# Genomic Characterization of a Halovirus Representing a Novel Siphoviral Cluster
## 3 Results  
### Biological Characteristics of YPHTV-1  
The 16S rRNA gene phylogenetic analysis of strain H5 isolated from Yipinglang Salt Mine showed that it was clustered with _H. titanicae_ BH1 (KY471040), with a similarity of 98.33%. Moreover, H5 and H5G were clustered together with a similarity of 99.93% (Figure 1). Thus, it was preliminarily identified that the strain H5 belonged to _Halomonas titanicae_.  
The lytic phage YPHTV-1 was isolated from the brine collected from Yipinglang Salt Mine using _H. titanicae_ H5 as the host. After 24 h, clear circular plaques formed, 1.5-2.0 mm in diameter, surrounded by a cloudy halo (Figure 2A). TEM showed that phage YPHTV-1 had an icosahedral head with a diameter of 49.12 \(\pm\) 0.15 nm (n = 5) and a long tail 141.7 \(\pm\) 0.58 nm (n = 5) in length (Figure 2A), indicating that it was a siphovirus. The full infection cycle of YPHTV-1 was 70 min, of which the latent period was 30 min and the rapid growing period was 40 min; the burst size was 69 PFU/cell (Figure 2B).  
Figure 1: Phylogenetic analysis of H5 based on the 16S rRNA gene sequence. Phylogenetic trees were constructed using the neighbor-joining method by Mega 7.0. All parameters were default except the Bootstrap value was 1000, the p-distance model was used to calculate the distance, and the Gap/Missing Data Treatment cutoff was 50%.  
Figure 2: Biological features of phage YPHTV-1. (**A**) Transmission electron micrograph of YPHTV-1. Scale bar, 100 nm. Inset shows plaques morphology of YPHTV-1; scale bar, 5 mm. (**B**) One-step growth curve of phage YPHTV-1. Error bars represent the standard deviation of three replicates.  
YPHTV-1 was most stable at pH 7.0. When the pH was lower than 5 or higher than 9, the survival rate of the phage YPHTV-1 dropped to close to 0 (Figure 3A). After incubation at 22 \({}^{\circ}\)C, 30 \({}^{\circ}\)C, and 37 \({}^{\circ}\)C for 20 min, the survival rate of the phage YPHTV-1 slightly decreased. After incubation at 50 \({}^{\circ}\)C and 60 \({}^{\circ}\)C for 20 min, the survival rate of the phage YPHTV-1 dropped to 0 (Figure 3B). These findings show that the phage YPHTV-1 is stable in a neutral environment and at 22-37 \({}^{\circ}\)C.  
A cross-infectivity test was performed to examine the host range of YPHTV-1 within an extensive collection of nine strains of species of the family _Halomonadaceae_. The phage YPHTV-1 only infected its host strain, H5; even H5G, isolated from Qiaohou Salt Mine and sharing high 165 rRNA gene sequence similarity with H5, was not infected by YPHTV-1 (Figure 1).  
The first step of infection was identification and adsorption. Phage YPHTV-1 had the highest adsorption rate to strain H5 and H5G. The adsorption rate to _H. ventosae_ QH52-2 was significantly reduced, and this reduction was more pronounced in _C. beijernickii_ F3 and C. _canadensis_ F7 (Figure 4). These results suggested that the inability of the phage YPHTV-1 to infect the H5G was caused by the steps after adsorption.  
Figure 4: The adsorption of phage YPHTV-1 to different cells. H5 and H5G belong to _H. titanizea_, QH52-2 belongs to _H. ventosae_, F3 belongs to _C. beijerinclii_, and F7 belongs to _C. canadensis_. The numerical value represents the adsorption rate of the virus on different cells. ANOVA analysis was used to test the difference in the viral adsorption rate between the host bacterium H5 and other cells (* 0.01 \(<\)_p_ < 0.05, **_p_ < 0.01).  
Figure 3: The pH (**A**) and thermal (**B**) stability of YPHTV-1. Error bars represent the standard deviation of three replicates.