# Genomic Characterization of a Halovirus Representing a Novel Siphoviral Cluster
## 3 Results
### Phage YPHTV-1 Represents a New Cluster  
BLAST showed that the genome sequence of YPHTV-1 was not significantly similar to other viruses, and only a few viruses had partial sequence similarity. The similarity between YPHTV-1 and the _Virgibacillus_ phage Mimir 87 (MK560763) was 70.08% (coverage is 22%). The phage Mimir 87 was isolated from worker bees and infected salt-tolerant _Bacillus_ sp. The similar regions of YPHTV-1 and Mimir 87 were genes encoding DNA replication/regulation and nucleotide metabolism and transcription (DNA replication, recombinase protein RecT, and AAA family ATPase) (Figure 6). In addition, the similarity between YPHTV-1 and the _Bacillus_ phage vB_BtS_BBMtP15 (KX190835.1) was 72.80% (coverage was 19%). The similar regions of YPHTV-1 and vB_BtS_BBMtP15 were genes encoding packaging and morphological structure (portal protein, terminase, and capsid family protein) (Figure 6). Twenty-three viral genome sequences in GenBank (six with established taxonomic status in ICTV) were compared with the YPHTV-1 to further clarify its evolutionary status. A total of 24 phages were used to construct a phylogenetic tree with amino acid sequences and a heat map with genome-wide ANI. The phylogenetic analysis showed that the phage YPHTV-1 was clustered with an unclassified _Bacillus_ phage (Figure 7). The ANI values of YPHTV-1 in comparison with 23 other viral genomes were 0-70.85% (Figure S1), among which the ANI values of the _Bacillus_ phages were higher (_Bacillus_ phage vB_BtS_BMBtP15 was 70.23%; _Bacillus_ phage vB_BtS_BMBtP1 was 70.85%). YPHTV-1 ORF25 (HNHI endonuclease), ORF27 (terminase large subunit), ORF29 (portal protein), ORF31 (major capsid protein), ORF1 (integrase), and ORF18 (MazG-like family protein) were selected for the phylogenetic analysis. The phylogenetic tree of the five proteins showed that YPHTV-1 clustered with the _Bacillus_ phages (Figure S2), consistent with the genome-wide analysis. It was demonstrated that YPHTV-1 shared homology with the _Bacillus_ phages in integrase, packaging, and structural proteins and shared homology with the _Paenibacillus_ phages in the MazG-like family protein. _Bacillus_ was also a predominant microbe in hypersaline environments and has been found in 220-million-year-old salt crystals [56]. The genome network analysis showed that 37 viruses were related with phage YPHTV-1, but phage YPHTV-1 did not cluster with any viruses (Figure 8). Thirty-three out of the 37 viruses were divided into three clusters, 27 phages in VC217, five phages in VC173, and one phage in VC211. The four independent phages,  
Figure 6: Genome comparison between YPHTV-1 and _Virgibacillus_ phage Mimir87 and _Bacillus_ phage vB_BtS_BMBtP15. The arrow indicates the direction of gene transcription. The gray bar represents similarity. VC173 and VC211, had no definite classification status in ICTV, while almost all viruses in VC217 infected _Paenibacillus_ and belonged to the _Ferrovirus_ genus under _Caudoviricetes_ (Figure 8, shown in the green box). The VIRIDIC analysis showed that among the three virus clusters, VC173 had the highest average similarity with YPHTV-1 (13.66%); similarity was also found with the _Bacillus_ phage vB_BtS_BMBtP15 (14.54%) and vB_BtS-HD29 phi (14.13%) in VC173, while that with VC217 was only 3.90%. This finding was consistent with the results of ANI analysis, indicating that YPHTV-1 clustered with viruses infecting _Bacillus_ and _Paenibacillus_. Notably, except for the _Bacillus cereus_ phage phBC6A52, YPHTV-1 and the other four viruses in VC173 were siphoviruses [57; 58]. However, the affinity was insufficient to support that the phage YPHTV-1 belonged to this currently known virus genus; YPHTV-1 may represent a new virus genus. Figure 8: YPHTV-1 genome network analysis. Genomic network analysis was performed by vConnACT2, ClusterONE, and Cytoscape. The reference database was Prokaryotic Viral RefSeq211-Merged  
Figure 7: Phylogenetic analysis of YPHTV-1 based on the genome sequences. Phylogenetic trees were constructed using the ViPTree.