Viral metagenomics reveals two novel anelloviruses in feces of experimental rats

Anelloviruses (AV) were first found in the blood of a Japanese patient in 1997 [1]. They have a small covalently closed single-stranded DNA genome. The complete DNA genome ranges from 2 to 3.9 kb in size [2, 3]. AVs are a family of small circular ssDNA viruses with a vast genetic diversity [3, 4]. The virus was first identified as the "TT virus" (TTV), meaning to the patient's initials. It was later called Torque teno virus after the Latin words “torques” (necklace) and “tenuis” (thin) referring to the organization of TTV DNA genome [5]. Since AVs lack their own DNA polymerase, genome replication is entirely dependent on the machinery of the host cell [6], where DNA polymerase forms a double-stranded DNA intermediate during the S (synthesis) process of the cell cycle [7]. As a result, gene replication and ssDNA to dsDNA translation take place in the nucleus [3]. The nucleotide sequence can be used to deduce two key open reading frames (ORFs), ORF1 and ORF2, as well as additional ORFs. These ORFs partly overlap, and their approximate sizes vary greatly among strains [8]. Transfection of TTV DNA into cell cultures, as well as bone marrow cells originating from a TTV positive human, has been used to examine the transcriptional and protein profile of AVs [6]. The study has shown that alternate translational initiation can be used to express at least 5–7 proteins with molecular weights ranging from 12 to 80 kDa [9]. At present, AVs have been identified in several mammals [10], such as pigs [11], dogs [12], masked palm civets [13], rodents [4, 14], bats [15], sea lions [16, 17] and primates [3, 18]. These viruses are highly common, with a mostly stable global spread and a high level of genetic heterogeneity [18]. Lupus, influenza, hematologic conditions, lung diseases, and myopathy are among the major diseases related to AVs [19]. They can be transmitted by sexual routes, blood transfusions, and the fecal–oral pathway [18, 20].

Rodents are widely distributed and are the natural reservoirs of a diverse group of zoonotic viruses [4, 21]. Thus, analyzing the viral diversity harbored by rodents could assist efforts to predict and reduce the risk of future emergence of zoonotic viral diseases. Furthermore, rodents are commonly used in animal testing, particularly mice and rats. Experimental rats are important animal models [22], and a history of pathogenic infections in these animals might directly affect the animal trial results. Viral metagenomics is an unbiased virus-detecting technique that is increasingly applied to non-specifically detect both already known and highly divergent viruses.

Although AVs have been identified in variety of mammal species, the pathogenicity of AV remains poorly understood owing to the lack of a suitable model cell line or animal to support the viral cycle. Here, using the viral metagenomic technique and bioinformatics analysis, we investigated the virome in the feces of experimental rats so as to finding possible AVs, which will be helpful for future establishing animal model for AV infection.

The fecal sample pool from 10 experimental rats generated a total of 4,460,218 unique sequence reads. Sequence reads were de novo assembled and compared to the GenBank nonredundant protein database using BLASTx. Results indicated that two contigs showed significant similarity to known AVs, which showed overlapping regions at the start and end of the contigs, confirming their circular genomes. The two complete viral genomes were named ratane153-zj1 (Fig. 1) and ratane153-zj2 (Fig. 2), respectively. The circular genomes of ratane153-zj1 and ratane153-zj2 are 2785 nt and 1930 nt long, with a G + C content of 50% and 46%, respectively. The ORF1, the largest ORF in these two AVs, encodes a 589 amino acid long putative capsid protein in ratane153-zj1 and 351 amino acids in ratane153-zj2, respectively.

To determine the relationship of these two novel viruses to other AVs, a phylogram was created based on the amino acid sequences encoded by ORF1. The phylogenetic analysis indicated that the two AVs identified in fecal samples of experimental rats were clustered into two distinct groups of AVs which were discovered from different wild species in the order Rodentia [14]. Besides, pairwise sequence comparisons based on the complete nucleotide sequences of ORF1 (nucleotides [nt] 384 to 2153, Fig. 1) showed that ratane153-zj1 shared the highest sequence identity of 59.81% to an unclassified AV (GenBank No. ATY37435) from wild Neodon leucuru (Fig. 3). Ratane153-zj1 closely clustered with members within the genus Wawtorquevirus and formed a separate branch (Fig. 3). While the complete nucleotide sequences of ORF1 of ratane153-zj2 (nt 335 to 1390, Fig. 2) had the highest sequence identity of 60.84% with an unclassified AV (GenBank No. ATY37438) from wild Chinchilla lanigera, and they clustered with one AV (GenBank No. QYD02305) belonging to the genus Etatorquevirus from Lynx rufus.

To investigate the prevalence of these 2 novel AVs in cohorts of experimental rats, two additional batches of fecal samples were collected with sample sizes of 13 and 15, respectively, from the same sampling site. A total of 38 samples (including 10 samples of the original samples) were subjected to PCR screening using primers showed in Table 1. For ratane153-zj1, 2 out of 10 samples from the original samples were positive while the other 28 samples were all negative. For ratane153-zj2, there was only one positive sample from the original samples and 2 out of the 28 samples collected after library construction. The gel electrophoresis bands were T-A cloned and sequenced by Sanger method. Sequencing results confirmed the existence of the AVs in these samples.

Virome investigation based on sampling from patients with immunosuppression and antiviral therapy revealed that the human plasma virome was composed of seven distinct viral families where the dominant virus family, the Anelloviridae, accounted for 68% of the total virus population and 97% of the AVs belonged to the Alphatorquevirus genus [30]. Here, we report two novel AVs, ratane153-zj1 and ratane153-zj2, that were discovered in feces of rats. Phylogenetic analysis showed that 153-zj1 clustered within a group that included the one AV from wild Neodon leucuru, and ratane153-zj2 clustered within a group that included the one AV from wild Chinchilla lanigera. AVs are subgrouped into Torque teno viruses (TTV), Torque teno mini virus (TTMV), Torque teno midi virus (TTMDV) and small anellovirus (SAV) in human [31]. Given their sequence divergence, two novel AVs identified in this study can be assigned as new rat AVs species. The latest published report (2020 Release) from the International Committee on Taxonomy of Viruses (ICTV), lists 155 species of AVs in the family Anelloviridae [32]. Based on the taxonomic classification criteria of ICTV (i.e. cut-off values for sequence divergence of entire ORF1: genera > 56%, species > 35%) [33], we deduce that ratane153-zj1 belongs to the genus Wawtorquevirus and ratane153-zj2 is a member within the genus Etatorquevirus.

AVs have been found in many vertebrate hosts including primates [3, 8, 24]. They have also been found in invertebrate mosquito, most likely because of mosquito blood feeding from vertebrate hosts [34, 35]. In addition, the Chicken anemia virus is a pathogen of chickens with a global spread that belongs to the Anelloviridae family [36]. Rodents act as important natural hosts for the transmission of a wide variety of viruses in the wild [21]. AVs were found in most rodent animals in China and UK, especially in the genera Rattus, Mus, Niviventer, and Apodemus, which had a higher AV-positive rate at the pool level [4, 14]. In this study, we describe the application of the high-throughput sequencing to examine the frequency of AVs in rats. Screening results showed these two AVs were detectable at a low frequency in these 38 feces of experimental rats. These results also provide a firm base for detecting and monitoring possible rodent-origin AVs.

The pathogenicity of AVs remains poorly understood because of the lack of a suitable model cell line or animal to support the viral cycle [14]. Large experimental animals such as pigs are costly to treat and maintain in terms of reagents and resources [11]. This study results indicated that experimental rat carries the rodent AVs, suggesting that laboratory rat be infected by these two novel AVs, which could be used in studying the viral cycle and pathogenicity.

Using viral metagenomics, two novel AVs were identified from fecal sample of experimental rats. The complete genomes sequence of these two AVs was determined and fully characterized, with strain name ratane153-zj1 and ratane153-zj2. Ratane153-zj1 closely clustered with members within the genus Wawtorquevirus and formed a separate branch based on the phylogenetic tree while ratane153-zj2 belongs to the genus Etatorquevirus. Conventional PCR with two sets of specific primers designed based the two genomes, respectively, showed that they were detectable at a low frequency in cohorts of experimental rats. Our study expanded the genome diversity of AVs and provided genetic background information of viruses existed in experimental rats, also provide a meaningful basis for the identification and tracking of potential rodent-origin AVs.