Virome analysis of rodent
A total of 432 wild rodents were captured during the study period, which were classified into nine species belonging to seven genera of two largest mammalian families (Cricetidae and Muridae) within the order Rodentia (Fig. 1 and Supplementary Fig. 1). Within each suburban district, 3 to 8 species of rodents were sampled, exhibiting moderate diversity (Supplementary Table 1). Notably, the abundance of each species varied significantly across three natural habitats. Within each natural habitat, four to six species were determined, with more than two-thirds having sample sizes > 20 individuals (Supplementary Table 1).
The samples were grouped into 21 pools based on the rodent species and natural habitats and subjected to next-generation sequencing on 21meta-transcriptome libraries (Supplementary Table 2). After the quality control process, we obtained over 7.5 billion sequence reads, and assembled de novo into 525,739 contigs, among them 2,060 contigs were identified as viral sequences. Although bacterial, fungal, and plant-associated viruses consisting of approximately 1,233 contigs were also detected, we excluded them from subsequent analyses.
Based on blast alignment and the RNA-dependent RNA polymerase (RdRp) homology for RNA viruses and replicase protein genes for DNA viruses, we identified a total of 142 viral species from 26 viral families (Fig. 2A, Supplementary Table 4), including 8 DNA virus species belonging to the family Anelloviridae, Circoviridae, Genomoviridae, Parvoviridae and 134 RNA virus species belonging to 22 RNA families (Astroviridae, Caliciviridae, Chuviridae, Coronaviridae, Dicistroviridae, Flaviviridae, Hantaviridae, Hepadnaviridae, Hepeviridae, Iflaviridae, Nairoviridae, Nodaviridae, Orthomyxoviridae, Paramyxoviridae, Peribunyaviridae, Phenuiviridae, Picobirnaviridae, Picornaviridae, Polycipiviridae, Qinviridae, Rhabdoviridae, Sedoreoviridae), along with unclassified viruses from Riboviria and the order Bunyavirales, Reovirales. The predominant part of the viruses is associated with vertebrates (n = 133) and the remaining 9 were associated with invertebrates (Supplementary Table 4).
An array of viral species was found among nine rodent species, with each rodent species hosting1-74 viruses. Among them, R. norvegicus harbored the highest number of viruses (74 viruses from 12 rodents), followed by Ni. confucianus (30 viruses from 153 rodent), Apodemus draco (14 viruses from 48 rodent), Cr. longicaudatus (12 viruses from 27 rodent), Ap. peninsulae (12 viruses from 46 rodent), Ap. agrarius (7 viruses from 75 rodent), My. rufocanus (4 viruses from 19 rodent) and Al. eversmanni (3 viruses from 25 rodent). The composition at the family level exhibited significant variation across the nine rodent species (adonis test, P < 0.01; Fig. 2B), as well as among three types of natural habitats where the rodents were collected (adonis test, P < 0.01; Fig. 2C). Principal coordinate analysis (PCoA) plot demonstrated that grassland-habitating rodents displayed distinct viral composition from those observed in woodland and bushland-habitating rodents.
In addition, a total of 62 complete viral genomes were obtained from 40 viral species in 13 viral families and unclassified Riboviria (Supplementary Table 5). Sixteen of the them were obtained from novel viral species belonging to 6 families (Astroviridae, Caliciviridae, Flaviviridae, Hepeviridae, Picobirnaviridae, and Picornaviridae) and unclassified Riboviria (Supplementary Table 5). No recombination events were detected in these viruses and simplot analysis revealed their high genetic distance from 16 novel viral species across the whole-genome sequence (Supplementary Fig. 2).
Zoonotic and spillover-risk viruses
The current study identified a total of 142 viruses, out of which 25 were classified as high-risk viruses derived from 16 families (Fig. 3A), encompassing 8 zoonotic viruses (red dot) and 17 spillover-risk viruses (blue dot) (Fig. 3B). Among 25 high-risk viruses, Avian orthoavulavirus and Influenza A virus (H9N2) were identified from the highest variety of orders (22 and 15 orders, respectively), followed by Lyssavirus rabies (9 order), Rotavirus A (7 order), Beiji nairovirus (4 order), Cardiovirus B (4 order) and Chicken picobirnavirus (4 order), based on all available data (Supplementary Fig. 3). Two spillover-risk viruses, Pigeon torque teno virus and Sichuan mosquito circovirus 3, although only documented in pigeon and mosquito respectively, were identified within the highest number of rodent species in our data (four out of totally nine rodent species), indicating these viruses might probably are of rodent viruses, as opposed to other wild animals (Fig. 3B). Notably, eight vertebrate viruses were identified in rodents for the first time (Nuomin virus, Shrew hepatitis B virus, Beiji nairovirus, Influenza A virus H9N2, Avian orthoavulavirus 1, Chicken picobirnavirus, Feline hunnivirus, Pigeon torque teno virus), along with nine viruses previously considered as specific to invertebrates, mostly known to be carried by ticks (6/9), including Mukawa virus, Sichuan mosquito circovirus 3, Tick-associated circular virus-6, Tick-associated genomovirus 1, Gakugsa tick virus, Onega tick phlebovirus, Sara tick phlebovirus, Lasius neglectus virus 1, and Lasius niger virus 1 (Fig. 3B). Particular noteworthy is the identification of three spillover-risk viruses for the first time in China: chicken picobirnavirus, Lasius neglectus virus 1, and Lasius niger virus 1 (Fig. 3C). Six viruses (Mossman virus, Rodent Paramyxovirus LR11-23, Apodemus peninsulae jeilongvirus, cardiovirus C1, Theiler's encephalomyelitis virus, Bastrovirus BAS-3), although not belonging to high-risk viruses, were detected for the first time in China, which is also worth noting (Fig. 3C and Supplementary Table 6).
We further compared the zoonotic pathogens carried by the currently studied nine rodent species. With the exception of Mu. musculus, all other eight rodent species harbored at least one high-risk viruses (Fig. 3D). Among these species, Ra. norvegicus exhibited the highest number of zoonotic viruses (n = 3), followed by Cr. longicaudatus (n = 2), Ni. confucianus (n = 2), Ap. agrarius (n = 2), Ap. draco (n = 1) and Ap. peninsulae (n = 1).
Diversification and evolution of viruses in rodents
We constructed phylogenetic trees based on the protein sequences of RdRp for RNA viruses and replicase protein for DNA viruses to validate the taxonomic phylogeny of the 67 known and 75 novel viral species (Fig. 4A-H, see Figures S4-29 for detailed phylogenies). The 75 novel viruses, belonging to five phyla (Cossaviricota, Duplornaviricota, Kitrinoviricota, Negarnaviricota, Pisuviricota), encompass putative members of 10 unclassified viruses and 15 distinct viral families (Fig. 4I): Anelloviridae, Astroviridae, Caliciviridae, Dicistroviridae, Flaviviridae, Hepeviridae, Iflaviridae, Nodaviridae, Parvoviridae, Peribunyaviridae, Picobirnaviridae, Picornaviridae, Polycipiviridae, Qinviridae, Sedoreoviridae. Among these families, Picobirnaviridae (n = 41), Qinviridae (n = 6), Parvoviridae (n = 3), Caliciviridae (n = 2), Hepeviridae (n = 2), and Picornaviridae (n = 2) are predominant, with others belonging to the Anelloviridae (Cricetulus longicaudatus anello-like virus), Astroviridae (Rattus norvegicus astro-like virus), Dicistroviridae (Apodemus draco aparavirus), Flaviviridae (Myodes rufocanus hepacivirus), Iflaviridae (Niviventer confucianus iflavirus), Nodaviridae (Apodemus agrarius nodavirus), Peribunyaviridae (Niviventer confucianus picobirna-like virus 20), Polycipiviridae (Niviventer confucianus sopolycivirus), Sedoreoviridae (Apodemus peninsulae orbi-like virus), and unclassified virus from order Bunyavirales (Rattus norvegicus bunyavirus).
Cossaviricota
A total of three novel viruses from single-stranded DNA viruses of family Parvoviridae were identified, including a new member of the genus Aveparvovirus (Niviventer confucianus aveparvovirus) and two unclassified parvoviruses (Cricetulus longicaudatus parvo-like virus and Myodes rufocanus parvo-like virus) (Fig. 4B, and Supplementary Fig. 5). These newly discovered parvoviruses were found to form a distinct clade in a phylogenetic analysis, clustering together with neighbor Tetraparvovirus genus, which is a newly established genus in the Parvoviridae family. A pairwise comparison of NS1 amino acid (aa) sequences revealed that Niviventer confucianus aveparvovirus shared 52.4% identity with Psittaciform aveparvovirus, while Cricetulus longicaudatus parvo-like virus and Myodes rufocanus parvo-like virus exhibited 65.2–74.1% aa sequence identity with Phoenicopterus roseus parvo-like hybrid virus.
Duplornaviricota
A total of six viruses (four novel viruses and two known viruses) were identified within the Reovirales order of the Duplornaviricota phylum (Fig. 4D and Supplementary Fig. 8). Among the four novel viruses, three belonging to the order Reovirales exhibited significant divergence from known viruses and could not be grouped into any currently recognized virus groups. One novel virus (Shidu orbi-like virus) was discovered in the family Sedoreoviridae, which clustered within the genus Orbivirus with < 80% aa sequence identity with other orbiviruses. Two known viruses, rotavirus A and Rattus norvegicus rotavirus, were also identified within the Sedoreoviridae family.
Kitrinoviricota
A total of 8 viruses, consisting of four novel viruses and four known viruses, were identified within Kitrinoviricota, which were classified into three families (Flaviviridae, Hepeviridae and Nodaviridae) (Fig. 4E, and Supplementary Figs. 9–11). Within the Flaviviridae family, one novel virus (Myodes rufocanus hepacivirus), and three previously characterized viruses (Wufeng Niviventer niviventer pegivirus 1, Wufeng Niviventer fulvescens pegivirus 1, and Rodent hepacivirus) were identified. These viruses, all possessing genomes ranging from 9–13 kb, were clustered with other species in the Pegivirus and Hepacivirus genera (Fig. 5 and Supplementary Fig. 9). The novel Myodes rufocanus hepacivirus shared a 73.3% aa identity with Hepacivirus myodae in terms of RdRp sequence and had a full-length polyprotein encoding viral helicase1, Peptidase, and RdRP domains (Fig. 5). Within the family Hepeviridae, two novel viruses (Allocricetulus eversmanni hepe-like virus and Cricetulus longicaudatus hepe-like virus) as well as one known virus (Apodemus peninsulae hepevirus) were identified. The two novel viruses showed < 75% aa identities with their closest known relatives in terms of RdRp sequence similarity, forming a distinct branch within this family (Supplementary Fig. 10). For all currently identified Hepeviridae family members, a conserved RNA replication module containing capping enzyme superfamily 1 helicase, along with an RdRp domain was observed (Fig. 5). Within the Nodaviridae family, a novel virus (Apodemus agrarius nodavirus) was identified, which shared approximately 53.7% aa sequence identity with Nodamura virus. These two viruses together formed a unique clade distinct from other alphanodaviruses in the Nairoviridae family (Supplementary Fig. 11).
Negarnaviricota
A total of 31 negative-sense single-stranded RNA [ssRNA (-)] viruses identified, all within the Negarnaviricota phylum (Fig. 4F and Supplementary Fig. 12–20). Eight were determined as novel viruses, including six from Qinviridae family, one from Peribunyaviridae family, and one virus (Rattus norvegicus bunyavirus) from the unclassified Bunyavirales order. The novel virus within the Peribunyaviridae family, Niviventer confucianus picobirna-like virus, exhibited < 40% aa sequence identity with other members of the Peribunyaviridae family and was most closely related to Nanchang Perib tick virus 1 (Supplementary Fig. 12). The six novel viruses within the Qinviridae family exhibited significant divergence from the only Yingvirus genus, thus forming separate branch in the Qinviridae family (Supplementary Fig. 13). For the 23 known viruses identified in the Negarnaviricota phylum, Influenza A virus belonging to genus Alphainfluenzavirus in family Orthomyxoviridae, and Lyssavirus rabies belonging to Lyssavirus genus from Rhabdoviridae, displayed high diversity (Supplementary Figs. 14–15).
Pisuviricota
A total of 49 novel viruses and 33 known viruses were identified within the Pisuviricota phylum and classified into eight families (Fig. 4G, and Supplementary Figs. 21–29). The 49 novel viruses were distributed across diverse clusters representing seven families within the Pisuviricota phylum: Picobirnaviridae (n = 41), Caliciviridae (n = 2), Picornaviridae (n = 2), Astroviridae (n = 1), Dicistroviridae (n = 1), Iflaviridae (n = 1), and Polycipiviridae (n = 1). The 33 known viruses were distributed across diverse clusters representing seven families within the Pisuviricota phylum: Picobirnaviridae (n = 11), Picornaviridae (n = 10), Astroviridae (n = 5), Caliciviridae (n = 2), Dicistroviridae (n = 2), Polycipiviridae (n = 2), and Coronaviride (n = 1) respectively. One novel virus (Rattus norvegicus astro-like virus) and three known viruses were identified in the unclassified members of the family Astroviridae (Supplementary Fig. 21). The novel Rattus norvegicus astro-like virus exhibited a 58.3% aa sequence identity with Avian associated bastrovirus 2, based on comparison of RdRP amino acid sequence. Two novel viruses (Rattus norvegicus sapovirus 1 and Rattus norvegicus sapovirus 2) and two known viruses (Sapovirus rat/S4-82 and Murine norovirus) were identified within the Caliciviridae family. These two novel caliciviruses, although most closely related to the Sapoviruse genus, only shared < 60% aa sequence identity with Sapovirus GII.8 and Murine sapovirus (Supplementary Fig. 22). Both novel viruses possess a full-length polyprotein encoding RNA_helicase and RdRP domains (Fig. 5).A total of 41 novel viruses and three known viruses belonged to the Picobirnavirus genus within the Picobirnaviridae family (Supplementary Fig. 23). The dsRNAv_Picobirnaviridae_RdRp domain was identified in the RdRp protein of picobirnaviruses (Fig. 5). One novel virus (Apodemus draco aparavirus) and two known viruses (Novo Mesto dicistrovirus 1 and Rattus norvegicus cripavirus) were identified within the Dicistroviridae family. The novel Apodemus draco aparavirus was closely related to the Aparavirus genus in the Dicistroviridae family, possessing a full-length polyprotein encoding RNA_helicase, Peptidase_C3G, and Dicistroviridae_RdRp domains (Fig. 5 and Supplementary Fig. 24). Only one novel virus was identified in the Iflaviridae family, i.e., Niviventer confucianus iflavirus, which clustered with Varroa destructor virus 2, and shared aa sequence identity of approximately 36.6% (Supplementary Fig. 25). One novel virus and two known viruses were identified in the family Polycipiviridae, demonstrating a close phylogenetic relationship to the Sopolycivirus genus (Supplementary Fig. 26). Two novel viruses were identified within family Picornaviridae, including Myodes rufocanus parabovirus classified under the genus Parabovirus; and Rattus norvegicus mischivirus that were categorized as Mischivirus (Supplementary Fig. 27). Both viruses possess a full-length polyprotein encoding rhv_like, RNA_helicase, and ps-ssRNAv_RdRp-like domains (Fig. 5).
Cross-species viruses in rodents
Based on a combination of our data and the virus records from the NCBI database, cross-species events in rodents were identified at the species, genus, and family level (Fig. 6A). Out of the 142 viruses identified, 33 belonging to 15 families were found in two or more rodent species. Among them, 22 viruses were determined in two or more rodent genera, and 7 viruses were found in two rodent families (Fig. 6A). Notably, three families, Picornaviridae, Hantaviridae, and Paramyxoviridae, accounted for nearly half of the cross-species viruses (48.5%, 16/33) (Fig. 6A). All five viral species belonging to the family Hantaviridae identified herein are known to be capable of cross-species transmission. Particularly noteworthy are Rodent hepacivirus and Orthohantavirus hantanense, which exhibit a broad host range and are widely detected in 50.9% (28/55) and 29.1% (16/55) of rodent species, respectively. Additionally, it appears that cross-species transmission occurs more frequently between individual rodents living in different habitats rather than those within the same habitat. Approximately half (16/33) of the cross-species viruses were observed among individuals from different natural habitats. Ap. agrarius and Mu. musculus were more likely to share similar viruses with six species circulating within both groups, whereas Al. eversmanni tended to share viruses within its own population (Fig. 6A). Furthermore, we constructed a tripartite network to visually represent associations among 142 viruses, 55 rodent species, and their potential non-rodent hosts derived from animals and vectors.
Of these viruses, only 33 were cross-species viruses, while the majority (n = 109) exhibited host specificity at the species level by exclusively infecting rodents. Among the known viral species in our dataset, over 56.72% (38/67) were found to be associated with only one rodent species (Fig. 6B); when combined with virus records from NCBI database, this percentage increased to 88.06% (59/X). Additionally, of the novel viral species identified in our study (n = 75), most (71) were also exclusively associated with one specific rodent species. (Fig. 6C). All these results suggested the host specificity of these viruses.
Finally, we evaluated patterns of viral transmission across rodents by constructing a host-virus correlation network between 33 cross-species transmitted viruses and their respective 55 host species in Rodentia order (Fig. 6D). Cytoscape analysis revealed an intricate network consisting of 88 nodes and 123 edges, notably, network revealed that 14 rodent species carried multiple cross-species transmitting viruses, including Ni. Confucianus, Ra. Norvegicus and Ap. draco, which carried 18, 10, and 9 cross-species transmitted viruses, respectively.