridae with other bat-hosted sequences ( Fig. 2 ). For genomes recovered, read support was sufficient (average support >88 reads/base pair in full genomes, average support >1764 reads/base pair in partial genomes) ( Supplemental Fig. 1 and 2 ). In all cases, support was lower in the 5’ and 3’ untranslated regions (UTRs) ( Supplemental Fig. 1 and 2 ). Cardiovirus (average support 12007 reads/base pair) recovered the most read support (although, only in a small portion of the genome) followed by Mischivirus (average support 712 reads/base pair), Sapovirus (average support 635 reads/base pair) and Kobuvirus (average support 174 reads/base pair) ( Fig. 2 , Supplemental Fig. 1 and 2 ). While lower, read support was sufficient for Teschovirus (average support 70 reads/base pair), Kunsagivirus (average support 29 reads/base pair), Sapelovirus (average support 16 reads/base pair), Hepatovirus (average support 15 reads/base pair), and bat picornaviruses (average support 13 reads/base pair) ( Fig. 2 , Supplemental Fig. 1 and 2 ). We further analyzed assembled contigs (contiguous sequences representing partial or full genomes submitted to NCBI) in their own genus-specific phylogenies ( Fig. 3A-I ). We recovered the most contigs from the genus Sapovirus , followed by Sapelovirus and bat picornaviruses ( Fig. 2 ). Though read support was high for Kobuvirus and Cardiovirus sequences identified, we found fewer discrete contigs representing unique virus species from different individuals for these taxa ( Fig. 2 ). Download figure Open in new tab Figure 2: Maximum-likelihood nucleotide phylogeny (RAxML-NG-MPI 57 ) of sequences from Picornaviridae and Caliciviridae genera from which novel sequences were classified from a 2900 bp overlapping region of the RdRp region of the genome, using a best-fit GTR+I+G4 nucleotide substitution model ( Supplemental table 1 ). Bootstraps were computed using Felsenstein’s method and are visualized on tree branches. Rings indicate number of contigs (red) and reads (blue and log10 scale) recovered for each novel sequence genera. Novel sequences are indicated by a yellow square. Tip shape indicates host in which the sequence was derived corresponding to the legend. Tree is rooted in Sindbis virus (accession NC_001547.1 ). Roots have been removed for ease of visualization. Branch lengths are scaled by nucleotide substitutions per site, corresponding to scalebar. Download figure Open in new tab Figure 3: Maximum-likelihood nucleotide phylogenies (RAxML-NG-MPI 57 ) of (A) Cardiovirus sequences across the whole genome, (B) Hepatovirus sequences across the whole genome, (C) Kobuvirus sequences across the whole genome, (D) Kunsagivirus sequences across the whole genome, (E) Mischivirus sequences across the whole genome, (F) Shanbavirus /unclassified bat picornavirus sequence across the whole genome, (G) Sapelovirus sequences across the whole genome, (H) Teschovirus sequences across the whole genome, and (I) Sapovirus sequences across the whole genome. Best fit nucleotide substitution models and overlapping base pair length per tree are summarized in Supplemental table 1 . Bootstraps were computed using Felsenstein’s method and are visualized on tree branches. Novel sequences are highlighted in yellow. Collapsed clades are represented by white squares. Tip shape indicates host in which the sequence was derived corresponding to the legend. Trees are rooted in Sindbis virus (accession NC_001547.1 ). Roots have been removed for ease of visualization. Branch lengths are scaled by nucleotide substitutions per site, corresponding to each scalebar. Picornaviridae The partial E. dupreanum cardiovirus sequence had high identity to a divergent orangutan-hosted encephalomyocarditis virus ( Supplemental table 2 ) and appeared to be phylogenetically basal to these primate-hosted cardioviruses. Due to the novelty of cardioviruses in bats, addition of more sequences would likely resolve the placement of this sequence in its own clade ( Fig. 3A ). E. dupreanum hepatovirus sequences formed a clade sister to a Cameroonian E. helvum -hosted hepatovirus (accession NC_028366 ), separate from insectivorous Hipposideros spp. bat-hosted hepatoviruses ( Fig 3B ). Consistent with previous reports 13 , human-hosted hepatoviruses form a monophyletic clade sister to a separate clade of animal-hosted hepatoviruses, and hedgehog and shrew-borne hepatoviruses form a paraphyletic clade basal to the African bat hepatoviruses ( Fig. 3B ). As mentioned previously, the E. dupreanum kobuvirus sequences described in this paper were determined to be genetic variants of the same virus previously described 52 . E. dupreanum kobuviruses were basal to other bat-hosted kobuviruses (namely Scotophilus / Rhinolophus sequences from Asia), in addition to canine and human-hosted kobuviruses, but were sister to Aichivirus F sequences which include Myotis and Miniopterus -hosted kobuviruses ( Fig 3C ). Few full-length Kunsagivirus sequences are published, but phylogenetically we can see that E. dupreanum kunsagivirus formed a monophyletic clade with E. helvum kunsagivirus B (accession NC_033818 ), separate from the kunsagivirus sequences found in other mammalian hosts ( Fig. 3D ). With high identity and phylogenetic clustering ( Fig. 3D , Supplemental table 2 ), E. dupreanum kunsagivirus appeared to be very similar to the kunsagivirus circulating in Cameroonian E. helvum bats. The most divergent virus, P. rufus mischivirus , formed a monophyletic clade with a H. gigas mischivirus C sequence from the DRC that is separate from all other bat-hosted mischiviruses that have been previously identified in Miniopterus spp. The long branch length separating these clades suggests considerable differences in nucleotide substitution rates as well ( Fig. 3E ). Without more sequences to resolve the phylogeny, it is unclear that geographical species distribution alone may be driving viral species differentiation. H. gigas is part of the family Hipposideridae , within the Yinpterochiroptera suborder (previously known as megabats), as is P. rufus . By contrast, Miniopterus spp. are part of the Yangochiroptera suborder (previously known as microbats) 75 . While these patterns suggest a co-speciation of viruses along host evolutionary lines, previous analyses have instead supported host-jumping mechanisms as a driver of Mischivirus diversity in bats 34 . R. madagascariensis picornaviruses formed a sister paraphyletic clade to other Rousettus bat picornaviruses from East Africa, and within their own clade separated into two smaller clades representing two species ( R. madagascariensis picornavirus 1 and 3) ( Fig. 3F ). R. madagascariensis picornavirus 2: accession OQ818346 , was a short sequence (<2000bp in length) and was therefore excluded from phylogenetic analysis. Shanbaviruses, a Picornaviridae genus first described from Chinese Miniopterus bats, and other unclassified bat picornaviruses ( bat picornavirus 7 and BtYS4) were basal to these African bat picornaviruses and phylogenetically distinct from a Shanbavirus A sequence from a Danish Myotis daubentonii , again suggestive of some host-virus co-speciation relationships, though further sampling will be needed to parse true evolutionary relationships ( Fig. 3F ). Bat sapeloviruses formed a monophyletic clade sister to marmot and Tasmanian-devil sapeloviruses - excepting one Myotis -hosted sapelovirus - with simian and porcine-hosted sapeloviruses basal in the phylogeny ( Fig. 3G ). Within the larger bat sapelovirus clade, two sister clades formed between Eidolon -hosted sapeloviruses and Rousettus / Eonycteris -hosted sapeloviruses ( Fig. 3G ). As seen with BLAST, E. dupreanum sapeloviruses 1 and 2, and R. madagascariensis sapelovirus 1 had respectively high identity to those viruses in Africa hosted by other bat species within the same genera ( Fig. 3G , Supplemental table 2 ). In a similar pattern as the Sapelovirus phylogeny, bat teschoviruses formed a divergent and distinct clade to those hosted by swine (porcine) – which are the usual hosts of teschoviruses 76 ( Fig. 3H ). Within the bat-hosted teschoviruses, viruses again clustered following species phylogenetics: R. madagascariensis teschovirus 1, R. madagascariensis teschovirus 2, and E. dupreanum teschovirus 1 grouped with similar viruses hosted by other bat species within the same respective genera ( Fig. 3H ). In general, in virus clades with prior, publicly-reported viruses for comparison, Malagasy bat Picornaviridae from E. dupreanum and R. madagascariensis were, respectively, phylogenetically closest to E. helvum and R. aegypticus -hosted Picornaviridae described from Cameroon, Kenya, and Uganda. Caliciviridae Bat sapoviruses, in the genus Sapovirus , are phylogenetically distinct from human and swine-hosted Sapporo viruses, which also fall within the Sapovirus genus ( Fig. 3I ). In our analyses, most bat sapoviruses formed a monophyletic clade proximal to the human and swine Sapporo viruses, Myotis spp. -hosted sapoviruses resolved as basal in the Sapovirus clade. This apparent paraphyly in bat Sapovirus sp. indicates a potential for host-switching events leading to the diversification of this genus ( Fig. 3I ). Novel E. dupreanum and R. madagascariensis sapoviruses again nest sister to closely related African E. helvum and R. aegypticus -hosted sapoviruses, respectively ( Fig. 3I ). Similarity analysis Picornaviridae Within the Picornaviridae family, many novel sequences had the highest identity (average >80% BLASTx 55 identity) to African (Cameroon, Kenya, and Uganda) E. helvum and R. aegypticus viruses ( Supplemental table 2, Fig. 3A-I ), which are sister host species to E. dupreanum and R. madagascariensis . When comparing Madagascar sequences against their closest matches in GenBank, we observed a consistent sharp drop in similarity in the 2A-to-2B and 3A-to-3B broad peptide regions of the genome ( Fig. 4A-E , Supplemental fig. 3 for amino acid similarity plots , Supplemental fig. 4 for nucleotide similarity plots). The border of the P1 and P2 regions (between VP1 and 2A, respectively) are thought to comprise a genomic region susceptible to recombination 25 , while the 3A region of Picornaviridae , known to be highly divergent across genera, is associated with host range determination and viral replication 77 , thus offering some explanation for the heightened genomic divergence in these regions. We also consistently observed drops in similarity in the 5’ and 3’ UTRs ( Fig. 4A-E , Supplemental fig. 3 and 4 ). The 5’ UTR of Picornaviridae is thought to play a role in antagonizing innate host immunity; indeed, work in enteroviruses shows that the development of mutations in this region can dampen replication competence of the virus 78 . As the 5’ UTRs of most of the novel Malagasy Picornavirales demonstrated very low similarity (<50%) to related reference sequences ( Fig. 4A-E ), it is possible that these Malagasy bat viruses employ different replication and immune evasion strategies than previously documented for this virus family ( Fig. 4A-E ). Download figure Open in new tab Figure 4: Amino acid similarity computed in PySimPlot 59 for novel full-length sequences. Similarity analyses with query sequence (A) E. dupreanum hepatovirus : accession PP766455 , (B) E. dupreanum kunsagivirus : accession OQ818217 , (C) R. madagascariensis picornavirus 1: accession OQ818325 , (D) E. dupreanum sapelovirus : accession OQ818321 , (E) R. madagascariensis teschovirus 1: accession OQ818323 , and (F) E. dupreanum sapovirus 1: accession PP766459 against similar sequences identified from BLAST and other matched novel sequences within the same genus. Novel sequences described in this study are starred with an asterisk. Line color corresponds to different virus sequences, with annotated regions of the genome below each plot. Plots were generated with a window size of 100aa and a step size of 20aa. Peptides in orange and corresponding grey shaded areas denote areas of interest for host interactions and immunogenicity, and blue peptides denote 5’ and 3’ UTRs. Amino acid similarity plots for Kobuvirus and Mischivirus are in Supplemental fig. 3 . Matched nucleotide similarity plots are in Supplemental fig. 4 . For example, novel E. dupreanum hepatovirus sequences were nearly identical to each other, and across the genome, demonstrated highest identity to Hepatovirus H2, from a E. helvum bat ( Fig. 4A ). Nonetheless, the Malagasy viruses showed a large dip in identity to the E. helvum virus in the 5’ UTR (<25% average identity), in addition to other dips across the 2A/2B peptides, the 3A/3B peptides, and within the 3’ UTR ( Fig. 4A ). E. dupreanum kunsagivirus mirrored these patterns, showing reduced identity to previously described kunsagiviruses in the 5’ UTR, 2A, 3A, and 3’ UTR regions, in addition to a slight reduction in identity in the 2C region ( Fig. 4B ). E. dupreanum kobuvirus sequences described in this study were nearly identical across the genome to an E. dupreanum kobuvirus sequence described previously 52 ( Supplemental fig. 3A ). P. rufus mischivirus had low identity to comparable sequences across the genome (<75%), with more dramatic drops observed in the 5’ UTR and the 3A peptide regions ( Supplemental fig. 3B ). R. madagascariensis picornavirus 1 sequences had high (∼99%) identity across the genome to each other, and R. madagascariensis picornavirus 3 was most similar to African Rousettus bat picornavirus accession PP711945 ( Fig. 4C ). Though R. madagascariensis picornavirus 1 and 3 were derived from the same host species, they nonetheless demonstrated <80% average identity to one another and demonstrated predictable divergence in the 2A, 3A, and 5’ and 3’ UTR regions, suggesting that these viruses likely had different replication and immune evasion strategies ( Fig. 3C ). Similar to R. madagascariensis picornaviruses, the novel E. dupreanum and R. madagascariensis sapeloviruses had high identity to sister species (e.g. E. helvum and R. aegypticus, respectively)-hosted viruses, but still differed in 5’ UTR, 2A peptide, and 3A peptide regions ( Fig. 3D ). R. madagascariensis teschovirus 1 was not as closely related to R. madagascariensis teschovirus 2 ( Fig. 3E ) as were all R. madagascariensis bat picornaviruses ( Fig. 3C ). However, E. dupreanum teschovirus 1 had highest identity to Rousettus bat picornavirus: PP711948, derived from a R. aegypticus host in Uganda ( Fig. 4E ). As before, we observed drops in similarity between the Malagasy bat teschoviruses and previously described sequences in the 3A peptide region, in addition to drops in similarity in VP2 and VP1 regions, as well ( Fig. 4E ). Caliciviridae For sapoviruses, we anticipated lower similarity in the VP1, NS4, and Vpg regions of the genome. VP1 is associated with host interactions and immunogenicity 79 , while Caliciviridae NS4 has been suggested to be a homolog for Picornaviridae 3A, since both families exhibit similar genome organization 80 . If true, then divergence in NS4 might be expected due to divergence in host range, though the function of NS4 is not well characterized. Vpg (or NS5 in some sources), is a nonstructural protein that primes genome replication 81 . Both novel E. dupreanum sapovirus 1 sequences were nearly identical to each other, but E. dupreanum sapovirus 1: accession OQ818319 is a partial genome and was missing the NS1/NS2 region of the genome, so it is possible some variation may exist in this region in the two sequences ( Fig. 4F ). The most similar virus, a Cameroonian E. helvum sapovirus , hovered around 70% identity to the novel query sequences across most of the genome, with three drops in similarity at the border of Vpg and Pro-Pol, on the border of Pro-Pol and VP1, and in VP1 ( Fig. 4F ). The drops in identity were not as dramatic as those witnessed in certain regions for other novel Malagasy bat picornaviruses; in general, E. dupreanum sapovirus 1 displayed an average around 50% identity to previously characterized viruses in this clade ( Fig. 4F ). As observed in Malagasy bat Picornaviridae ( Fig. 4A-E ), dips in identity across the genome corresponding to areas involving host interactions and immune responses could indicate that while E. dupreanum and R. madagascariensis -hosted sapoviruses were respectively most similar to African E. helvum and R. aegypticu s-hosted sapoviruses (range from ∼67% to ∼90% identity) ( Supplemental table 3 ), these novel viruses likely use different replication and immune evasion strategies. All R. madagascariensis sapoviruses were partial genomes, so were not included in genome wide similarity analysis or subsequent recombination analysis. RDP4 recombination analysis Picornaviridae Recombination analysis performed on novel Malagasy Picornaviridae indicated that there is evidence for genetic exchange with viruses hosted by African E. helvum and R. aegypticus ( Fig. 5 , Supplemental table 6 ). Analyses using full genome novel Picornaviridae and high identity reference sequences identified from BLAST 55 ( Supplemental table 2 ), phylogenetic analysis ( Fig. 2 and Fig. 3A-I ), and genome-wide similarity scans ( Fig. 4 ), indicated that the following genera were most likely to be under recombination pressure: Hepatovirus , Sapelovirus , and Teschovirus ( Supplemental table 5 ). No significant recombination pressure was observed in Cardiovirus , Kunsagivirus , Kobuvirus , and Mischivirus ( Supplemental table 6 ). Some recombination pressure was observed in R. madagascariensis picornaviruses and Sapovirus but was not further analyzed due to either fewer than four significant tests for recombination and/or no novel sequence identified as a recombinant sequence or major parent sequence in the alignment ( Supplemental table 6 ). Download figure Open in new tab Figure 5: Bootscan plots computed in RDP4 60 for potential recombinant sequences (A) Bat sapelovirus Bat/CAM/Sap-p24/2013: accession NC_033820 and (B) Rousettus bat picornavirus 29A/Kenya/BAT3/2015: accession PP711934 . Line color corresponds to pairwise alignments between the potential recombinant sequence, major parental sequence, and the minor parental sequence. Asterisks denote novel sequences described in this study. Horizontal dashed line refers to a 70% cutoff bootstrap percentage, and grey bars indicate regions identified as significant areas of recombination (P<0.05) across at least 5 analyses within RDP4 60 (RDP, GENECONV, Bootscan, Maxchi, Chimaera, and 3Seq). Nucleotide bootscan plots were generated using a window size of 200bp and a step size of 20bp. Genome maps are below each plot, peptides in orange denote areas of interest for host interactions and immunogenicity, and blue peptides denote 5’ and 3’ UTRs. RDP4 60 statistics are reported in Supplemental table 6 . Through RDP4 analysis, E. dupreanum hepatoviruses (both accession PP766455 and PP766457 ) were identified as possible minor parental sequences to recombinant E. helvum hepatovirus M32EidHel2010 (accession NC_028366 ), with breakpoints across the genome; however, bootstrap support was too weak to indicate any other parental sources of genomic material ( Supplemental fig. 5A ). Within Sapelovirus , the strongest bootstrap support indicated that Cameroonian bat sapelovirus Bat/CAM/Sap-P24/2013 (accession NC_033820 ) was the likely recombinant sequence with genomic material from E. dupreanum sapelovirus 1 (major parental sequence) ( Fig. 5A ). The highest support across most of the genome was for E. dupreanum sapelovirus 1 as a major parental sequence excepting the P2 region (2A, 2B, and 2C) and the 3A peptide where higher bootstrap support indicated that Kenyan Eidolon bat picornavirus 6A/Kenya/BAT606/2015 was the minor parental sequence (accession PP711943 ) ( Fig. 5A ). There was additional evidence that E. dupreanum sapelovirus 2 was a recombinant sequence with Kenyan Eidolon bat picornavirus 6A/Kenya/BAT606/2015 as a minor parental sequence across most of the genome, with highest bootstrap support for this genomic history in the P2 (2A, 2B, 2C) region and lower bootstrap support in P1 region (VP4, VP2, VP3, and VP1) ( Supplemental fig. 5B ). Evidence for recombination was highest overall within the Teschovirus genus ( Fig. 5B and Supplemental fig. 5C and 5D ), where the most likely recombinant was identified as Kenyan Rousettus bat picornavirus 29A/Kenya/BAT3/2015 (accession PP711934 ), with genomic material contributed from R. madagascariensis teschovirus 2 (major parental sequence) and R. madagascariensis teschovirus 1 (minor parental sequence), with higher support for the major parental sequence in the P1 region (VP4, VP2, VP3, and VP1) and higher support for the minor parental sequence among the rest of the genome ( Fig. 5B ). Additional analysis with a consensus sequence of R. aegypticus teschovirus sequences (accessions PP711948 and PP711934 ) further supported the involvement of R. madagascariensis teschovirus 1 and 2 in recombination, but with lower bootstrap support across the whole genome ( Supplemental fig. 5C and 5D ). Support for R. madagascariensis teschovirus 1 as a recombinant sequence with R. aegypticus teschovirus clade as a minor parental sequence was highest in the 5’UTR/3D region ( Supplemental fig. 5C ). The opposite pattern was observed in the 3’UTR in analysis of R. aegypticus teschovirus clade as a recombinant sequence with R. madagascariensis teschovirus 1 as a major parental sequence ( Supplemental fig. 5D ). VP1-VP3 peptides in Picornaviridae are thought to play a role in generation of neutralizing antibodies 82 , and as previously mentioned, the P1-P2 junction, 3A/3B peptides, and 5’ UTR also play a role in determination of host range and immune response 25 , 77 , 78 . DISCUSSION Using mNGS data, we analyzed the diversity of Picornavirales , namely Picornaviridae and Caliciviridae , finding evidence of high viral similarity between sequences from Malagasy fruit bats ( E. dupreanum and R. madagascariensis ) with those from their sister species ( E. helvum and R. aegypticus , respectively) in the mainland African countries of Cameroon, Kenya, and Uganda, although it is possible these countries simply represent the most intensely sampled localities to date. Further, we find evidence that recombination between these similar sequences may have led to the diversification of these viral genera in Malagasy fruit bats through phylogenetic clustering and recombination analyses. We have previously demonstrated potential cross-continental viral genetic exchange between African and Malagasy fruit bat-hosted nobecoviruses, so it is possible these Picornaviridae and Caliciviridae are diversifying in similar manners 46 . We identified 13 full-length and 37 partial-length (ranging from 1014 to 8379 bp in length) genomic sequences (14 Caliciviridae and 36 Picornaviridae sequences) from Malagasy fruit bats: E. dupreanum , P. rufus , and R. madagascariensis . Multiple Picornaviridae and Caliciviridae species were identified within the same sampling dates at E. dupreanum and R. madagascariensis roost sites, suggesting that many of these diverse viruses shed simultaneously within a representative bat population, a pattern that has been reported before for fruit bats 83 . Bats captured at a site within the same sampling session could reflect that roost’s viral population dynamics at a single timepoint, as they often shared similar virome profiles. Indeed, viral diversity was significantly different between roost sites dominated by E. dupreanum versus R. madagascariensis , and sapeloviruses, teschoviruses, and sapoviruses all resolved into disparate host species-specific clades. R. madagascariensis picornaviruses, which displayed the lowest identity to previously-known sequences (∼80% to known Ugandan/Kenyan R. aegypticus -hosted picornaviruses 63 ), formed their own clade distinct from their Rousettus -hosted viruses described elsewhere, which further separated into two separate clades corresponding to two different viral species. Many of the novel sequences we identified in this analysis displayed reduced identity to previously-described sequences in genomic areas that determine host range and immune response (5’ UTR, 2A/2B peptides, and 3A/3B peptides in Picornaviridae 25 , 77 , 78 , NS4 and VP1 peptides in Caliciviridae 80 , 81 ). These regions of reduced similarity indicate that, despite relatedness to sister species-hosted viruses in Africa, Malagasy bat-hosted viruses could employ different replication and immune evasion mechanisms. Our recombination analysis did not identify the 3A region, which is highly diverse in Picornaviridae and likely contributes to host range 77 , to be under recombination pressure. To our knowledge, recombination analysis has not been previously performed on any African bat Picornavirales sequences 35 , 36 , 63 , 84 . While no evidence for local recombination between the novel Malagasy viruses was found, analyses presented here suggest that recombination likely contributed to the diversification of this clade in bat hosts, and in some cases may have predated dispersal of these viruses between Madagascar and mainland Africa. Of note, we observed evidence of more virus genetic exchange between E. dupreanum and R. madagascariensis vs. P. rufus . The former two bat species are known to co-roost in caves in our system 46 , while the latter species is tree-dwelling, highlighting the importance of host proximity in viral dispersal, exchange, and diversification. Cave co-roosting has been previously shown to support recombination and diversification in bat coronavirus systems 85 . As E. dupreanum and R. madagascariensis are known to co-roost with insectivorous bats on occasion 86 , additional screening for these viral families in more Malagasy bat species may further support evidence for high fidelity to a single host species. In Madagascar and some areas of mainland Africa, humans hunt bats for food 87 – 89 . While no bat-borne zoonosis has been linked to this practice in Madagascar, undiagnosed fevers are common, and it is possible that bat virus zoonoses may be occurring undetected 90 , 91 . Enteric viruses described from Cameroonian hunters demonstrated a high diversity of Picornavirales , including some sequences which share evolutionary ancestry with bat- or other animal-hosted viruses 84 . Nonetheless, human-hosted Picornavirales in Cameroon segregate phylogenetically from animal viruses, suggesting that, while zoonosis may be possible in this clade, these cross-species emergence events are relatively rare 84 . Indeed, discrete host-species relationships appear to drive most of the observed diversification within the Picornavirales clade. While we lack Picornavirales sequences from humans in Madagascar to test these hypotheses, the bat sequences described here group with other animal (particularly bat)-derived viruses from related host species 63 . As recombination events can precede more dramatic host switches, including zoonoses, understanding of these processes within diverse viral clades is critical to assessing potential zoonotic risk. ETHICS STATEMENT The animal study was reviewed and approved by UC Berkeley Animal Care and Use Committee and Madagascar Ministry of Forest and the Environment under guidelines posted by the American Veterinary Medical Association. DATA AVAILABILITY All full and partial length genome sequences were submitted to NCBI and assigned accession numbers OQ818316 - OQ818318 , OQ818320 - OQ818324 , OQ818328 , OQ818329 , PP766456 , PP766459 , PP766469 (full-length genomes), and OQ818319 , OQ818325 , OQ818337 , OQ818340 , OQ818342 - OQ818348 , PP766449 - PP766455 , PP766457 , PP766458 , PP766460 - PP766468 , PP766470 - PP766477 (partial-length genomes). Detailed descriptions of analyses done in this paper, including scripts used to generate figures, are available on our GitHub ( https://github.com/brooklabteam/mada-bat-picornavirus ). AUTHOR CONTRIBUTIONS CEB conceived of the project and acquired the funding, in collaboration with J-MH, VL, and PD. Field samples were collected, and RNA extracted by CEB, HCR, SA, AA, TR, GK, and VR. AK led the mNGS at CZB, with support from VA, HCR, TR, CEB, and JLD. ARH led the mNGS at NIH, with support from FL, LL, RD, SG, CEB, and DCD. GK analyzed the resulting data and wrote the original draft of the manuscript with CEB, which all authors edited and approved. SH and ECR provided noteworthy edits to the manuscript and contributed to finalization of the manuscript. FUNDING Research was funded by the National Institutes of Health (1R01AI129822-01 grant to J-MH, PD, and CEB and 5DP2AI171120 grant to CEB, 5DP2AI171120-S1 to CEB and GK), DARPA (PREEMPT Program Cooperative Agreement no. D18AC00031 to CEB), the Bill and Melinda Gates Foundation (GCE/ID OPP1211841 to CEB and J-MH), the Adolph C. and Mary Sprague Miller Institute for Basic Research in Science (postdoctoral fellowship to CEB), the Branco Weiss Society in Science (fellowship to CEB), Department of Education Graduate Assistance in Areas of National Need (P200A210054 fellowship funding to GK), and the Chan Zuckerberg Biohub. This work was funded in part by the intramural program of the National Institute of Allergy and Infectious Diseases. ACKNOWLEDGMENTS The authors acknowledge Anecia Gentles, Kimberly Rivera, Fifi Ravelomanantsoa, and Sarah Guth for help in the field and lab. We acknowledge the Virology Unit at the Institut Pasteur de Madagascar. We thank Amy Kistler, Cristina M. Tato, Maira Phelps, Vida Ahyong, Angela Detweiler, Michelle Tan, Norma Neff, and Joseph L. DeRisi of the Chan Zuckerberg Biohub (CZB) for logistical support. We additionally thank Angela Detweiler, Michelle Tan, and Norma Neff of the CZB genomics platform for mNGS support and thank the Brook lab at the University of Chicago for helpful contributions to the manuscript. This work was completed in part with resources provided by the University of Chicago’s Research Computing Center. CITATIONS 1. ↵ Le Gall , O. , et al. Picornavirales, a proposed order of positive-sense single-stranded RNA viruses with a pseudo-T = 3 virion architecture . Arch. Virol . 153 , 715 ( 2008 ). 2. ↵ Cao , G. , Jing , W. , Liu , J. & Liu , M . 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Longtine , Sucheta Godbole , Sophia Horigan , Emily Cornelius Ruhs , Vololoniaina Raharinosy , Tsiry Hasina Randriambolamanantsoa , Vincent Lacoste , Jean-Michel Heraud , Philippe Dussart , Daniel C. Douek , Cara E. Brook bioRxiv 2024.12.31.630946; doi: https://doi.org/10.1101/2024.12.31.630946 Share This Article: Copy Citation Tools Picornaviridae and Caliciviridae diversity in Madagascar fruit bats is driven by cross-continental genetic exchange Gwenddolen Kettenburg , Hafaliana C. Ranaivoson , Angelo Andrianianina , Santino Andry , Amy R. Henry , Rachel L. Davis , Farida Laboune , Elizabeth R. Longtine , Sucheta Godbole , Sophia Horigan , Emily Cornelius Ruhs , Vololoniaina Raharinosy , Tsiry Hasina Randriambolamanantsoa , Vincent Lacoste , Jean-Michel Heraud , Philippe Dussart , Daniel C. Douek , Cara E. 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