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 .
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 .
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
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  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 .
S10 Table. Coronavirus strains and Genbank accession numbers of sequences used in the
full genome phylogeny.