Results

Sample collection from rodents

In this study, between May 2017 and June 2021, we collected 588 throat and anal swabs of 326 rodents belonging to the order Rodentiafrom Haikou City (CDC, Fucheng, Yanfeng, and Longtang), Baisha County (Yinggeling, Huangjingjiaoling, and Bangxi) Dongfang City, Lingao County, Tunchang County, Lingshui County, Ledong County, Chengmai County, and Sanya City in the Hainan Province, which is the only tropical island province in China (Figure 1 and Table S1). Rodent species were morphologically identified and confirmed by mt-Cyt bgene sequences, including Rattus norvegicus ( 23.53%),Rattus tanezumi (38.24%), Rattus andamanensis(6.18%), Rattus losea (6.65%), Niviventer fulvescens (19.41%), and Leopoldamys edwardsi (5.00%). The species from Rattus under the Muridae are clustered on the evolutionary tree and show a close distance to the Cricetulus of the Circetidae and a far distance to the dipus andrhizomys . The 588 samples were combined into 28 pools according to rodent species, swab types, and sample locations.

NGS sequencing

According to species, a total of 136G GB of nucleotide data (7,553,938 valid reads,150 bp long) were obtained from 28 pools. Archaea, bacteria, microbial eukaryotes, and invalid reads with no significant similarity to any amino acid sequences in the viral NR protein database were excluded. A comparison of the total number of contigs and the proportion of viral reads from 6 rodent species and 15 sampling locations is shown in Figure 2a-d. The viral composition of each pooled sample is shown in Figure 2e. The readings were filtered into 57,980 contigs, and the virus-associated contigs that best matched the viral ORF proteins available in each pool were screened.

Metagenomic analysis and virome overview

An overview of collection region, rodent species, and virus-related contigs is shown in Table S3. Mammalian virus-associated contigs were assigned to 15 families (Table S4) and were composed of double-stranded (ds) DNA, dsRNA, single-stranded (ss) DNA, ssRNA viruses and retro-transcribing. Viral reads were assigned toFlaviviridae ,Coronaviridae , Arenaviridae , Astroviridae ,Adenoviridae , Parvoviridae , Papillomaviridae ,Herpesviridae ,Picornaviridae , Hepeviridae, and some arthropod viruses, including Phenoviridae and Panviridae , as well as a large group of unclassified RNA viruses. The heatmap shown in Figure 3 indicates that the viromes hosted by rodents might be related to their geographic distribution. Flaviviruses and picornaviruses are widely distributed in Chengmai, Lingao, and Baisha, with high abundance. Coronaviruses, herpesviruses, and astroviruses are widely distributed throughout the world. Pestivirus and Bocavirus are present only in the mountainous forest area of Baisha. The origins, host, structure information and accession numbers of the viruses identified in this study are shown in Table S5.

Characteristics of negative-stranded RNA viruses

Arenaviridae

Arenaviruses (AreVs; order Bunyavirales , familyArenaviridae ) are a group of enveloped RNA viruses whose genome contains at least two single negative-sense RNA segments totalling approximately 10.5kb. AreVs are currently classified into four genera:Antennavirus, Hartmanivirus, Mammarenavirus, and Retarenavirus.Mammarenavirus cause central nervous system disease and haemorrhagic fever in humans in Africa and Latin America [23-24]. Human pathogenic arenaviruses include the Tacaribe Complex (New-World) and LCMV-LASV Complex (Old-World), which include the LASV and LCMV.
To identify partial genomes, representative reads of AreVs were obtained from Rattus tanezumi collected from Lingshui (Yelin). Primers were designed for PCR amplification and using Sanger sequencing to cover the gaps. We obtained a WENV partial large segment (L) termed AreV-HMU-2 (Table S5). The preliminary experiment in our study obtained a novel WENV complete sequence fromRattus norvegicus collected from Haikou (Daoke) in 2015, termed the HMU virus. AreV-HMU-2 were detected in 9% (2/21) of the throat swabs whereas the HMU virus was detected in 4% (1/22) of the anal swabs. AreV-HMU-2 and HMU viruses showed 95.45% identity with each other and 94.8% and 93.92% identity with the WENV 3, respectively. Both viruses showed ≤ 79% identity with other mammalian pathogenic arenaviruses (Table S6). The genome of the HMU virus comprises 7,147 bp and shares high sequence similarity with the WENV isolate Rn-242 [19]. Additionally, AreV-HMU-2 and HMU virus phylogenetic analyses based on partial L showed that both viruses were closely related to WENV and LORV and formed an independent clade within the genus Mammarenavirus (Figure 4).

Characteristics of positive-stranded RNA viruses

Coronaviridae

Coronaviruses (CoVs; orderNidovirales , suborder Cornidovirineae , familyCoronaviridae ) are a group of enveloped RNA viruses with unusually large positive ssRNA genomes of, 26-32kb [25,26]. The 2020 report of the International Committee on the Taxonomy of Viruses (ICTV11https://talk.ictvonline.org/) states that CoVs can be divided into four recognised genera: Alphacoronavirus ,Betacoronavirus , Gammacoronavirus , and Deltacoronavirus . CoVs are etiological agents in animals and humans that result in respiratory, hepatic, and enteric diseases such as cold, severe acute respiratory syndrome (SARS) and (COVID-19) [27-29].
To identify partial genomes, representative reads were obtained for CoVs in Rattus tanezumi collected from Haikou (Fucheng) and Lingao and Haikou (Yanfeng), andNiviventer fulvescenscollected from Baisha (Yinggeling). We obtained a novel CoV strain complete genome sequence, named CoV-HMU-1, and three CoV RNA-dependent RNA polymerase (RdRp) sequences and spike S segments, termed CoV-HMU-2, CoV-HMU-3, and CoV-HMU-4, respectively. CoV-HMU-1 and CoV-HMU-2 were detected in 40% (10/25) and 3% (1/32) of anal swabs, respectively. CoV-HMU-4 and CoV-HMU-3 were both detected in 10% (2/20) of the throat swabs (Table S5). Sequence similarity and phylogenetic analyses of RdRp and S revealed that the identified CoVs could be classified asEmbecovirus under the genus Betacoronavirus , with RdRp and S ORFs showing 96.1-99% and 95.2-97.9% nucleotide (nt) identities, respectively (Table S7). The tree topology showed that the RdRp of CoV-HMU-1 clustered withRattus norvegicus Betacoronavirus (KM349744), CoV-HMU-3 clustered with Betacoronavirus 1 GCCDC4 (MW773844), and CoV-HMU-2 and CoV-HMU-4 clustered with Longquan Rl rat coronavirus (KF294371) (Figure 5a).
The two domains of the S protein of CoVs are related to the invasive ability of the virus into the host cell; the S1 domain is involved in cellular receptor binding and the S2 domain promotes the fusion between the viral capsid and the cell membrane. The putative S1 region was located at residues 493–873 for CoV-HMU-1, 463–775 for CoV-HMU-2 and CoV-HMU-4, and 202–442 for CoV-HMU-3. CoV-HMU-1 S1 shared 94.49% amino acid (aa) identity with Betacoronavirus HKU24, and 59.62–59.26% aa identity with lineages 2 and 3 CoVs (Figure 5b). CoV-HMU-2 and CoV-HMU-4 shared high aa identities with lineage 2 CoVs in the S1 region (88.46–96.31%) and showed 95.19% aa identities with each other. CoV-HMU-3 shared 95.00% aa identity with Betacoronavirus 1 and 66.49-59.26% with CoV-HMU-2 and CoV-HMU-4. The putative S2 region was located at residues 1081–1560 for CoV-HMU-1, 880–1408 for CoV1-HMU-2 and CoV-HMU-4, and 880–1419 for CoV-HMU-3.

Flaviviridae

Flaviviruses (orderAmarillovirales , familyFlaviviridae ) are a group of enveloped RNA viruses with positive-sense ssRNA genomes of 9-13 kb.Flaviviridae can be classified into four genera, Flavivirus , Hepacivirus ,Pegivirus , and Pestivirus , which include many important arboviruses and mammalian viruses that are responsible for various mild to severe infectious diseases in humans, primates, bats, horses, and rodents [30-32]. Yellow fever, dengue, and Zika viruses are also important human pathogens [33]. Pestiviruses (PestVs), including the economically important bovine viral diarrhoea virus and classical swine fever virus [34].
We obtained two novel complete PestV genome sequences, PestV-HMU-1 and PestV-HMU-1, using representative reads for pestiviruses inLeopoldamys edwardsi collected from Baisha and Rattus tanezumi collected from Chengmai County. The Nest-PCR was designed to screen for novel PestVs in the pool with read clues. PestV-HMU-1 and PestV-HMU-2 were detected in 12% (2/17) and 6% (1/18) of throat swabs, respectively (Table S5). Additionally, pairwise alignment revealed that the RdRp (non-structural protein 5, NS5, or NS5B) of PestV-HMU-1 and PestV-HMU-2 showed less than 62.39% and 93.33% aa identity with other PestV members (Table S8), respectively. Phylogenetic analysis based on RdRp aa sequences suggested that the two PestVs could be assigned to two different lineages compared with known PestVs; PestV-HMU-1 clustered with Kioloa rodent PestV (OL452246) and PestV-HMU-2 clustered with Norway rat pestV (NC025677) but appeared to represent a separate evolutionary lineage (Figure 6). Sequence similarity and phylogenetic analysis revealed that both novel sequences could be classified within the genus Pestivirus under Flaviviridae .

Astroviridae

Astroviruses (AstroVs; order Nidovirales , familyAstroviridae ) are non-enveloped RNA viruses with positive-sense ssRNA genomes of 6.8-7 kb. The AstroVs contained three overlapping ORF.Mamastrovirus es and astroviruses are members of the Astroviridae family. AstroVs infect humans and animals, causing diarrhoea, fever, and vomiting; the major clinical symptom is watery diarrhoea. AstroVs may be associated with meningitis, aseptic encephalitis, and meningoencephalomyelitis in humans and animals [35].
We obtained a novel AstroV strain complete genome sequence termed AstroV-HMU-4, and two AstroVs RdRp and capsid proteins, termed AstroV-HMU-3 and AstroV-HMU-5, using representative reads for AstroVs inRattus tanezumi collected from Lingao and Chengmai. AstroV-HMU-3, AstroV-HMU-4, and AstroV-HMU-5 were detected in 11% (2/18), 22% (4/18), and 96% (24/25) of the anal swabs, respectively, and AstroV-HMU-5 was detected in 68% (17/25) of throat swabs (Table S5). The identity analysis of RdRp showed that the three identified AstroVs had ≥97.59% aa identity with other known AstroVs (Table S9). Moreover, evolutionary trees were constructed for complete protein sequences of RdRp and CP. Phylogenetic analysis of RdRp and CP showed that the three AstroVs clustered with Civet astrovirus (OM451116) and rodent astrovirus (KT946730) within the genus Mamastrovirus ; however, AstroV-HMU-3 appeared to represent a separate evolutionary lineage (Figure 7).

Caliciviridae

Calicivirus virions are positive-sense, non-enveloped ssRNA viruses belonging to the family Caliciviridae . Partial Calicivirus RdRp (CaliV-HMU-1) was obtained from Rattus norvegicus in Sanya (Table S5). However, the sequence was difficult to compare at the nucleotide level and showed aa identity with other known caliciviruses. Therefore, we suggest that CaliV-HMU-1 belongs to a new species under Sapovirus , showing 68.06% aa identity with the human Sapovirus GI.

Hepeviridae

Hepeviruses include enterically transmitted small quasi-enveloped viruses with positive-sense RNA genomes. Hepatitis E is an important causative agent of acute sporadic viral hepatitis worldwide. We obtained a partial RdRp sequence of picornavirus from HaikouRattus norvegicus and named it HepeV-HMU-1 (Table S5). Sequence similarity of partial RdRp indicated that HepeV-HMU-1 showed 92.16% nt identity with Paslahepevirus isolated from Guangdong Rattus norvegicus .

Picornaviridae

Picornaviruses are members of the Picornaviridae family and are small, non-enveloped, and positive ssRNA viruses. Picornavirus can cause skin, mucous membrane, intracranial, heart, liver, nervous, and respiratory diseases in many vertebrate hosts [36]. We obtained a partial RdRp sequence of the picornavirus from Chengmai Rattus tanezumi and named it PicoV-HMU-1 (Table S5). Sequence similarity of partial RdRp indicated that PicoV-HMU-1 shared 93.14% nt identity with kobuvirus 1 isolated from Guangdong Rattus losea.

Characteristics of DNA viruses

Parvoviridae

Parvoviruses (ParVs; order Piccovirales , familyParvoviridae ) are small, non-enveloped, non-segmented, ssDNA viruses with an average genome size of 4-6 kb [37]. ParVs are classified into three subfamilies: Densovirinae ,Hamaparvovirinae , and Parvovirinae . ParVs infect many different animal hosts, including bovines, canines, bats, rodents, and nonhuman primates. Cross-species transmission of Parvovirinae among carnivores has been previously reported [38]. ParV B19 causes hydrops fetalis in fetuses, erythema infectiosum in children, arthritis in adults, and aplastic crisis in patients with hemoglobinopathies.
To identify the genomes, we used representative reads for ParVs inRattus tanezumi collected from Chengmai and Haikou (Fucheng),Rattus andamanensiscollected from Baisha (Bangxi), and Leopoldamys edwardsicollected from Baisha (Huangjingjiaoling) and obtained two novel ParV complete genome sequences, ParV-HMU-1 and ParV-HMU-2. Moreover, three ParV partial NS1 strains were obtained and named ParV-HMU-3, ParV-HMU-4, and ParV-HMU-5, respectively. The analysis revealed that 14% (3/21), 11% (2/18), and 20% (4/20) of the anal swabs were positive for ParV-HMU-1, ParV-HMU-4, and ParV-HMU-5, respectively. Moreover, 8% (3/25) and 11% (2/18) of throat swabs were positive for ParV-HMU-2 and ParV-HMU-3, respectively (Table S5). Genome identity analysis of NS1 revealed that ParV-HMU-4 and ParV-HMU-5 share ≥99.09% aa identity with other known ParVs and with each other (Table S10). Pairwise alignment of NS1 indicated that ParV-HMU-1, ParV-HMU-2, and ParV-HMU-3 showed high diversity (66.36 %, 68.79 %, and 75.58% aa identity with other known ParVs, respectively). Evolutionary trees constructed for NS showed that the five ParVs were clustered with rat bocavirus (KT454512) underbocavirus ; however, ParV-HMU-1, ParV-HMU-2, and ParV-HMU-3 appeared to represent a separate evolutionary cluster (Figure 8a).

Papillomaviridae

Papillomaviruses (PVs; orderZurhausenvirale s, family Papillomaviridae , subfamilyFirstpapillomavirinae , and Secondpapillomavirinae ) are a diverse group of small, non-enveloped viruses with dsDNA genomes of 5-8 kb. However, ancestral PVs are composed of four major ORFs that encode early (E1, E2) and late (L1 and L2) proteins. PVs can cause persistent infections in the skin and mucosal membranes in humans and mammals and may also cause epidermal proliferative lesions.
Representative reads for PVs in Rattus norvegicus collected from Tunchang (Nandian), Rattus tanezumi collected from Haikou (Yanfeng), and Niviventer collected from Baisha (Yinggeling) were used to obtain two novel PV strains with complete genome sequences, named PV-HMU-5 and PV-HMU-6, and two PVs L1 sequences, named PV-HMU-3 and PV-HMU-4. PV-HMU-3 was detected in 45% (9/20) of throat swabs whereas PV-HMU-5 was detected in 61% (11/18) of throat swabs and 55% (10/18) of anal swabs. PV-HMU-6 was detected in 50% (9/18) of throat swabs and 38% (7/18) of anal swabs whereas PV-HMU-4 was detected in 10% (2/20) of throat swabs (Table S5). Pairwise alignment revealed that the L segments of PV-HMU-3 and PV-HMU-4 showed 98.98% and 100.00% aa identity with known PVs (Table S11), respectively. Moreover, genome identity analysis of L revealed that PV-HMU-5 and PV-HMU-6 showed 89.96% and 89.63% nt identities with all other known PVs, respectively. Phylogenetic analysis based on the L nucleotide sequences revealed that four PVs were assigned to the genusFirstpapillomavirinae ; however, PV-HMU-3, PV-HMU-4, and PV-HMU-5 clustered with each other; PV-HMU-6 clustered with Mastomys natalensis papillomavirus 1 (MRU01834) and separated from the members of the PVs (Figure 8b).

Characteristics of unclassified RNA viruses

In the present study, we identified seven unclassified RNA viruses in swabs of different rodent species from different sites on Hainan Island (Table S4). The seven unclassified virus families mainly belonged to the unclassified Picornavirales underPisuvricota . After annotating the complete or partial ORF or RdRp coding regions of these fully or partially sequenced viruses, many unclassifiedPicobirnaviridae and Picornaviridae reads belonged to pools from the hosts ofRattus and Niviventer . Moreover, reads ofRhabdoviridae ,Dicistroviridae , and Chuviridae appeared in pools from Rattus hosts.Data analysis showed that rodents carry a larger proportion of viral RNA and a wider range of phylogenetic diversity. Therefore, further research on rodent-borne RNA will have important implications for expanding our understanding of the viral spectrum and pathogenicity of rodent-borne viruses.