3.4 Genetic features of DYPV and LSPV
To further characterize both viruses, primers were designed based on the sequences obtained here and those of related viruses described previously. In this manner we were able to recover the complete genomes of DYPV from 1-Dongyang and the ticks (A. javanense) collected from this animal. The genetic features of DYPV are described in Fig. 2a. Notably, viruses obtained from the pangolin and ticks were closely related to each other, although still exhibited almost 2 per cent nt sequence difference. In addition, both viruses were genetically and phylogenetically distinct from known viruses of the genus Pestivirus, with >43 per cent nt and >48 per cent aa differences (Supplementary Table S5). As with classical pestiviruses, the genome of DYPV encodes twelve proteins. All known cleavage sites of the NS3 protease were observed in DYPV (Supplementary Fig. S3). In addition, the NS4A-NS4B cleavage site, located at Position L2426/A2427 in BVDV1 and L2336/A2337 in CSFV, appeared at Position L2315/K2316 site in DYPV (Supplementary Fig.S3).
Figure 2.  Schematic of the annotated DYPV and LSPV genomes. (a) Genome comparison between DYPV and known pestiviruses. (b) Genome comparison between LSPV and known coltiviruses. Notably, despite the use of both meta-transcriptomic and PCR methods, only nine genome segments (1–5 and 8–11) were obtained from the novel coltivirus—in animal 2-Lishui—and our attempt to recover segments 6, 7, and 12 failed. Details of the genetic features of LSPV are described in Fig. 2b. All recovered segments had the consensus sequences (GAG/AUU/A) at the 5ʹ-terminus and (G/CAGUC) and the 3ʹ-terminus (Fig. 3a), respectively. However, the genome sequences of LSPV were distinct from those of recognized coltiviruses, with <75 per cent aa similarity (Supplementary Table S6). Like other coltiviruses, LSPV Segment 9 also contained two ORFs. However, the sequences adjacent to the UGA stop codon, which are the signal of read-through, were different from known coltiviruses. Notably, however, a similar pattern was observed in SHLV (Fig. 3b), the closest relative of LSPV, that was sampled from ticks in Australia.
Figure 3.  Characteristics of the LSPV genome. (a) Sequence conservation of the 5ʹ- and 3ʹ-terminal 10 nts in genomic segments of the LSPV genome were analyzed and visualized using Weblogo. (b) Sequence information around the stop codon of segment VP9 of LSPV and known coltivirus.