Introduction The order Rodentia is the single largest group of mammalian species accounting for 40% of all mammal species [1]. There are ca 2200 living rodent species, including mice, rats, voles, squirrels, prairie dogs, beavers, chipmunks, and guinea pigs. Many rodents have mixed diets but some eat mostly seeds or green vegetation. Rodents are known to vector more than 60 known human infectious diseases [2]. Some rodents live in close association with humans offering numerous opportunities for cross-species viral transmission through their urine, feces, or their arthropod ectoparasites such as ticks, mites, and fleas [2]–[8]. Rodents have been associated with numerous viruses including members of the Arenaviridae, Reoviridae, Togaviridae, Picornaviriade, and Flaviviridae families [2], [9]–[12]. The hantavirus pulmonary syndrome (HPS), an infection with an exceptionally high mortality first identified in the southwestern United States, was recognized as a zoonotic viral infection with Sin Nombre virus (SNV) in the Hantavirus genus in the Bunyaviridae family originating from deer mouse (Peromyscus maniculatus) [13]. Since then, HPS has been identified throughout the United States [14]–[17] with SNV responsible for most cases [18]–[23]. Deer mice captured in Montana showed an SNV antibody prevalence of approximately 11% [24]. Other members of the Hantavirus genus transmitted from rodents include Hantaan, Dobrava-Belgrade, Seoul, and Puumala viruses, causing hemorrahagic fever with renal syndrome (HFRS) worldwide [25]–[31]. HFRS was endemic in 28 of 31 provinces of China and is considered a major public health concern. Over 1,200 HFRS cases occurred in 2007 in China [32], [33]. It was reported that there were approximately 8,300 patients with HFRS in Inner Mongolia and 261 (3.14%) died during 1955–2006 [33]. Recently, Seoul virus was detected in 47 of 649 Norway rats (Rattus norvegicus) [34]. Several vole species (Microtus arvalis, Pitymys subterraneus, and M. subterraneus) have been linked with Tula virus also in the Hantavirus genus [35]–[39]. In Switzerland, acute infection with Tula virus was found in a 12-year-old boy after a rodent's bite [40]. Tick-borne encephalitis virus (TBEV) in the Flavivirus genus of the family Flaviviridae can cause fatal encephalitis in humans [41]–[43]. Several rodent species such as voles (Microtus agrestis and Myodes glareolus), field mice (Apodemus agrarius) are natural hosts of ticks that cause TBE [43], [44]. Lassa fever, an acute viral hemorrhagic fever first described in 1969 in Nigeria, is caused by Lassa virus, a member of the family Arenaviridae [45]. Its primary animal host is the Multimammate mouse (Mastomys [Praomys] natalensis) [3]. Lassa fever, endemic in West Africa, causes 30,000–500,000 cases and 5,000 deaths annually [46]. Because of their health and economic impact there is a growing awareness of emerging (and re-emerging) zoonotic infections [2], [7]. Increased interactions between rodents and humans occur when people build homes in wildlife habitat or conduct more recreational activities there [2], [47], [48]. Irruptions in density of rodent hosts, as occurred prior to the SNV infections in the southwestern US, also increase the risk of human viral exposure [49]. Viral surveys in wild and domesticated animals with extensive contacts with humans can be used to monitor for the presence of known zoonotic viruses or closely related viral species and to provide a baseline of the viruses present to help detect future changes associated with disease outbreaks. The identification of animal viruses closely related to human viruses also provides information regarding past successful zoonoses. To assist in these goals we performed an initial characterization of the fecal viromes of rodents from two locations in the US. Results Viral metagenomic overview Viral particles in fecal samples were purified by filtration, digested with DNase and RNase enzymes to remove unprotected nucleic acids, amplified by random RT-PCR, and subjected to 454 pyrosequencing. A total of 1,441,930 sequence reads with an average of 177-bp were generated from the extracted nucleic acids in the present study and the sequences from each animal were assembled de novo into contigs of variable length. Both singlets and contigs longer than 100-bp were then classified using BLASTx and BLASTn as likely virus, phage, bacteria, or eukaryota based on the taxonomic origin in the annotation of the best-hit sequence (E score 90% similarity at the amino acid level with known viruses, the majority exhibited 40%, >40% and >50% amino acid similarity in P1, P2 and P3 regions respectively. The similarities over these three regions in Mosavirus were less than 40% at the amino acid level to those of any reported piconaviruses. Mosavirus is therefore proposed as a novel genus in the family Picornaviridae. 10.1371/journal.ppat.1002218.g006 Figure 6 Phylogenetic analysis of Mosavirus and Rosavirus. Phylogenetic tree obtained from amino acid sequences of complete 3D proteins of all taxonomic genera in the family Picornaviridae. Mosavirus and Rosavirus are labeled with black circles. A second proposed new picornavirus genus In the same house mouse feces where Mosavirus was found, we also found another picornavirus temporarily named Rosavirus for rodent stool associated picornavirus. A genome fragment of 3,956-bp was sequenced, including a partial 2C gene, the complete P3 region and a complete 3′ UTR (GenBank JF973686). The 2C segment contained the conserved NTPase motif GXXGXGKS (GGPGCGKS) and helicase motif DDLGQ typical of picornaviruses. The P3 region of Rosavirus was hypothesized to be cleaved at 3A/3B (E↓G), 3B/3C (Q↓I) and 3C/3D (Q↓G). The 3A (106-aa) and 3B (31-aa) proteins had typical lengths but did not show any detectable sequence similarity to other picornaviruses. 3B did have a conserved tyrosine at position 3 and another conserved glycine at position 5, consistent with its function as the genome-linked protein, VPg. The 3C, with 206 amino acids, had the H-D-C catalytic triad at positions 47, 90, and 158, followed by 15 amino acids downstream of the GIH motif, similar to the substrate-binding site of chymotrypsin-like proteases. By BLAST, 3C contained the peptidase C3 superfamily and was genetically closest to turkey hepatitis virus, sharing 30% amino acid similarity. Rosavirus 3D contained conserved RdRp motifs KDELR, YGDD and FLKR. Interestingly, the 3D protein in Rosavirus has a mutated motif GAMPSG compared with conserved GG[LMN]PSG in other picornaviruses. The 3D protein in Rosavirus was most closely related to the 3D of avian turdivirus 2, sharing 44% amino acid similarity. In addition, Rosavirus had the longest reported 3′ UTR of 795-bp in length. The P3 region in Rosavirus showed very low amino acid similarity ( 10−5 were deemed unclassifiable. Complete genome sequencing of circular DNA viruses Complete circular DNA viral genomes were amplified using inverse PCR (iPCR) with specific primers designed from 454 derived short-sequence fragments. iPCR amplicons were then directly sequenced by primer walking. PCR reactions contained 2.5 U of LA Taq polymerase (Takara) in 2.5 µl of 10X LA PCR buffer (Takara), 4 µl of 2.5 mM dNTP (Takara), 2.5 µl of forward and reverse primers (10 pmol/µl), and 2.5 µl of nucleic acids (for first round) or 1 µl of the first-round PCR product (for second PCR round) as a template in a 25 µl total volume. PCR was performed at 94°C for 1 min, followed by 30 cycles of 98°C for 10 s, 68°C for 4–10 min depending on the sizes of the expected amplicon, and a final extension at 72°C for 10 in, and then held at 4°C. Genome acquisition and detection of linear RNA viruses All primers are described in Table S6. Sequence reads showing significant BLASTn or BLASTx hits to Aichi virus were linked together using RT-PCR. The 5′ and 3′ rapid amplification of cDNA end (5′ and 3′ RACE) was used to acquire the 5′ and 3′ extremities of the Aichi-like virus genome [89], [145]–[147]. For the complete genome of astrovirus, pairs of specific reverse (Ast-R1 and Ast-R2) and forward primers (Ast-F1 and Ast-F2) designed from an initial astrovirus-like-sequence of 414-bp were used in 5′ and 3′ RACE [89], [145]–[147] to amplify ∼1.5-kb and 5-kb PCR products, respectively. In a mouse (Peromyscus crinitus) fecal specimen two small picornavirus-like-genome fragments (300-bp and 251-bp, respectively) were detected. PCR failed to link these two fragments together, suggesting that they belonged to two different viruses. For the complete genome of the Mosavirus, specific reverse (Mosa-R1 and Mosa-R2) and forward primers (Mosa-F1 and Mosa-F2) were used in 5′ and 3′ RACE [89], [145]-[147] to amplify ∼1.5-kb and 6-kb PCR products, respectively. For the Rosavirus, a pair of specific forward primers (Rosa-F1 and Rosa-F2) were used in 3′ RACE [89], [146], [147] to amplify ∼4-kb amplicon. 5′ RACE was not successful. To investigate the prevalence of Aichi-like virus, consensus primers were used for PCR screening designed on a nucleotide alignment of the 2C-3B region of all human Aichi virus genotypes available in GenBank and the mouse Aichi-like virus strain characterized here. For the RT reaction, 10 µl of extracted RNA was added to 10 µl of RT mixture containing 4 µl of 5X First-Strand buffer (Invitrogen), 1 µl of 10 mM dNTP (Fermentas), 1 µl of random primer, 1 µl of SuperScript III Reverse Transriptase (Invitrogen), 1 µl of RNase inhibitor (Fermentas), and 1 µl of DEPC-treated water. The RT reaction mixture was incubated at 25°C for 5 min, 50°C for 60 min, 70°C for 15 min to inactivate the enzyme, and then held at 4°C. For the first round PCR, 2.5 µl of cDNA template was mixed with 2.5 µl of 10X ThermoPol Reaction buffer (New England Biolabs), 0.5 µl of 10 mM dNTP (Fermentas), 2.5 µl of each primer (10 pmol/µl) (Ai-Deg-F1 and Ai-Deg-R1), targeting the Achi-like virus, 0.4 µl of Taq DNA Polymerase (New England Biolabs). DEPC-treated water was added up to a 25 µl total volume. The PCR condition was as follows: denaturation at 95°C for 5 min, 35 cycles of 95°C for 30 s, 63°C for 30 s and 72°C for 1 min, a final extension at 72°C for 10 min, and then held at 4°C. The second round of amplification was performed using the same conditions except that the annealing temperature was 60°C, and inner primers Ai-(Deg-F2 and Ai-Deg-R2). The second round PCR amplification resulted in the amplicon size of 735-bp. Phylogenetic analysis and genomic structure prediction Reference viral sequences from different viral families were obtained from GenBank. Sequence analysis was performed using CLUSTAL X with the default settings. Sequences were trimmed to match the genomic regions of the viral sequences obtained in the study. A phylogenetic tree with 100 bootstrap resamples of the alignment data sets was generated using the neighbor-joining method [148]. The genetic distance was calculated using Kimura's two-parameter method (PHYLIP) [149]. Sequence identity matrix was measured using BioEdit. GenBank accession numbers of the viral sequences used in the phylogenetic analyses were shown in the trees. Putative ORFs in the genome were predicted by NCBI ORF finder. The circular genome architectures were predicted using Vector NTI 11.5 Advance (Invitrogen) with the following conditions: minimum ORF size of 100 codons, start codons ATG and GTG, stop codons TAA, TGA and TAG. To identify stem-loop structures, nucleotide sequences were analyzed with Mfold. Full and partial genome sequences are at GenBank accession numbers JF755401-JF755427, and JF973686-JF973687. The 454 pyrosequencing data is in the short read archive at SRA030869. Supporting Information Figure S1 Phylogenetic analysis of mouse Sapelovirus. Phylogenetic tree obtained from partial P1 protein of the genera Enterovirus, Sapelovirus, and Hepatovirus in the family Picornaviridae. The novel Sapelovirus is labeled with a black circle. (PDF) Click here for additional data file. Figure S2 Phylogenetic analysis of mouse adeno-associated virus (AAV) and adenovirus. A. Phylogenetic tree obtained from partial VP7 protein of AAVs. The novel AAV is labeled with a black circle. B. Phylogenetic tree obtained from hexon protein of adenoviruses. The novel adenovirus is labeled with a black circle. (PDF) Click here for additional data file. Table S1 PmPV1 nucleotide (amino acid) sequence similarity (%) to other papillomaviruses belonging to the different genera. (PDF) Click here for additional data file. Table S2 Genomic characteristics of novel circular DNA viruses detected in rodents. (PDF) Click here for additional data file. Table S3 Coding potential/putative proteins of the genome of mouse kobuvirus and comparison of amino acid sequence similarity (%) of the eleven proteins of a newly discovered mouse kobuvirus, human Aichi virus and bovine kobuvirus that belong to the Kobuvirus genus in the family of Picornaviridae . (PDF) Click here for additional data file. Table S4 Pairwise amino acid sequence similarity (%) between P3 regions of Mosavirus, Rosavirus and their closely-related picornavirus genera. (PDF) Click here for additional data file. Table S5 Pairwise amino acid sequence similarity (%) between novel mouse astrovirus, its closely related astroviruses, and rat astrovirus. The upper right and lower left were ORF2 (capsid) and ORF1b (RdRp) similarities, respectively. (PDF) Click here for additional data file. Table S6 Primers used in the study. (PDF) Click here for additional data file.
