A total of 250 reads with a mean length of 125 bp from five pools of fecal samples identified by high-throughput sequencing had maximum nt sequence similarity to those of HBOVs, based on the analysis of customized informatics. Finally, 16 contigs (150–796 bp) were generated by assembling these positive reads, and these contigs showed ~74.8%–78.0% nt identity to HBOVs, indicating the possible presence of novel BOVs related to known HBOVs.
The sequence from one contig targeting the conserved NS1 gene of BOV was selected for screening for BOV primers. Seven (1.75%) of the 400 fecal samples from Macaca mulatta were positive for BOV (tentatively named MmBOV) in hemi-nested PCR. The sequences from the seven BOV-positive samples showed 100.0% nt identity with each other. The information details of positive samples are provided in a Supplementary Table S2.
The almost complete MmBOV nt sequence was 4, 831 nucleotides in length. Based on similarity to other known BOVs and with use of the NCBI's ORF finder, the MmBOV genome was predicted to contain three ORFs. ORF1 (638 aa) encodes the nonstructural protein NS1, ORF2 (662/533 aa) encodes the two overlapping structural proteins VP1 and VP2, and ORF3 (212 aa) encodes the highly phosphorylated nonstructural protein NP1 (Fig. 1A). The NP1 gene of MmBOV shared a short overlapping sequence with the VP1 gene, but was separated by the NS1 gene, similar to HBOVs. Also similar to HBOVs, a splicing site in the NS gene was identified in MmBOV, indicating that it also contained the spliced NS2 protein (Fig. 1B). Like HBOVs, the MmBOV genome had a characteristically low G/C content, no GC3 content, and high (> 40) ENC values, with the latter somewhat higher than those in HBOVs (Hussain et al. 2019) (Fig. 1C). Conserved motifs, including rolling circle replication, helicase, and ATPase (421–429 aa), were identified in the predicted NP1 protein of MmBOV, similar to closely related HBOVs; the Ca2+ binding loop (Y18LGPF) and the catalytic center (H41DXXY) of phospholipase A2 in the unique region of VP1 (VP1u), which are required for parvovirus infectivity (Zadori et al. 2001), were also identified in the VP1 of MmBOV (Fig. 1D). The sequence of MmBOV has been deposited in GenBank (accession numbers: MN091929).
Figure 1. Genomic organization of MmBOV. (A) Genomic organization of MmBOV and known primate bocaparvoviruses. (B) Partial NS1 nucleotide sequence alignment of MmBOV and known primate bocaparvoviruses. (C) Genomic features of MmBOV and other primate bocaparvoviruses. (D) Amino acid sequence alignment of partial VP1 proteins of MmBOV and other primate bocaparvoviruses. The phospholipase A2 motif, consisting of the calcium binding region and catalytic residues, are shown. HBOV human bocaparvovirus, GBOV gorilla bocaparvovirus, CPZh2 central chimpanzee bocaparvovirus.
A BLASTN analysis of the complete genome revealed that MmBOV shared the highest nt identity with HBOV4 (72.0%; MG383446.1). The NS1, NP1, and VP1 sequences of MmBOV also had high degrees of similarity (68.2%/ 69.2%, 73.3%/67.6%, and 70.4%/73.1% at the nt/aa levels) with those of HBOV4 (Table 1). Table 1 lists the similarities between MmBOV and other BOVs. Simplot analysis revealed that MmBOV shared identities with human and gorilla/chimpanzee BOVs, supporting the pairwise identities shown in Table 1 and Fig. 2A. Therefore, these results suggested no obvious recombination in MmBOV, based on the known HBOV and GBOV sequences.
HBOV1 HBOV2 HBOV3 HBOV4 GBOV1 GBOV2 CPZh2 Othersa Note NS1 67.4–67.6 67.2–67.8 67.0–67.2 67.6–68.2 66.7 68.4 67.4 NT 69.7–69.8 65.8–70.8 67.5–68.3 68.3–69.2 67.7 70.5–70.6 67.0 ≤ 45.7 AA NP1 70.9–71.1 73.0–73.1 69.9–72.1 72.9–73.3 69.2 72.9 68.6 NT 63.1–63.8 66.7–66.8 62.7–67.6 66.2–67.6 61.3 67.1 60.9 ≤ 50.6 AA VP1 69.0–69.2 70.2–70.5 69.8–70.0 70.1–70.4 69.2 69.3 56.1 NT 69.7–70.0 72.1–72.5 72.3–72.7 72.8–73.1 70.5 70.3 55.2 ≤ 48.7 AA HBOV human bocaparvovirus, GBOV gorilla bocaparvovirus, CPZh2 central chimpanzee bocaparvovirus.
aAll other known bocaparvoviruses. NT and AA represent pairwise comparison of nucleotide and amino acid, respectively.
Table 1. Nucleotide and amino acid identity (%) between MmBOV and other bocaparvoviruses.
Figure 2. Comparative sequence analysis of MmBOV. A Almost complete genomic sequence identity of MmBOV and other known primate bocaparvoviruses, determined by Simplot analysis. B Variable region-based sequence alignment of MmBOV and other primate bocaparvoviruses. VR-Ⅲ, VR-Ⅵ-Ⅷ, and VR-Ⅸ are indicated in pink color. The identical residues among the human bocaparvoviruses are highlighted by gray color.
Previous structural analysis of HBOVs suggested the variable region Ⅲ (VR-Ⅲ) as a potential determinant of the tissue tropism of HBOVs, and VR-Ⅵ–Ⅷ and VR-Ⅸ as possible host range determinants (Kailasan et al. 2015; Mietzsch et al. 2017). Sequences analysis showed that the VR-Ⅲ of MmBOV was identical to that of HBOV2–4, but distinct from that of HBOV1 (Fig. 2B). In terms of host determinants, VR-Ⅵ–Ⅷ of MmBOV were similar to those of HBOVs, whereas MmBOV differed from HBOVs in the deletion of three aas in VR-Ⅸ (Fig. 2B).
To determine the relationship of MmBOV to other known BOVs, phylogenetic analyses were performed for the NS1, NP1, and VP1 nt sequences. From the almost complete genome, we observed that MmBOV formed a single branch between primate bocaparvovriruses and the cluster including dromedary camel/ bovine porcine BOVs in the BOV genus, but was more closely related to the Primate bocaparvovirus than to other animal BOVs (Fig. 3). Phylogenetic trees of the NS1, NP1, and VP1 genes consistently showed that MmBOV formed a monophyletic peripheral branch in the primate BOVs, but clustered most closely to those of the Primate bocaparvovirus 2 group (Fig. 4), which is consistent with our sequence analysis results.
Figure 3. Phylogenetic tree of MmBOV and other bocaparvoviruses based on almost complete nucleotide sequences. The tree of complete nucleotide sequences was reconstructed using the maximum-likelihood method with 1000 bootstrap replicates. The viruses from species Primate bocaparvovirus 1, 2, 3 are shown by green, blue and pink, respectively. The position of MmBOV is marked by ■.
Figure 4. Phylogenetic tree of MmBOV in bocaparvoviruses based on three complete coding sequences. The MmBOV trees were constructed based on the complete nucleotide sequences of NS1 (A), NP1 (B), and VP1 (C), aligned with those of other primate bocaparvoviruses using the maximum-likelihood method with datasets of 1000 replicates using MEGA 6.0 software. The viruses from species Primate bocaparvovirus 1, 2, 3 are shown by green, blue and pink, respectively. The position of MmBOV is marked by ■.