Novel <i>Neoromicia</i> picornavirus.

<p><b>A)</b> Overview of the confirmed picornavirus contig alignment positions in reference to a typical picornavirus genome. The dark grey contig (1077bp) of the P1/P2 region was used to construct a Bayesian phylogeny. <b>B)</b> The maximum clade credibility tree constructed in BEAST v1.8 used the GTR plus invariant sites and gamma distribution substitution model. The genera are indicated on the right side of the sequence names (with GenBank accession numbers); the <i>Neoromicia</i> picornavirus from this study is indicated with a black circle. Posterior probability values of less than 50% were omitted.</p

Map of Africa depicting the overlapping distributions of dromedary camels and cape serotine bats as hosts of MERS and related coronaviruses.

<p>The map was constructed in ArcMap v.10.4.1. The geographic distribution of dromedary camels are depicted with horizontal lines, with seroprevalence data of MERS antibodies detected from surveillance activities in camels shown with crossed lines. The distributions of <i>N</i>. <i>capensis</i> were taken from museum collections (point data) and thus extrapolated as modelled data. Clipart images of camels or bats show where viral RNA of MERS and MERS-related strains and have been reported.</p

Strategies for sample pooling and data analysis workflows.

<p><b>A)</b> The workflow depicts the sample pooling strategies utilized in the study. Faecal and rectal samples were primarily pooled for initial viral metagenomic sample processing. Subsequently, remaining sample material (faecal homogenates, rectum, or intestine) available for individual bats were pooled according to species and location to associate selected viral families with specific species. A further step was implemented to determine specific host individuals harbouring coronaviruses in order to clarify exact sampling locations. <b>B)</b> Bioinformatics data analysis of Illumina reads with a developed workflow in CLC genomic workbench that trims data for quality, removes adaptors and depletes host reads based on set mapping parameters. Remaining reads were taxonomically assigned with BLASTn and MEGAN v6. <b>C)</b> Shows the BLASTn assignments of reads according to virus genome types: ssRNA, ssDNA and dsDNA viruses.</p

<i>Phenuiviridae</i> sequence identified from the <i>Neoromicia</i> virome.

<p><b>A)</b> Alignment of the <i>Phenuiviridae</i> contig in reference to a typical L segment gene. <b>B)</b> The phylogenetic tree was constructed with a 267 bp region of the bunyavirus L genome segment using BEAST v1.8 with the GTR substitution model plus invariant sites. Relevant genera are shown on the right and GenBank accession numbers of each sequence are provided; the novel <i>Neoromicia</i> bunyavirus sequence from this study is indicated with a black circle. SFTSV = Severe fever with thrombocytopenia syndrome virus.</p

A metagenomic viral discovery approach identifies potential zoonotic and novel mammalian viruses in <i>Neoromicia</i> bats within South Africa

<div><p>Species within the <i>Neoromicia</i> bat genus are abundant and widely distributed in Africa. It is common for these insectivorous bats to roost in anthropogenic structures in urban regions. Additionally, <i>Neoromicia capensis</i> have previously been identified as potential hosts for Middle East respiratory syndrome (MERS)-related coronaviruses. This study aimed to ascertain the gastrointestinal virome of these bats, as viruses excreted in fecal material or which may be replicating in rectal or intestinal tissues have the greatest opportunities of coming into contact with other hosts. Samples were collected in five regions of South Africa over eight years. Initial virome composition was determined by viral metagenomic sequencing by pooling samples and enriching for viral particles. Libraries were sequenced on the Illumina MiSeq and NextSeq500 platforms, producing a combined 37 million reads. Bioinformatics analysis of the high throughput sequencing data detected the full genome of a novel species of the <i>Circoviridae</i> family, and also identified sequence data from the <i>Adenoviridae</i>, <i>Coronaviridae</i>, <i>Herpesviridae</i>, <i>Parvoviridae</i>, <i>Papillomaviridae</i>, <i>Phenuiviridae</i>, and <i>Picornaviridae</i> families. Metagenomic sequencing data was insufficient to determine the viral diversity of certain families due to the fragmented coverage of genomes and lack of suitable sequencing depth, as some viruses were detected from the analysis of reads-data only. Follow up conventional PCR assays targeting conserved gene regions for the <i>Adenoviridae</i>, <i>Coronaviridae</i>, and <i>Herpesviridae</i> families were used to confirm metagenomic data and generate additional sequences to determine genetic diversity. The complete coding genome of a MERS-related coronavirus was recovered with additional amplicon sequencing on the MiSeq platform. The new genome shared 97.2% overall nucleotide identity to a previous <i>Neoromicia</i>-associated MERS-related virus, also from South Africa. Conventional PCR analysis detected diverse adenovirus and herpesvirus sequences that were widespread throughout <i>Neoromicia</i> populations in South Africa. Furthermore, similar adenovirus sequences were detected within these populations throughout several years. With the exception of the coronaviruses, the study represents the first report of sequence data from several viral families within a Southern African insectivorous bat genus; highlighting the need for continued investigations in this regard.</p></div

<i>Neoromicia</i> adenovirus sequences.

<p><b>A)</b> Overview of confirmed mastadenovirus contigs from the <i>Neoromicia</i> virome created with Velvet and CLC assemblers as they align to a characteristic mastadenovirus genome. The dark grey contig was used in B, along with the amplicons produced by conventional PCR (depicted by the checkered block). <b>B)</b> Bayesian phylogenetic tree of a 237 bp region of the DNA polymerase gene. The phylogeny was constructed in BEAST v1.8 using the Hasegawa, Kishino and Yano (HKY) substitution model plus gamma distribution model suggested by J-model test. The MCMC chain was set to 20,000,000 generations sampled every 2000 steps, with a 10% burn-in of the first generated trees. Adenovirus sequences detected from this study are shown with black circles, and bat species from which adenoviruses originated are indicated on the right side of the sequence names. Posterior probability values of less than 50% were omitted. GenBank accession numbers are shown next to sequences.</p

<i>Betacoronavirus</i> full genome phylogeny.

<p><b>A)</b> The full genome phylogeny of 4 lineages (A-D) of the genus <i>Betacoronavirus</i> constructed using BEAST software with the GTR substitution model using invariant sites and gamma distribution. The MCMC chain was set to 15,000,000 generations sampled every 1500 steps, with a 10% burn-in of the first generated trees and displayed as a radial tree in Figtree. The lineages are indicated with clipart images of host species. Also displayed are the averaged pairwise similarities between lineages as well as highlighted similarities between human coronaviruses and related viruses identified in bats (and other animals). <b>B)</b> Close-up of the external nodes of the lineage B phylogeny to show relative distances of human and civet SARS-CoV strains and SARS-related <i>Rhinolophus</i> strains (WIV1, Rp3, Rm1 and HKU3). <b>C)</b> Close-up of the lineage C external nodes depicting the human and camel MERS strains with the bat MERS-related viruses (BtCoVNeo5038 from this study is indicated with a star). Sequence abbreviations and GenBank accession numbers are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0194527#pone.0194527.s011" target="_blank">S10 Table</a>.</p

Novel <i>Neoromicia</i> cyclovirus.

<p><b>A)</b> The circular genome organization of NeoCycloV-1 (19681/RSA) is shown with the rep and cap genes in opposing directions. The characteristic <i>Cyclovirus</i> nonamer is indicated at nucleotide position 99. <b>B)</b> Bayesian phylogeny of representative species of the <i>Circoviridae</i> using complete genomes. The phylogenetic tree was constructed in BEAST v1.8 using GTR plus invariant sites and gamma distribution substitution model. The genera are indicated on the right side of the sequence names (with indicated GenBank accession numbers), and the <i>Neoromicia</i> cyclovirus from this study is indicated with the black circle. Chicken anemia virus from the <i>Anelloviridae</i> family was used as an outgroup. Posterior probability values of less than 50% were omitted.</p

Map of <i>Neoromicia</i> sampling locations.

<p><b>A)</b> Sampling locations of <i>Neoromicia</i> species from 2007 to 2015 in the north eastern regions in South Africa. The map was plotted according to GPS coordinates in QGIS 2.0.1. <b>B)</b> Proportion and number of <i>Neoromicia</i> species collected per province for the <i>Neoromicia</i> virome analysis. LP = Limpopo, MP = Mpumalanga, GP = Gauteng, KZN = KwaZulu-Natal, NW = North West.</p

<i>Neoromicia</i> herpesvirus sequences.

<p><b>A)</b> Region of the herpesvirus DNA polymerase targeted with the molecular detection assay [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0194527#pone.0194527.ref038" target="_blank">38</a>]. <b>B)</b> The phylogenetic tree of a conserved 215 bp segment the herpesvirus DNA polymerase gene. The phylogeny was constructed in BEAST v1.8 using the GTR plus invariant sites and gamma distribution substitution model as suggested by J-model test. The <i>Neoromicia</i> herpesvirus sequences from this study are indicated with the black circles, and the species from which they originated are shown. The subfamily that compared sequences may possibly belong to, are indicated with GenBank accession numbers on the right side of the phylogenetic tree.</p