Discussion
With
economic globalisation and environmental changes, the scope of human
activities continues to expand, which increases the possibility of
pathogens jumping from rodent hosts to humans and domestic animals. The
risk of zoonotic EID is elevated in tropical forest areas experiencing
land-use change and wildlife biodiversity (mammal species richness),
based on the prediction of demographic, environmental, and biological
correlates associated with EID events [39]. Traditional virus
detection methods rely on cell culture based on visible cytopathic
effects and molecular detection of known viral genome sequences.
However,
the application of NGS technology enables the sequencing of pathogenic
genomes in one sample, including unknown pathogens. Hainan Island is an
important trade hub and tropical monsoon area with large animal species
diversity. Therefore, virome and phylogenetic analyses of rodent-borne
viruses in Hainan Island can provide insights into the prevention and
control of rodent-borne diseases in China.
The virome data obtained in this study revealed that the risk of
rodent-borne pathogens on Hainan Island should not be
underestimated. In this study, we
collected swab samples from six species and three genera of Muridae,
covering nine districts of Hainan Province, to analyse the viral
diversity carried by rodents living next to humans and living in natural
reserves.
The novel CoVs, PestVs, AstVs,
ParVs, and PVs identified in this study increase our knowledge of the
viral classification of each viral
family.
Many rodent-borne viruses detected
in this study were not only found on Hainan Island but also in the
southwestern (Yunnan, Hunan) and northern (Jilin, Ningxia, Inner
Mongolia, Xinjiang) regions of China [20], Thailand, Lao PDR,
Cambodia, and other parts of the world
[18].
Moreover,
we also identified widely distributed respiratory tract or enteric RNA
viruses, such as AstroV, pestivirus, papillomavirus, picornaviruses,
rotavirus, and picobirnavirus,
which is consistent with a previous
study [20]. However, the
abundance of these viruses in the metagenomic analysis in the present
study was different from that previously reported in rodent lung samples
[18]. Our data was not abundant
in viruses that are transmitted through blood or bodily fluids, such as
phleboviruses, hepaciviruses, Hantaan viruses, and
arteriviruses. This phenomenon may
be due to the different tissue tropisms of these viruses.
The results of the viral positivity
rate suggest that these viruses infect hosts primarily through the
respiratory tract, such as CoVs and PestVs. Moreover, some viruses were
positive in anal swabs but negative in throat swabs. These results may
be attributed to insufficient collection of virus-infected exfoliated
host cells during the collection of swabs or to the different
replication characteristics of faecal/oral- or respiratory-transmitted
viruses, which may be detected at different locations in different
cycles [19]. Some viruses, such
as AstroV-HMU-5, were detected in both throat and anal swabs, indicating
that the host may carry the virus for a long time and may transmit it
via the faecal-oral route (food- or water-borne routes)
[40].
Additionally, PV-HMU-5 was detected with PV-HMU-6 in the same host, and
throat and anal swabs showed PV positivity in multiple hosts, indicating
that this host group carried the
PVs for a long time, which
increases the probability of the virus becoming a potential pathogen.
The murine hepatitis virus in
murine coronaviruses was first isolated in 1949 [41], and a variant
named sialodacryoadenitis coronavirus was detected in rats in 1970
[42]. Coronavirus recombination
events are highly common, such as feline CoV type I and canine
coronavirus recombination events
[43]. Human CoVs recombine to
produce three genotypes [44] and SARS-CoV constantly undergoes
evolutionary recombination [45].
The results of the phylogenetic
analysis of the RdRp and S protein strains of CoVs were consistent.
The
four CoVs identified in this study
shared high
RdRp and S protein identities with
known murine CoVs and clustered with murine CoVs under the subgenus
Embecovirus in the evolutionary tree. CoV-HMU-4, CoV-HMU-2, CoV-HMU-3,
and mouse hepatitis virus showed a relatively close distance in the
evolutionary tree, especially CoV-HMU-3 and mouse hepatitis virus, under
the same branch. Based on the
rodent hosts of known and newly discovered CoVs, our results suggest
that the same virus can be detected in different regions and rodents,
indicating that rodents carrying CoVs may not exhibir geographic and
host specificity. The high similarity of CoVs observed in our study
suggests that each lineage shares a common ancestor. Additionally,
beta-CoVs were detected in the throat and anal swabs of rodents and only
a few clues were found in rodent lungs, suggesting that these viruses
primarily infect the upper respiratory tract whereas the absence of
alpha-CoVs in the two types of samples indicates that alpha-CoVs may not
be the main cause of lower respiratory tract infections in rodents.
Pestiviruses
may cause clinical illnesses, such as acute diarrhoea, acute
haemorrhagic syndrome, wasting disease, and transplacental infection,
leading to fetal death [46], and posing serious health and economic
burden. PestV genomic sequences have been detected in bat and rat
samples using NGS [8, 47].
In
the present study,
pairwise
alignment revealed that the RdRp of PestV-HMU-1 and PestV-HMU-2 showed
low aa identities with known pathogens, suggesting that they may be
novel species, and rodents have not
been previously reported to carry these viruses. PestV-HMU-1 was carried
by the Leopoldamys edwardsi collected from the
Huanjinjiaoling reserve and
PestV-HMU-2 was carried by the Rattus tanezumi collected from the
Chengmai. Moreover, PestV-HMU-1 was
also present with ParV-HMU-2 in Leop oldamys edwardsi from the
Huanjinjiaoling reserve of Hainan. The abundance and diversity of
viruses are high in the central mountainous areas of Hainan.
Multiple infectious ParVs have been
detected in animals; porcine ParV causes reproductive failure in pigs
[48] whereas canine and feline panleukopenia ParVs are pathogenic to
dogs and cats, respectively [49]. ParV infection can also cause
serious harm to humans; toad virus,
tusavirus, and cutavirus are mainly detected in children with diarrhoea
[50]. ParV-HMU-1 and ParV-HMU-2,
carried by Rattus andamanensi and Leopoldamys edwardsicollected from the two nature reserves in Baisha, showed low identities
with known pathogens. Overall, the virome reported in this study is
highly diverse. These results extend our knowledge of viral taxonomy and
host range and show that in inaccessible areas, there are still highly
diverse viruses that have evolved independently in their unique wildlife
hosts. If these viruses cross the host barrier, they are highly likely
to cause zoonosis.
Our
results provide a profile of the composition of the rodent-borne virome
and a baseline for rodent-borne viruses on Hainan Island, China. We
detected PestVs and bocaviruses with low identities that were not
previously reported to be carried by rodents. The associated viral
genome sequences obtained in our study were corroborated, allowing us to
further understand the phylogenetic relationship of the viruses on
Hainan Island to other regions and obtain data on the diversity and
independent evolutionary profiling of rodent-borne pathogens. This study
suggests that mammals still harbour a large number of uncharacterised
viruses, which require further investigation.
This study provides basic data for
the prevention and control of rodent-borne infectious diseases,
evaluates the potential pathogenicity of newly discovered
microorganisms, and provides a baseline for future virome studies and
identification of potential pathogens in EIDs.