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Background Rodents are widely distributed and are the natural reservoirs of a diverse group of zoonotic viruses. Thus, analyzing the viral diversity harbored by rodents could assist efforts to predict and reduce the risk of future emergence of zoonotic viral diseases. Rodents are commonly used in animal testing, particularly mice and rats. Experimental rats are important animal models, and a history of pathogenic infections in these animals will directly affect the animal trial results. The pathogenicity of Anellovirus (AV) remains poorly understood due to the lack of a suitable model cell line or animal to support the viral cycle. This study aimed to discover possible anelloviruses from the virome in feces of experimental rats by viral metagenomic technique. Methods Fecal samples were collected from 10 commercial SD rats and pooled into a sample pool and then subjected to libraries construction which was then sequenced on Illumina MiSeq platform. The sequenced reads were analyzed using viral metagenomic analysis pipeline and two novel anelloviruses (AVs) were identified from fecal sample of experimental rats. The prevalence of these two viruses was investigated by conventional PCR. Results The complete genomic sequence of these two AVs were determined and fully characterized, with strain name ratane153-zj1 and ratane153-zj2. The circular genomes of ratane153-zj1 and ratane153-zj2 are 2785 nt and 1930 nt in length, respectively, and both include three ORFs. Ratane153-zj1 closely clustered with members within the genus  Wawtorquevirus  and formed a separate branch based on the phylogenetic tree constructed over the amino acid sequence of ORF1 of the two AVs identified in this study and other related AVs. While the complete amino acid sequences of ORF1 of ratane153-zj2 (nt 335 to 1390) had the highest sequence identity with an unclassified AV (GenBank No. ATY37438) from  Chinchilla lanigera , and they clustered with one AV (GenBank No. QYD02305) belonging to the genus  Etatorquevirus  from  Lynx rufus.  Conventional PCR with two sets of specific primers designed based on the two genomes, respectively, showed that they were detectable at a low frequency in cohorts of experimental rats. Conclusion Our study expanded the genome diversity of AVs and provided genetic background information of viruses existed in experimental rats.

Viral metagenomics reveals two novel anelloviruses in feces of experimental rats