Background
Methods
Sample processing
Viral metagenomics analysis
Species annotation and analyses of abundance
Passage of fecal supernatants in BHK-21 cells
Detection of astrovirus in CPE-positive cells
Phylogenetic analysis
Statistical analysis
Results
Sequencing and quality control
Sample | Insert size (bp) | Seq strategy | Raw data | Clean data | Clean_Q20 | Clean_Q30 | Clean_GC (%) | Effective (%) |
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Myodes rufocanus | 350 | (150:150) | 2,504.06 | 2,384.93 | 76.53 | 65.45 | 58.69 | 95.242 |
Apodemus peninsulae | 350 | (150:150) | 2,234.18 | 2,090.33 | 75.94 | 64.76 | 57.70 | 93.561 |
Apodemus agrarius | 350 | (150:150) | 2,045.27 | 1,994.32 | 87.12 | 78.68 | 52.56 | 97.509 |
Tamias sibiricus | 350 | (150:150) | 2,459.21 | 2,391.77 | 83.63 | 73.78 | 52.87 | 97.258 |
Sciurus vulgaris | 350 | (150:150) | 2,155.58 | 2,007.30 | 73.76 | 62.15 | 59.05 | 93.121 |
Cricetulus triton | 350 | (150:150) | 2,270.82 | 2,148.70 | 78.59 | 67.93 | 55.53 | 94.622 |
Viral communities in fecal samples, based on family-level classifications
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(1) Single-stranded RNA viruses(Orthomyxoviridae, Picobirnaviridae, Bunyaviridae, and Arenaviridae)The members of the family Orthomyxoviridae can cause cyclical pandemics throughout the world in various species[13].In this study, they are assigned to the genus Influenzavirus A and the species influenza A virus.The reads related to the family Orthomyxoviridae occupied the largest proportion of viruses. The percentage of this family of viruses in each sample was: 45.04% (MR), 51.57% (AP), 41.08% (AA), 41.9% (TS), 27.59% (SV), and 22.1% (CT) (Fig. 2A).The family members of Picobirnaviridae cause a wide variety of mucocutaneous, encephalic, cardiac, hepatic, neurological, and respiratory diseases invertebrate hosts [14]. The Picorbirnaviridae family viruses were found in all six samples. The viruses were assigned to the genus picobirnavirus, the species human picobirnavirus, Microtus picobirnavirus V-111_USA_2008, and fox picobirnavirus. It is worth mentioning that the human picobirnavirus occupied the larger proportion of viruses in the SV sample (2.64%) (Figure 2C).The family Bunyaviridae have strong infectivity, wide distribution, a high fatality rate, and can cause serious infectious diseases in humans and animals [15], Most of the members of this family, such as Rift valley fever virus, Crimean-Congo hemorrhagic fever virus, La Crosse encephalitis virus, and Hantavirus, cause deadly diseases in humans. The natural hosts for Hantavirus are rodents, and it can cause hemorrhagic kidney fever. The virus was present in all samples of the six forest rodents. Among these, the abundance in fecal of SV (3.49%) was higher than that in the other samples. The viruses of this family were assigned to the genus, Orthobunyavirus, and the species, Shamonda virus (Fig. 2B, C).Arenaviridae is an enveloped RNA virus found worldwide. The Lassa fever virus, Junin virus, and Machupo virus can cause severe diseases with a high mortality rate[16]. Thus, the prevalence of infectious diseases is closely related to the local dynamic distribution of rodents. In this study, the virus was detected in the fecal of all six species of mice.The viruses in this family are assigned to the genus, Mammarenavirus, and the species, Lassa mammarenavirus.
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(2) DNA viruses (Herpesviridae)Viruses of the Herpesviridae family are enveloped, double-strand DNA viruses, divided into three genera based on phylogenetic clustering: α-herpesvirus, β-herpesvirus, and ɣ-herpesvirus [17]. This family was detected in fecal samples of all six species of rodent. The viruses in this family were assigned to the genera, Cytomegalovirus, Varicellovirus, Mardivirus, and the species, Cercopithecine herpesvirus 5 and Gallid herpesvirus 2, respectively (Fig. 2B, C).
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(3) Other rare viruses (Nodaviridae, Baculoviridae, Tombusviridae, Myoviridae)Insect viruses (Nodaviridae, Baculoviridae), plant viruses (Tombusviridae), and phages (Myoviridae) were identified in the fecal samples. The viruses in the family of Nodaviridae were assigned to the genera, Alphanodavirus and Betanodavirus, and the species, Pariacoto virus and Barfin flounder nervous necrosis virus, respectively. The viruses in the family of Tombusviridae were assigned to the genera, Tombusvirus, and no virus in the family of Tombusviridae was assigned to the species among the top 10 most widely distributed. It is worth mentioning that AA and CT did not contain any members from Tombusviridae.No virus in the family of Baculoviridae and Myoviridae was assigned to the genera, and species among the top 10 most widely distributed.
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(4) Unclassified virusesCurrently, there is little information about unclassified viruses and their evolution in forest rodents. In our data, many reads are classified as "unclassified virus sequences" in all samples, likely to be previously unidentified viruses that have not been studied. The identification and characterization of these unclassified viruses will provide insight into the evolutionary histories of other clinically important viruses, as well as the genetic basis behind their infectivity and virulence in humans and other animals. Such information is important for the development of future treatment options and vaccine research (Fig. 2A).
Phylogenetic analysis of zoonotic
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(1) Influenza virus AThe results of bioinformatics analysis showed that a consistent sequence of Influenza virus A was obtained in six voles, and a PB1 fragment of 2341 bp was obtained after splicing, which was named Rodent Influenza virus A CHNDB/2019. The results of the sequence alignment analysis revealed that this strain had 96.79–98.72% nucleotide sequence similarity with virus strains found in environments or other organisms; among them, the similarity to GQ325637.1 Influenza A virus (A/environment/Dongting Lake/Hunan/3–9/2007(H10N8)) mouse-adapted strain was the highest. The results of the phylogenetic tree of the PB1 segment showed that the virus was on the same tree branch as A/environment/Dongting Lake/Hunan/3–9/2007(H10N8), and the genetic distance between them was the closest (Fig. 3A).
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(2) Shamonda virusThe results of bioinformatics analysis showed that a completely identical splicing sequence of Shamonda virus was obtained in six different species of rodents. An M fragment of 4314 bp was obtained through splicing, named Rodent Shamonda virus CHNDB/2019. Due to the relatively few Shamonda virus sequences published by NCBI at present, the homology alignment results showed that very few sequences were highly homologous to the genome of this virus. The nucleotide sequence similarity with Shamonda virus isolate Ib An 5550 was the highest (96.38%), and the similarity with other virus strains was 86.21%–96.20%. Phylogenetic tree analysis showed that this virus's M gene has low homology to the genes of known viruses (Fig. 3B).
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(3) MammarenavirusSamples from AA, TS, SV, CT, and AP rodents were sequenced, and a consistent spliced Lassa mammarenavirus L fragment, with a sequence length of 7178 bp, named Rodent mammarenavirus CHNDB/2019/01, was obtained. On the other hand, a 7121 bp fragment was obtained from the MR rodent, by sequencing Fragment L; it was named Rodent mammarenavirus CHNDB/2019/02. Sequence analysis showed that the nucleotide sequence similarity between the Rodent mammarenavirus CHNDB/2019/01 L fragment and the Wenzhou Mammarenavirus isolate MYR-039 was the highest at 98.59%, while that between the Rodent mammarenavirus CHNDB/2019/02 L fragment and the rat mammarenavirus isolate RnYM3-2016 was the highest at 97.89%. Phylogenetic tree analysis of L fragment was consistent with the sequence similarity results. Rodent mammarenavirus CHNDB/2019/01 and Wenzhou Mammarenavirus MYR-039 isolate were clustered on one branch, while Rodent mammarenavirus CHNDB/2019/01 and rat mammarenavirus isolate RnYM3-2016 were clustered on another branch (Fig. 3C).