Viral metagenomics revealed diverse CRESS-DNA virus genomes in faeces of forest musk deer

In 2016, 85 forest musk deer faeces samples were collected from Chengdu, Sichuan province, China. Samples were collected by disposable materials and transported to the laboratory on dry-ice and store in the − 80 °C refrigerator. Samples were put into 1.5 ml tubes containing phosphate buffered saline (PBS). The supernatants of fecal samples were collected after vigorous eddy current for 5 min and centrifugation for 10 min (15,000 g) [36, 37].

500 μl of supernatant was filtered through a 0.45 μm filter (Millipore) to remove eukaryotic and bacterial cell sized particles. The viral particle enrichment filtrate was then treated with uncleases to digest nonparticle protected nucleic acid at 37 °C for 90 min [38]. Remaining total nucleic acid, protected from digestion with in viral capsids, were then extracted using the QiaAmp Mini Viral RNA kit (Qianen) according to manufacturer’s protocol [37, 39, 40]. Eight separate pools of nucleic acids from 85 faecal specimens were generated randomly, of which six contained ten faecal apecimens, the other one contained 12 faecal specimens and another one contained 13 faecal specimens. These eight viral nucleic acid pools, containing both DNA and RNA viral sequences, were then subjected to RT reactions with SuperScript III reverse transcriptase (Invitrogen) and 100 pmol of a random hexamer primer, followed by a single round of DNA synthesis using Klenow fragment polymerase [37, 41]. Eight libraries were constructed using Nextera XT DNA Sample Preparation Kit (Illumina) and sequenced using the MiSeq Illumina platform with 250 bases paired ends with dual barcoding for each library. The data is processed using an internal analysis pipeline running on a 32-nodes Linux cluster. Clonal reads were removed, and low quality sequence tails were trimmed with Phred quality score ten as the threshold. The adapter is trimmed using of VecScreen’s default parameters, NCBI BLASTn, with specialized parameters designed for adapter removal [42]. After deleting repeated reads and reads less than 50 in length followed by de novo assembly [43]. The contigs and singlets were matched against an internal viral proteome database using BLASTx with an E-value cutoff of < 10–5. BLASTx were used to identify viral sequences in order to annotated viral proteins available in GenBank’s viral RefSeq database [44].

Putative open reading frames (ORFs) in the circular genomes were predicted by Geneious software version 2019.0.3 [45], and the stem-loop in the circular genomes were located through the The Mfold [24] (Table 1 and Fig. 1b). If the whole genome sequence of the virus was not obtained through sequence reads analysis, inverse PCR was needed. Two whole genomes of UJSL004 and UJSL005 were acquired by screen PCR and inverse PCR. Primers are shown in an additional file (see Additional file 1). The PCR conditions in screen PCR were: 95 °C for 5 min, 31 cycles 95 °C for 30 s, 50 °C (for the first round) or 57 °C (for the second round) for 30 s and 72 °C for 40 s, a final extension at 72 °C for 5 min, resulting in an expected amplicon of 300 bp–500 bp. The PCR conditions in inverse PCR of UJSL004 were: 95 °C for 5 min, 35 cycles 95 °C for 30 s, 50 °C (for the first round) or 55 °C (for the second round) for 30 s and 72 °C for 1.5 min, a final extension at 72 °C for 5 min, resulting in an expected amplicon of 1000 bp. The PCR conditions in inverse PCR of UJSL005 were: 95 °C for 5 min, 35 cycles 95 °C for 30 s, 50 °C (for the first round) or 51 °C (for the second round) for 30 s and 72 °C for 1.5 min, a final extension at 72 °C for 5 min, resulting in an expected amplicon of 1000 bp.

The Rep protein sequences of these novel virus were homology alignment with the reference sequences in GenBank using the ClustalW program in MEGA7.0. Phylogenetic analyses were constructed using full-length rep protein of novel virus and other genetically close relatives [22, 46]. Save the aligned sequence as a Nexus form file, which was used to construct the phylogenetic tree using Bayes’ theorem in Mrbayes3.2.7 program. Using mixed models and Markov chain Monte Carlo (MCMC) methods. In phylogenetic analyses, tree samples are typically most divergent, so we introduced the average standard deviation of split frequencies (ASDSF) in MrBayes to allow quantitative evaluation of similarity among these samples. MrBayes allow users to set cut-off frequency (default value 0.10, [47‐49]). We used the “sump” and “sumt” commands to get more detailed diagnostic information after the run has completed.

Four complete CRESS-DNA genomes showing the highest sequence identity to CRESS-DNA virus. Genomes were 3518 nt (UJSL001, from library 2), 3212 nt (UJSL003, from library 3), 2148 nt (UJSL006, from library 7) and 3025 nt (UJSL017, from library 1) in length. As shown in Fig. 1a, the genomes of UJSL001, UJSL003 and UJSL017 contained two bidirectional ORFs while UJSL006 is in the same direction, encoding the putative Rep and Cap proteins. BLASTp search in GenBank based on the protein sequence of Rep showed UJSL001 shared the highest identity of 48.76% to unclassified circular virus (KY487934.1), UJSL003 shared the highest sequence identity of 44.00% to unclassified ssDNA viruses (MH617688.1), UJSL006 shared the highest sequence identity of 58.86% to an unclassified circular DNA viruses (MK858258.1) and UJSL017 shared the highest identity of 62.54% to unclassified ssDNA viruses (KU043411.1) (Table 2).

Three complete CRESS-DNA genomes showing the highest identity to Smacovirus. Genomes were 2665 nt (UJSL002, from library 3), 2866 nt (UJSL004, from library 5) were obtained through inverse PCR, and 2526 nt (UJSL007, from library 9) in length, respectively. Figure 1a manifested the genomic organization of UJSL002, UJSL004 and UJSL007, where the predicted Rep and Cap of the three viruses are differently arranged. BLASTp search in GenBank based on the protein sequence of Rep showed UJSL002 shared the highest sequence identity of 69.09% to a Bovine faeces associated smacovirus5 (NC_030125.1), UJSL004 shared the highest sequence identity of 75.39% to a Bovismacovirus (NC_039054.1) and UJSL007 shared the highest sequence identity of 95.54% to two Porprismacovirus (MH500284.1 and MH500317.1) (Table 2).

A complete CRESS-DNA genome showing the highest sequence identity to Circoviridae. Genome was 3852 nt (UJSL005, from library 6) in length. UJSL005 genome was acquired through inverse PCR based on a large contigs from library 6 and Sanger sequencing. Figure 1a indicated the genomic organization of UJSL005, where the predicted Rep and Cap of the UJSL005 in the opposite direction. BLASTp search in GenBank based on the protein sequence of Rep showed UJSL005 shared the highest sequence identity of 32.63% to unclassified Circoviridae (NC_026635.1) (Table 2).

Based on the alignment of the Rep amino acid sequences herein detected with the best matches of BLASTp search in GenBank and those of representative CRESS-DNA genomes including 6 groups of unclassified CRESS-DNA virus (CRESSV1–6), two GasCSV-like viruses, Bacterial plasmids (pCRESS1–9) and a small group of Eukaryotic plasmids (P. pulchra plasmids) from GenBank, a phylogenetic tree was constructed [50‐52]. For phylogenetic analyses, we used a dataset with 672 sequences of the Rep amino acid (Fig. 2) (Additional file 2).

UJSL001, UJSL003 and UJSL017 fall into the branch of unclassified CRESS-DNA virus (CRESSV1–2), UJSL001 and UJSL003 belong to the cluster of CRESSV2, UJSL001 showing close relationship with CRESS_AUM21936, UJSL003 showing close relationship with CRESS_AXH77830 (Fig. 3a) (see Additional file 3) and UJSL017 belong to the cluster of CRESSV1, showing close relationship with CRESSV1_KJ206566 and CRESSV1_KU043411 (Fig. 3e) (see Additional file 7). UJSL002, UJSL004 and UJSL007 belong to the cluster of Smacoviridae (Fig. 3b) (see Additional file 4), UJSL005 fall into the branch showing close relationship with pCPa-like (pCRESS4–8) clusters (Fig. 3c) (see Additional file 5) and UJSL006 fall into pPAPh2-like (pCRESS9) clusters, showing close relationship with pCRESS9_KXT29032 (Fig. 3d) (see Additional file 6).

The viral genomes described in detail here were deposited in GenBank under the following accession numbers: MN604398, MN621468- MN621470, MN621480- MN621482 and MN621476.