Additional file 4: of Whole genome sequencing and identification of Bacillus endophyticus and B. anthracis isolated from anthrax outbreaks in South Africa

Table S1. Plasmid comparison of the four sequenced Bacillus endophyticus strains (3618_1C, 3631_9D, 3631_10C, 3617_2C) with B. endophyticus Hbe603 strain. (DOC 18 kb

Additional file 1: of A reservoir of ‘historical’ antibiotic resistance genes in remote pristine Antarctic soils

Table S1. Environmental factors of the 17 sampled sites. Table S2. The selected ARGs chosen from noradab, including the names, gene and ARG description, and ARG families. Figure S1. The number of unique ARGs and number of unique AR hosts per site. Linear model indicated in red and lowess in blue (Pearson’s correlation r = 0.89, P = 1.62e-06). Figure S2. ARG host frequencies across sampled sites. The number of different ARG hosts is indicated in green with the number of unique ARG hosts displayed in red, axis on the left. The black line represents the relative abundance, axis on the right. Figure S3. ARG redundancy analysis. The only environmental factor to display a significant impact was percentage N (P =0.024). (DOCX 129 kb

Additional file 2: of Whole genome sequencing and identification of Bacillus endophyticus and B. anthracis isolated from anthrax outbreaks in South Africa

Figure S2. Phenotypic electron microscopic examination of the morphology of B. endophyticus strains after 24 h incubation on nutrient agar containing 0.8% sodium bicarbonate stained using copper sulphate. (TIFF 4206 kb

Additional file 1: of Whole genome sequencing and identification of Bacillus endophyticus and B. anthracis isolated from anthrax outbreaks in South Africa

Figure S1. (1) Colony morphology of (a) Bacillus endophyticus that is small circular, wet and non-mucoid and (b) B. anthracis appear circular, mucoid on nutrient agar supplemented with sodium bicarbonate at 5% CO2 after incubation at 37 °C.  Colony morphology of B. endophyticus and B. anthracis on sheep blood agar incubated at 37 °C. B. anthracis shows the characteristic shiny, rough with ground-glass appearance compared to the white slimy and smooth colonies of B. endophyticus. (TIFF 2652 kb

<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

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

<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

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

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

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