31 research outputs found
A metagenomic viral discovery approach identifies potential zoonotic and novel mammalian viruses in Neoromicia bats within South Africa
Species within the Neoromicia 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, Neoromicia capensis 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 Circoviridae family, and also identified sequence data from the Adenoviridae, Coronaviridae,
Herpesviridae, Parvoviridae, Papillomaviridae, Phenuiviridae, and Picornaviridae
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 Adenoviridae, Coronaviridae,
and Herpesviridae 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 Neoromicia-associated MERS-related virus, also from South Africa. Conventional PCR analysis detected diverse adenovirus and herpesvirus sequences that were widespread
throughout Neoromicia 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.S1 File. Virome sequence data information from the Parvoviridae and Papillomaviridae
families (with references and figures).S1 Table. Neoromicia samples collected to investigate the South African Neoromicia virome.S3 Table. Neoromicia samples pooled for molecular detection of selected viruses.S4 Table. Alpha- and Betacoronavirus genera hemi-nested RT-PCR primers.S5 Table. Pairwise similarities inferred from distance estimations of full length genomes
of the Circoviridae family. Sequence similarities of viruses in the Circoviridae family inferred
from estimated evolutionary divergence calculated from pairwise distances. Full genomes were
aligned and trimmed to 1075 overlapping positions. All ambiguous positions were removed
for each sequence pair. Analyses were conducted in MEGA7 [41].S6 Table. Pairwise similarities inferred from distance estimations of an L gene region
between selected Bunyavirales. The table shows pairwise sequence similarities inferred from
evolutionary divergence estimates of 249 positions of compared Bunyavirales. The number of
base differences per site from between sequences were converted to percentage of similarities.
Standard errors for distance estimates are shown above the diagonal. Codon positions
included were 1±3 as well as noncoding. Ambiguous positions were removed for each
sequence pair as per pairwise deletion. Estimates were analysed in MEGA7 [41].S7 Table. Pairwise similarities inferred from distance estimations of a 605bp conserved
segment of the coronavirus RNA dependent RNA polymerase gene. The table shows
pairwise sequence similarities inferred from evolutionary divergence estimates of 605 positions
of compared coronaviruses. The number of base differences per site from between
sequences were converted to percentage of similarities. The sequences from this study are
highlighted in grey and closest similarities to sequences from other studies are indicated in
bold. Standard errors for distance estimates are shown above the diagonal in grey text.
Codon positions included were 1±3 as well as noncoding and ambiguous positions were
removed for each sequence pair as per pairwise deletion. Estimates were analysed in MEGA
7 [41].S8 Table. Genome annotation of BtCoVNeo5038 with similarities to compared lineage C
betacoronaviruses. The percentage similarities were inferred from pairwise distance estimates
of the base pair and amino acid differences per site for each gene and of the overall genome.
The estimates were calculated in MEGA7 [41] using pairwise deletion to treat gaps. All percentage
similarities are given in comparison to BtCoVNeoV5038. Accession number of compared
betacoronaviruses are listed in order: KC869678.4, KX574227, EF065505.1, EF065509.1,
JX869059.2, KF958702.1, KF917527.1, and KJ477102.1.S9 Table. Pairwise similarities inferred from distance estimations between betacoronavirus
full genomes. The table shows pairwise similarities inferred from evolutionary divergence
estimates of betacoronavirus full genomes. The number of base differences per site
between sequences were converted to percentage similarities. Within lineage similarities are
indicated in shaded blocks. Standard errors for distance estimates are shown above the diagonal
in grey text. Codon positions included were 1±3 as well as noncoding and ambiguous positions
were removed for each sequence pair as per pairwise deletion. Estimates were analysed in
MEGA 7 [41].S10 Table. Coronavirus strains and Genbank accession numbers of sequences used in the
full genome phylogeny.This work was financially supported in
part by the National Research Foundation (NRF) of
South Africa: the South African Research Chair
held by WMgrant no. 98339, as well as grant
numbers 92524, 85756, and 91496. The opinions,
findings and conclusions expressed are those of
the authors alone, and the NRF accepts no liability
in this regard for research supported. Additional
support was obtained byWMfrom the
Poliomyelitis Research Foundation (grant number:
12/14). The Research Trust of the National Health
Laboratory Service and the Medical Research
Council was awarded to JW. This research was
partially supported by the Grant or Cooperative
Agreement Number [5 NU2GGH001874-02-00],
funded by the Centers for Disease Control and
Prevention. Its contents are solely the
responsibility of the authors and do not necessarily
represent the official views of the Centers for
Disease Control and Prevention or the Department
of Health and Human Services. MG was supported
by funding from the NRF's Innovation bursary
award (grant UID: 79409), the Poliomyelitis
Research Foundation (grant no. 13/48), and the
postgraduate study abroad bursary program of the
University of Pretoria, which funded a research visit
to the Los Alamos National Laboratory in New
Mexico.http://www.plosone.orgam2018Mammal Research InstituteMedical Virolog
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>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
<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
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
Conserved sequences of identified <i>Neoromicia</i> coronaviruses.
<p><b>A)</b> Region of the coronavirus ORF1ab (within the RdRp gene) targeted with the molecular detection assay. <b>B)</b> The coronavirus phylogenetic tree generated using the 605bp region was constructed in BEAST v1.8 using the GTR plus invariant sites and gamma distribution substitution model. The MCMC chain was set to 20,000,000 generations, sampled every 2000 steps, with a 10% burn-in of the first generated trees. Coronavirus genera and <i>Betacoronavirus</i> lineages are indicated on the right side of the sequences. GenBank accession numbers are shown with each sequence and coronaviruses detected in this study are indicated with a black circle. Posterior probability values of less than 50% were omitted.</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
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
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