3.1. Sequencing and quality control
Sequencing of DNA and RNA extracted from lung tissues and gut samples of 3 wild Myodes rufocanus organisms was performed. The length of the insert size was 350 bp. Bases showing overlapping information and low mass, and bases that were not measured were excluded and the total numbers of clean data obtained from the six samples (three lung and three gut samples) were 238493, 209033, 199432, 239177, 200730, and 214870, respectively. Sequencing data quality was distributed in the quality score Q20 so as to ensure a normal order of the subsequent advanced analysis. The clean sequence tags were subjected to redundancy processing using the mothur software to obtain unique sequence tags. The percentages of effective sequences of the six samples were 95.242%, 93.561%, 97.509%, 97.258%, 93.121%, and 94.622%, respectively (Table 1).
3.2. Distribution of the samples based on family-level classification
Pretreated Clean Data of all the samples was compared with the reference genome of viruses in the NCBI database, NT database, and ACLAME database to obtain an annotation to each level (from Kingdom to Species).
In total, 82 families of mammalian viruses, plant viruses, phages, insect viruses and fungal viruses were found. An overview of the reads of the top 35 families of viruses in each sample is shown in Fig. 1. In addition, an overview of the classification of the identified mouse viruses in each sample from Kingdom to Species is shown in Fig. 2 (a-f).
The top 10 most widely distributed families of viruses were Orthomyxoviridae, Picobirnaviridae, Herpesviridae, Nodaviridae, Bunyaviridae, Arenaviridae, Myoviridae, Tombusviridae, Unclassified, and Baculoviridae. The reads related to the family of Orthomyxoviridae comprised the largest proportion of viruses, especially in lung tissue samples, most of which were classified into the genus Influenzavirus A. The diverse reads related to these families occupied 25–45% of the total viral sequence reads. It is worth mentioning that lung1 and lung2 did not contain any members from Tombusviridae (Fig. 3a,b).
3.3. Single-stranded RNA viruses identified in mouse samples (Orthomyxoviridae, Picornaviridae, Bunyaviridae, and Arenaviridae)
The family Orthomyxoviridae is a group of large enveloped and segmented viruses with negative-sense single stranded RNA genomes (~ 13.6 kb in length). The members of this family can cause animal acute upper respiratory tract infection, and can spread quickly by air. Thus, there are cyclical pandemics throughout the world of species from the Orthomyxoviridae family [17]. The host of influenza viruses and their route of transmission is also important in human health, though mice can also be infected with influenza virus. In this study, the reads related to the family Orthomyxoviridae comprised the largest proportion of viruses with their percentages in each sample being: lung1: 45.04%, lung2: 51.57%, lung3: 41.08%, gut1: 41.9%, gut2: 27.59%, and gut3: 22.1%. The family were assigned to the genus of Influenzavirus A, with species of influenza A virus.
Picornaviridae are small, non-enveloped, positive single-stranded RNA viruses with a genome of 7–9 kb in size. The members of the family of Picornaviridae cause a wide variety of vertebrate hosts mucocutaneous, encephalic, cardiac, hepatic, neurological and respiratory diseases [18]. The Picornaviridae family viruses existed in all six samples and were assigned to the genus of Picobirnavirus, with species of human picobirnavirus, microtus picobirnavirus V-111_USA_2008, and fox picobirnavirus.
The family of Bunyavirus is a group of spherical enveloped viruses with negative-sense single-stranded RNA genomes (250–450 kd in length). These viruses have strong infectivity, wide distribution, and high fatality rate, and can cause serious infectious diseases in humans and animals[19]. As one of the biggest RNA virus families, it includes five genera: Orthobunyavirus, Hantavirus, Nairovirus, Phlebovirus, and Tospovirus. 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 host of Hantavirus is rodents, and it can cause kidney hemorrhagic fever. The virus was present in all samples of the three Myodes rufocanus organisms. Among these, the abundance in the gut3 (3.49%) sample was higher than in the others. In samples, the viruses of this family were assigned to the genus of Orthobunyavirus, and the species Shamonda virus.
Arenavirus is an enveloped RNA virus found worldwide. The virus genome is divided into two segments. Five human pathogenic viruses of this family have been found, including Lassa fever virus (LASV), lymphocytic choriomeningitis virus, Tacaribe virus, Junin virus, and Machupo virus. Among these, Lassa fever virus,Junin virus and Machupo virus can cause severe disease with a high mortality rate. Lassa fever virus is highly contagious and its infection is easily passed on from person to person[20]. The family of Arenavirus are commonly associated with rodents, so the prevalence of the infectious disease is closely related with the local dynamic distribution of rodents. In this study, the virus was detected in the lung tissue and feces of the 3 mice. The viruses in this family were assigned to the genus Mammarenavirus, and species Lassa mammarenavirus.
3.4. Bat main DNA viruses (Herpesviridae)
Herpesviridae family are enveloped, double-strand DNA viruses which are divided into three genera based on phylogenetic clustering: alphaherpesvirus, betaherpesvirus, and gammaherpesvirus. α-herpes viruses proliferate quickly and can cause pathological changes within cells; they include herpes simplex virus and varicella zoster virus. The growth cycle of β-herpes virus is longer and these viruses infect cells to form giant cells, such as cytomegalovirus. Target cells of ɣ-herpes virus infection are lymphoid cells, and infection can lead to lymphoid hyperplasia, such as the case with Epstein-Barr virus. This family can be detected in the lung tissues and gut samples of the 3 mice. The viruses in this family were assigned to the genera: Cytomegalovirus, Varicellovirus, and Mardivirus, and the following species: Cercopithecine herpesvirus 5 and Gallid herpesvirus 2.
3.5. Other rare mouse viruses (Nodaviridae, Baculoviridae, Tombusviridae, Myoviridae, and Unclassified)
Insect virus (Nodaviridae, Baculoviridae), plant virus (Tombusviridae), phages (Myoviridae) and unclassified viruses were identified in the samples. The viruses in the family of Nodaviridae were assigned to the genus Alphanodavirus and Betanodavirus, and species of Pariacoto virus and Barfin flounder nervous necrosis virus. The viruses in the family of Tombusviridae were assigned to the genus Tombusvirus.