Characterization and Genomic Analyses of dsDNA Vibriophage vB_VpaM_XM1, Representing a New Viral Family

Characterization and Genomic Analyses of dsDNA Vibriophage vB_VpaM_XM1, Representing a New Viral Family | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Characterization and Genomic Analyses of dsDNA Vibriophage vB_VpaM_XM1, Representing a New Viral Family Zuyun Wei, Xuejing Li, Chunxiang Ai, Hongyue Dang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4560493/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Vibrio parahaemolyticus has been a leading cause of foodborne disease outbreaks and infectious diarrhea cases in coastal areas, antibiotic resistance has increased significantly due to widespread antibiotic abuse, bacteriophages (phages) are viruses that specifically infect bacteria, it is necessary to isolate and characterize new phages to broaden our understanding of the ecology, evolution, and diversity of both phages and their bacterial hosts further. Results A novel vibriophage vB_VpaM_XM1 (XM1) was described in the present study. The morphological analysis revealed that phage XM1 had Myoviridae -like morphology, with an oblate icosahedral head and a long contractile tail. The genome size of XM1 is 46,056 bp, with a G + C content of 42.51%, encoding 69 open reading frames (ORFs). Moreover, XM1 showed a narrow host range only lysing Vibrio xuii LMG 21346 (T) JL2919, Vibrio parahaemolyticus 1.1997, and Vibrio parahaemolyticus MCCC 1H00029 among the tested bacteria. One-step growth curves showed that XM1 has a 40-minute latent period and 264 plaque-forming units (PFU)/cell burst size. In addition, XM1 exhibited broad pH, thermal, and salinity stability, as well as strong lytic activity, even at a multiplicity of infection (MOI) of 0.001. Multiple genome comparisons and phylogenetic analyses showed that phage XM1 is grouped in a clade with three other phages, including Vibrio phages Rostov 7, X29, and phi 2, and is distinct from all known viral families that have ratified by the standard genomic analysis of the International Committee on Taxonomy of Viruses (ICTV). Conclusions Therefore, the above four phages might represent a new viral family, tentatively named Weiviridae. The broad physiological adaptability of phage XM1 and its high lytic activity and host specificity indicated that this novel phage is a good candidate for being used as a therapeutic bioagent against infections caused by certain Vibrio parahaemolyticus strains. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Background Vibrio parahaemolyticus is a Gram-negative halophilic bacterium widely distributed in estuarine, coastal, and marine environments [ 1 – 3 ]. This bacterium is predominantly associated with various sea animal food, including fish and shellfish such as shrimps, lobsters, crabs, and oysters[ 4 ]. V. parahaemolyticus has also been found as a causative agent of acute gastroenteritis in humans resulting from the consumption of undercooked or raw seafood [ 5 , 6 ]. In China, V. parahaemolyticus has been a leading cause of foodborne disease outbreaks and infectious diarrhea cases in coastal areas [ 7 ]. Bacteriophages (phages) are viruses that specifically infect bacteria and represent the most abundant biological entity on the planet. They can kill nearly half of the bacterial population every two days and play a critical role in bacterial control in the natural environment [ 8 – 10 ]. Bacteriophages are highly diverse morphologically and genetically, and they can facilitate horizontal gene transfer and, therefore, are crucial to bacterial diversity and evolution [ 11 ]. Furthermore, phage genomes contain many new genes with unknown functions, so they may be one of the largest unexplored gene pools [ 12 , 13 ]. Antibiotic resistance has increased significantly due to widespread antibiotic abuse, leading to the emergence of multidrug-resistant bacteria in marine aquaculture and natural environments, causing the spread and therapeutic difficulty of bacterial pathogens [ 14 – 16 ]. Phage therapy has been considered a promising method to control antibiotic-resistant bacterial pathogens [ 17 – 20 ]. Bacteriophages with high host specificity, high lytic activity, and eco-friendly properties are beneficial candidates as bio-control agents [ 21 – 23 ]. Indeed, specific phages have been successfully applied as biocontrol agents to control foodborne pathogens [ 24 , 25 ]. However, the phage resources are still very minimal. As of 30 June 2023, only 122 Vibrio and 26 V. parahaemolyticus phage genomes were recorded in the NCBI RefSeq database ( http://www.ncbi.nlm.nih.gov/genome ). It is necessary to isolate and characterize new phages to broaden our understanding of the ecology, evolution, and diversity of both phages and their bacterial hosts further. Bacteriophage classification and genetic backgrounds are critical for the application of phage therapy [ 26 , 27 ]. In earlier studies, phages were classified mainly according to their morphological similarity and nucleic acid composition [ 28 ]. Specific conserved genes of phages are also used for phage taxonomic analyses, such as those encoding the large subunit of terminases and the major capsid proteins [ 29 ]. Sequencing technology is getting more advanced, and phage taxonomic classifications based on genomes, transcription mechanisms, and gene contents are becoming more accurate [ 30 ]. Based on virus taxonomic classification by the International Committee on Taxonomy of Viruses (ICTV), Duplodnaviria currently contains one kingdom (Heunggongvirae), two phyla (Peploviricota and Uroviricota), two classes (Herviviricetes and Caudoviricetes), eight orders, and 66 families including those that do not belong to any defined orders. Tailed phages all belong to class Caudoviricetes according to the latest nomenclature rules of ICTV. In contrast, they had previously been classified as Myoviridae, Podoviridae, and Siphoviridae based on their tail morphology. With the development of genome sequencing and phylogenetic analysis, phage taxonomy has changed [ 31 ]. At the time of writing, the ICTV has abolished the phage nomenclature of Myoviridae, Podoviridae, and Siphoviridae. The present study reported a new vibriophage isolated from V. parahaemolyticus ( i.e. , vB_VpaM_XM1). The morphology, host range, one-step growth curve, stabilities against pH, salinity, and thermal changes of vB_VpaM_XM1 were evaluated. Based on genomic annotation and comparative genomic and phylogenetic characterizations, vB_VpaM_XM1 and three other phages represent a new viral family. Results and discussion Biological characterization of XM1. The phage vB_VpaM_XM1 (XM1 in short) was isolated from temporary maintenance water of marketed marine fish using V. parahaemolyticus as the host bacteria. It was able to form clear, circular, and boundary-smooth plaques (Figure 1A). As shown in Figure 1B, transmission electron microscope (TEM) analysis reveals that XM1 carries an icosahedral head (76.92±2.65 nm long and 64.10±1.36 nm wide) and a long contractile tail (130.77±5.68 nm). The lytic cycle of XM1 was determined with a one-step growth curve at 0.01 multiplicity of infection (MOI). The latent period of phage XM1 was about 40 min, and its burst size was approximately 264 PFU/cell (Figure 2A). Previously, phage vB_VPAP_DE10 infecting V. parahaemolyticus has been shown to have a latent period of approximately 0-25 min, with a burst size of 19 PFU/cell [32]. Phages F23s2 and H256D1 showed a latent period of 0-20 min and 0-5 min, respectively, with a burst size of 12 PFU/cell and 131 PFU/cell, respectively [33]. Vibrio phage VP06 has a latent period of 30 min and a burst size of 60 PFU/cell [34]. Compared with these previously studied V. parahaemolyticus -infecting phages, phage XM1 exhibited a longer latent period and a much larger burst size. To examine the host range of phage XM1, a spotting test was performed against 58 bacterial strains isolated from various environments (Table S1). The results showed that XM1 only lyses V. xuii LMG 21346 (T) JL2919, V. parahaemolyticus 1.1997, and V. parahaemolyticus MCCC 1H00029, suggesting that XM1 has a narrow range of and high specificity to its hosts. Furthermore, the effects of pH, temperature, and salinity on the stability of phage XM1 were tested. XM1 maintained activity from pH 4 to 10, with the highest lytic activity at pH 9 (Figure 2B). The optimal pH for V. parahaemolyticus phages CA8 and BA3 was from pH 5 to 7 and pH 6 to 7, respectively [12], while V. parahaemolyticus phage R18L exhibited stability from pH 6 to 11 [35]. Thermal stability tests showed that phage XM1 was stable at 4 ℃ to 60 ℃ for 3 h with a decreasing stability trend with increasing temperature and a total loss of activity at 70 ℃ (Figure 2C). Phages CA8 and BA3 were stable only at temperatures ranging from 20 ℃ to 40 ℃ [12], while phage R18L was stable from 4 ℃ to 40 ℃ [35]. Phage XM1 in SM buffer at 3% salinity showed the most significant activity but could not grow at 0% salinity (Figure 2D), indicating coastal and marine environments as XM1’s habitat. V. parahaemolyticus phage VB_VpP_BT-1011 can survive at 0% to 3% NaCl [36], indicating this phage also includes estuarine environments as its habitat. Results from the current study show that phage XM1 is stable at broad pH, temperature, and salinity ranges, indicating its tolerance and adaptation to various environmental stresses. V. parahaemolyticus was infected with XM1 at different MOIs to investigate its effect on bacterial growth. The killing curve (Figure 3) indicated that increasing the MOI increased bacterial growth inhibition. There was an initial increase in the concentration of host bacteria. However, with the release of phages, the concentration of host bacteria began to decrease, with no observable difference in the XM1 inhibitory effects observed after 11 h of infection, regardless of different MOIs. The intense bactericidal activity indicates that XM1 is a potent candidate for use in phage therapy. Genome sequence of vibrio phage XM1. Genome analyses revealed that Phage vB_VpaM_XM1 is a double-stranded DNA virus belonging to the Duplodnaviria realm in the ICTV. Its genome size is 46,056 bp with a total G + C content of 42.51%, containing 69 predicted open reading frames (ORFs). The size of XM1 protein-coding sequences (CDSs) ranges from 51 to 825 amino acid residues (207.74 on average). Among the 69 ORFs, 66 are transcribed in the forward direction, while the other 3 ( i.e. , ORF4, ORF35, and ORF36) are transcribed in the reverse direction. The 69 predicted genes primarily encode viral structure proteins and proteins for DNA packaging, DNA metabolism and replication, and host lysis (Figure 4). Nearly two-thirds of the predicted genes (45/69) can be assigned functions according to their homology to known sequences of other phages. Concretely speaking, 19 predicted genes (ORF5, ORF8, ORF9, ORF11, ORF12, ORF13, ORF15, ORF16, ORF18, ORF19, ORF20, ORF21, ORF22, ORF23, ORF24, ORF26, ORF27, ORF29, and ORF30) are related to viral structure proteins, 3 predicted genes (ORF2, ORF3, and ORF6) are associated with DNA packaging, and 14 predicted genes (ORF1, ORF7, ORF10, ORF37, ORF40, ORF41, ORF42, ORF44, ORF46, ORF48, ORF56, ORF58, ORF59, and ORF64) are connected with DNA replication and metabolism. On the other hand, the remaining predicted genes (24 ORFs) encode hypothetical proteins with unknown functions. Specifically, protein sequences encoded by ORF4, ORF51, ORF60, and ORF66 show no homology to any known protein sequences in the database, potentially indicating their sequence and functional novelties. The predicted terminase large subunit protein (ORF3) plays an important role in the late stage of viral DNA packaging [37]. The predicted portal protein (ORF6) is involved in dsDNA viral genome packaging and release [38]. Three ORFs are predicted to encode host lysis proteins, including a lysozyme (encoded by ORF25), a sporulation-specific N-acetylmuramoyl-L-alanine amidase (encoded by ORF32) that belongs to one of the four families of peptidoglycan hydrolases [39], and a Rz-like spanin (encoded by ORF36) that interacts with bacterial outer membrane and plays a role in the final step of host lysis [40]. Additionally, ORF45 encodes a site-specific integrase that may be a lysogen-related protein [41]. Moreover, no virulence gene or factor was found in the genome of phage XM1. Therefore, phage XM1 may be safe for use in phage therapy. Phylogenetic and comparative genomic analyses of phage XM1. To determine the phylogenetic taxonomy of phage XM1, a proteomic tree based on viral whole genomes was generated using VipTree ( https://www.genome.jp/viptree , accessed on 10 Jun 2023)[42]. The result shows that XM1 is closely clustered with Vibrio phages Rostov 7 (accession: MK575466.1)[43], X29 (accession: NC_024369.2)[44], and phi2 (accession: KJ545483.2)[45] (Figure 5A and 5B), indicating that these four phages may form a new taxonomic group of viruses. Through BLASTN in the NCBI database, phage XM1 has the highest genomic sequence identity to Vibrio phages Rostov 7, X29, and phi2, with a 77.76%, 77.60%, and 77.54% identity scores and 33%, 25%, and 25% query coverage, respectively (accessed on 10 Jun 2023). The ANI value of the XM1 genome with the viral genome of Rostov 7, X29, and phi 2 was 73.35%, 72.22%, and 72.23%, respectively (Figure 6A). However, phage XM1 showed no genomic match with other NCBI viral genomes. Phage XM1 shares a similar overall genomic organization with Vibrio phages Rostov 7, X29, and phi 2 (Figure 6B). The predicted genes of XM1 showed 66% to 100% sequence identities to genes of the other three Vibrio phages, except several genes that had no sequence match. To further confirm the taxonomic novelties of phage XM1 and its closest relatives ( i.e. , Vibrio phages Rostov 7, X29, and phi 2), a whole-genome phylogenetic tree (Supplementary Figure 1, Figure 7) was constructed with 154 representative viral genomes selected from all the 66 families of Duplodnaviria currently defined by ICTV (Accessed on 9 Jun 2023). The phylogenetic tree showed that the phages Rostov 7, X29, and phi 2 belong to Duplodnaviria, Heunggongvirae, Uroviricota, Caudoviricetes. In addition, the analytic result indicated that phages XM1, Rostov 7, X29, and phi 2 are phylogenetically grouped and form a unique viral cluster not affiliated with any known viral families. Therefore, we tentatively name Weiviridae as the new family name for these four novel Vibrio -lysing phages. The terminase large subunit is a relatively conserved protein used as a marker for establishing phage phylogenetic relationships [46]. Similarly, the major capsid protein, the primary component of phage capsid, is conserved among phylogenetically related phages and is frequently used in phage classification [47]. In this study, a phylogenetic tree was constructed based on the protein sequences of phage terminase large subunit and phage capsid protein, respectively (Figure 8). Our analyses show that phage XM1 is grouped with Vibrio pahges Rostov 7, X29, and phi 2 and phylogenetically distant from other phage families. The genome phylogenetic tree also showed similar results (Figure S2). The gene sequences selected in these two phylogenetic trees were based on Blast in NCBI (Accessed on 10 Jun 2023). Values at the nodes indicate the bootstrap support calculated from 1000 replicates. According to phylogenetic trees (Figures 5, 7, and 8), the Vibrio phages Rostov 7, X29, and phi 2 belong to class Caudoviricetes. However, none of them are classified into any known phage families. Phage XM1 and these phages are grouped as a new clade and different from previously described phages. These results indicate that phages XM1, Rostov 7, X29, and phi 2 can be classified as a new phage family. Conclusions This study isolated and fully characterized a new phage, vB_VpaM_XM1, infecting V. parahaemolyticus and having a large burst size and narrow host range. XM1 has a broad range of temperature, pH, and salinity adaptability, and exhibits strong lytic activity. These indicated that XM1 has great potential as a novel antibacterial agent for the biological control of vibriosis in aquaculture. Moreover, the complete XM1 genome sequence was determined and compared with its phage relatives. Furthermore, phylogenetic analyses revealed that XM1 clusters a new clade with vibrio phages Rostov 7, X29, and phi 2, and should belong to a new viral family named Weiviridae. Our report provides an in-depth analysis of phage at the genomic, phylogenetic, and ecological levels and provides a potential antimicrobial candidate for pathogenic V. parahaemolyticus . Materials and methods Phage isolation and purification. V. parahaemolyticus 1.1997 was used as the bacterial host [ 48 ]. It was grown in a rich organic (RO) medium with a shaking speed of 160 rpm/min at 28 ℃. Firstly, 1 mL of water sample from seafood markets (Xiamen, China) was added into 10 mL of an exponentially growing culture of V. parahaemolyticus 1.1997 and incubated for 24 h. The mixed culture was then passed through a 0.22 µm filter membrane ( Millipore, Bedford, MA, USA) to remove bacterial cells. The filtrate was diluted and mixed with exponential host cultures to get phage plaque using the double-layer agar method [ 49 ]. After the above-mentioned steps, the well-separated plaque was removed and stored in the storage medium (SM) buffer (50 mM Tris-HCl, 0.1 M NaCl, and 8 mM MgSO 4 , pH 7.5) at 4 ℃ for later use. Phage enrichment. To obtain highly concentrated phage, 1 L phage lysate was treated with DNase I and RNase A at room temperature for 1 h until the final concentration reached 1µg/mL, then 1 M NaCl was supplied for 30 min at 4 ℃ to promote the separation of phage particles and cell debris. Finally, the solution was mixed with 10% polyethylene glycol (PEG 8000) and stored for 3 d at 4 ℃ to precipitate virions. Viral particles were subsequently collected by centrifugation (12,000×g, 60 min, 4 ℃) and resuspended in 6 mL of SM buffer. The phage suspensions were prepared via cesium chloride gradient centrifugation (1.3, 1.5, 1.7 g/mL) centrifuged at 200,000×g for 24 h at 4 ℃ using an Optima L-100 XP ultracentrifuge (Beckman Coulter, CA, USA). The visible phage band was extracted and future dialyzed through 30-kD super-filters (Millipore, MA, USA) [ 50 ]. Morphology observation. The phage morphology was observed using a JEM-2100 transmission electron microscope (JEOL, Tokyo, Japan) at an acceleration voltage of 80 kV. To prepare the samples for observation, 20 µL of high-titer phage concentrate was plated on 200-mesh formvar-coated copper electron microscope grids and allowed to absorb for 10 min, then negatively stained with 1% phosphotungstic acid for 1 min, followed by air drying for 10 min. The size of phage particles was measured from at least five TEM images using ImageJ software [ 51 ]. Host range. The host range of XM1 was determined by spot testing and confirmed by the double-layer agar method [ 52 ]. 1 mL of exponentially growing bacteria (10 8 CFU/mL) was mixed with 5 mL of the pre-warmed (50°C) semisolid liquid medium, then poured onto a solid agar plate immediately. After 10 min of air drying, 5 µL of purified phage solution was spotted on the host bacterial lawn. The plate was then incubated at 28 ℃ for 24 h. Phage infection was determined by visual examination of the plates for plaques. The used bacteria included 58 strains in the genera Vibrio , Idiomarina , Pseudoalteromonas , Photobacterium , and Shewanella (listed in Table S1). One-step growth curve. The one-step growth curve of phage XM1 was determined using the previously described method [ 53 ]. Briefly, 1 mL of exponentially growing bacteria (10 8 CFU/mL) was exposed to phages at a MOI of approximately 0.01, then placed in the dark for 10 min. Bacteria were then pelleted (6,000×g, 5 min), and the non-adsorbed phages in the supernatant were discarded. The pellet was then washed twice and resuspended in 100 mL RO medium, and the culture was then incubated at 28 ℃ with a shaking speed of 160 rpm/min. Every 10 min, subsamples were collected, and the viral abundance was detected using the double-layer agar method. The burst size was calculated as the ratio between the number of virions at the growth plateau and the initial number of infected host cells [ 54 ]. pH, temperature and salinity tolerance. A series of 3 experiments were designed to determine the influence of pH, temperature, and salinity on the stability of phage XM1. In all experiments, the double-layer agar method was applied to estimate the infection activity of the phage. In the pH experiment, the pH of the SM buffer was adjusted from 2 to 12 with HCl or NaOH solution. The phage concentrate was added to the SM buffer so that the final concentration was 10 14 PFU/mL, and then all treatments were incubated at 4 ℃ for 3 h, 24 h, and 48 h. For the experiment that investigated the thermal stability of the phage, phage in all treatments was incubated for 3 h, with incubation temperatures set at 4 ℃, 24 ℃, 37 ℃, 50 ℃, 60 ℃, and 70 ℃, respectively. As for the salinity tolerance experiment, solutions with salinity ranging from 0 to 5% were used for phage incubation (incubation time: 12 h). Growth curve experiment. The phage XM1 was mixed with the host V. parahaemolyticus at different MOIs (0.001, 0.01, 0.1, 1, 10) and incubated at 28 ºC. Meanwhile, V. parahaemolyticus at the same MOI level but without phage was used as a positive control. The growth curves were monitored over 12 h, and optical density (OD 600 ) measurements were recorded every 1 h. Three independent assays were carried out for each assay. DNA extraction and sequencing. Viral genomic DNA was extracted using the Takara MiniBEST Viral RNA/DNA Extraction Kit according to the manufacturer's protocol. In brief, 200 µL viral concentrate was mixed with 200 µL Buffer VGB, 20 µL Proteinase K, and 1 µL Carrier RNA, then incubated at 56 ℃ for 10 min. After that, 200 µL of ethanol was added to the mixture before a 2-min centrifugation (12,000×g). Next, 500 µL RWA was added, and the solution was centrifuged at 12,000×g for 1 min. Following that, 700 µL RWB was added and the mixture was centrifuged at 12,000×g for 1 min, and this step was repeated twice. Finally, 30 µL RNase-free dH 2 O was added into the centrifuge tube and incubated for 5 min at room temperature before the final centrifugation was conducted (12,000×g for 2 min). The extracted DNA was stored at -20 ℃. Phage genome sequencing was performed using the Illumina Nova platform by the Shanghai Hanyu Bio-Tech Co., Ltd (Shanghai, China). Genome annotation and phylogenetic analysis. The opening reading frames (ORFs) of the XM1 genome were predicted by the Glimmer3/GeneMarkS/Prodigal online server and annotated by BLASTp search against the National Center for Biotechnology Information (NCBI) nonredundant (nr) protein sequences (Accessed on 27 Feb 2023) [ 55 , 56 ]. Gene map was created based on the genome annotations using CGView-Circular Genome Viewer ( https://proksee.ca ) [ 57 ]. Genomic structures and comparison maps of phages belonging to the same categories were made using EasyFig [ 58 ]. A phylogenetic tree based on genome sequence similarities computed by tBLASTx was constructed using the Viral Proteomic Tree server (VipTree, https://www.genome.jp/viptree/ , accessed on 10 Jun 2023) [ 42 ]. OrthoFinder was used to compare the genomic similarity by orthology (OrthoANI), which was calculated using the BLASTp analysis [ 59 ]. To explore the phage taxonomic status, the complete nucleotide sequence of phage XM1 and its related viral genomic sequences were submitted to the virus classification and tree building online resource (VICTOR) ( http://ggdc.dsmz.de/victor.php , accessed on 9 Jun 2023) for phylogenetic analysis, with the recommended settings of genome BLAST distance phylogeny (GBDP) method being used [ 60 ]. The terminase large subunit protein and capsid protein sequences of XM1 were used to construct phylogenetic trees to analyze its evolutionary relationships, and a Neighbor-joining method in the MEGA 6.0 software package with 1000 bootstrap replicates was used to construct the phylogenetic tree (Accessed on 10 Jun 2023) [ 61 , 62 ]. Declarations Acknowledgements We thank Jie Wu and Lanfeng Dai for helpful guidance on the experimental methods. We also thank Jan Meier-Kolthoff of the Leibniz Institute DSMZ scientific community for his help in the construction of the GBDP tree. Author contributions Z.W., C.A., and H.D. developed ideas and organized the study; Z.W. performed the experiments; Z.W. and X.L. analyzed the data; Z.W. wrote the manuscript. H.D., X.L. and C.A. made revisions to the manuscript. All authors have read and agreed to the published version of the manuscript. Funding This work was supported by the National Key Research and Development Program of China (grant No. 2020YFA0608302) and the National Natural Science Foundation of China (grant Nos. 42076111, 42141003, 42188102, and 41861144018). Availability of data and materials Not applicable. Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests. References Su YC, Liu C. Vibrio parahaemolyticus: a concern of seafood safety. Food Microbiol. 2007;24(6):549–58. Ceccarelli D, Hasan NA, Huq A, Colwell RR. 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JMBio. 2002;316(3):547–61. Prevelige PE, Cortines JR. Phage assembly and the special role of the portal protein. Curr Opin Virol. 2018;31:66–73. Moak M, Molineux IJ. Peptidoglycan hydrolytic activities associated with bacteriophage virions. Mol Microbiol. 2004;51(4):1169–83. Berry J, Summer EJ, Struck DK, Young R. The final step in the phage infection cycle: the Rz and Rz1 lysis proteins link the inner and outer membranes. Mol Microbiol. 2008;70(2):341–51. Thorpe HM, Smith MCM. In vitro site-specific integration of bacteriophage DNA catalyzed by a recombinase of the resolvase/invertase family. Proc Natl Acad Sci U S A. 1998;95(10):5505–10. Nishimura Y, Yoshida T, Kuronishi M, Uehara H, Ogata H, Goto S. ViPTree: the viral proteomic tree server. Bioinformatics. 2017;33(15):2379–80. Gaevskaya NE, Pogozhova MP, Vodopyanov AS, Pisanov RV, Romanova LV, Anoprienko AO, et al. Biological and genetic characteristics of cholera bacteriophage Rostov 7. Bacteriology. 2019;4:27–30. 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A Novel Alteromonas Phage Lineage with a Broad Host Range and Small Burst Size. Microbiol Spectr. 2022;10(4):e0149922. Schneider CA, Rasband WS, Eliceiri KW. NIH Image to ImageJ: 25 years of image analysis. Nat Methods. 2012;9(7):671–5. Feng X, Yan W, Wang A, Ma R, Chen X, Lin TH, et al. A Novel Broad Host Range Phage Infecting Alteromonas. Viruses. 2021;13(6):987. Li X, Liang Y, Wang Z, Yao Y, Chen X, Shao A, et al. Isolation and Characterization of a Novel Vibrio natriegens-Infecting Phage and Its Potential Therapeutic Application in Abalone Aquaculture. Biology (Basel). 2022;11(11):1670. Ma R, Shao S, Wei S, Ye J, Yang Y, Jiao N, Zhang R. A Novel Phage Infecting the Marine Photoheterotrophic Bacterium Citromicrobium bathyomarinum. Viruses. 2022;14(3):512. Delcher AL, Bratke KA, Powers EC, Salzberg SL. Identifying bacterial genes and endosymbiont DNA with Glimmer. Bioinformatics. 2007;23(6):673–9. Besemer J, Lomsadze A, Borodovsky M. GeneMarkS: a self-training method for prediction of gene starts in microbial genomes. Implications for finding sequence motifs in regulatory regions. Nucleic Acids Res. 2001;29(12):2607–18. Grant JR, Stothard P. The CGView Server: a comparative genomics tool for circular genomes. Nucleic Acids Res. 2008;36(Web Server issue):W181-4. Sullivan MJ, Petty NK, Beatson SA. Easyfig: a genome comparison visualizer. Bioinformatics. 2011;27(7):1009–10. Lee I, Ouk Kim Y, Park SC, Chun J. OrthoANI: An improved algorithm and software for calculating average nucleotide identity. Int J Syst Evol Microbiol. 2016;66(2):1100–3. Meier-Kolthoff JP, Goker M. VICTOR: genome-based phylogeny and classification of prokaryotic viruses. Bioinformatics. 2017;33(21):3396–404. Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. Mol Biol Evol. 2013;30(12):2725–9. Saitou N, Nei M. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol. 1987;4(4):406–25. Additional Declarations No competing interests reported. Supplementary Files supplementary.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4560493","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":318809196,"identity":"79431ce9-bbba-4311-98aa-a58107ed7846","order_by":0,"name":"Zuyun Wei","email":"","orcid":"","institution":"Xiamen University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Zuyun","middleName":"","lastName":"Wei","suffix":""},{"id":318809209,"identity":"7c6b2d6a-4c61-43d3-8135-5dfa11bd1f90","order_by":1,"name":"Xuejing Li","email":"","orcid":"","institution":"Xiamen University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xuejing","middleName":"","lastName":"Li","suffix":""},{"id":318809211,"identity":"9bdc2e6d-c430-4960-91c5-65b23264f386","order_by":2,"name":"Chunxiang Ai","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAuklEQVRIiWNgGAWjYDACCQgpB+GxkaDFmGQtDIkNRGvhn91j9pinxiJ9w7UzBgwfyg4DRRoIWHLnjLnhjGMSuTNn5xgwzjh3GChyAL8WA4kcM4kPbBK5/dI5Bsy8bYeBIglEaEn4J5HOBtLyl2gtH9skEvhBWhiJ0SJxI61McmafhOHM2WkFB3vOpfNI3CCghX9G8jZpnm918ga3kzc++FFmLcc/g4AWFHAAiHlIUD8KRsEoGAWjABcAABBEOg5DBRFmAAAAAElFTkSuQmCC","orcid":"","institution":"Xiamen University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Chunxiang","middleName":"","lastName":"Ai","suffix":""},{"id":318809215,"identity":"c0a93b68-f3cb-4120-b1d6-242ad072896a","order_by":3,"name":"Hongyue Dang","email":"","orcid":"","institution":"Xiamen University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Hongyue","middleName":"","lastName":"Dang","suffix":""}],"badges":[],"createdAt":"2024-06-11 01:23:23","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4560493/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4560493/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":59173463,"identity":"667d2a63-c289-4f1f-bea6-760be6772b8e","added_by":"auto","created_at":"2024-06-27 08:58:14","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":594306,"visible":true,"origin":"","legend":"\u003cp\u003eMorphology of phage vB_VpaM_XM1.\u003c/p\u003e\n\u003cp\u003e(A) Plaques of vB_VpaM_XM1 infecting \u003cem\u003eV. parahaemolyticus\u003c/em\u003e 1.1997.\u003c/p\u003e\n\u003cp\u003e(B) Transmission electron micrograph of vB_VpaM_XM1. The scale bar represents 100 nm.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-4560493/v1/e837666cd661370c3108a804.png"},{"id":59173458,"identity":"372fda69-8b85-43c1-b4c5-4a3a7f72b3d7","added_by":"auto","created_at":"2024-06-27 08:58:14","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":79856,"visible":true,"origin":"","legend":"\u003cp\u003eBiological properties of phage vB_VpaM_XM1.\u003c/p\u003e\n\u003cp\u003e(A) One-step growth curve of phage vB_VpaM_XM1.\u003c/p\u003e\n\u003cp\u003e(B) pH stability curve of phage vB_VpaM_XM1.\u003c/p\u003e\n\u003cp\u003e(C) Stability of phage vB_VpaM_XM1 in different temperatures.\u003c/p\u003e\n\u003cp\u003e(D) Stability of phage vB_VpaM_XM1 in different salinity. The data shown are average values from triplicate experiments, and error bars indicate standard deviations.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-4560493/v1/646513ce9f5c59df4f838579.png"},{"id":59174013,"identity":"6f7f2794-5ada-479b-b986-028a15bead87","added_by":"auto","created_at":"2024-06-27 09:06:14","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":272125,"visible":true,"origin":"","legend":"\u003cp\u003eKilling curves of \u003cem\u003ev. parahaemolyticus\u003c/em\u003e by phage vB_VpaM_XM1 at various MOIs (0.001, 0.01, 0.1, 1, and 10).\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-4560493/v1/6366f3cf161b250f9a1c4d35.png"},{"id":59173464,"identity":"586739ab-c03e-490c-bddc-7363b87b9076","added_by":"auto","created_at":"2024-06-27 08:58:14","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1098992,"visible":true,"origin":"","legend":"\u003cp\u003eAnnotated genome map of phage vB_VpaM_XM1. The 69 ORFs are represented by colored arrows, and the direction of each arrow represents the direction of transcription.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-4560493/v1/c75b65bb0192fb8be4d83578.png"},{"id":59174015,"identity":"a60a43b7-6c82-4211-bfc1-101f255b33d3","added_by":"auto","created_at":"2024-06-27 09:06:15","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2327537,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic analyses of phage vB_VpaM_XM1.\u003c/p\u003e\n\u003cp\u003e(A) A circular proteomic tree constructed with phage vB_VpaM_XM1 and other phage genomic sequences using VipTree.\u003c/p\u003e\n\u003cp\u003e(B) The viral proteomic tree, including vB_VpaM_XM1 and its 17 nearest phage relatives. The phages selected were a part of a rectangular tree of the whole genome. The left color line indicates the viral taxonomic families (The left color line is blank because there is no specific virus family for these viruses) , and the right color line indicates the host groups.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-4560493/v1/013c5d462349cf0e774af69c.png"},{"id":59173459,"identity":"9ead0e6a-561a-4405-a704-d1eba2bf28c7","added_by":"auto","created_at":"2024-06-27 08:58:14","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":634737,"visible":true,"origin":"","legend":"\u003cp\u003eComparative genomic analyses of phage vB_VpaM_XM1.\u003c/p\u003e\n\u003cp\u003e(A) The genome-wide tree based on the average nucleotide identity (ANI) from 10 phages. Ten phages were selected based on the phages that showed the closest relationships to vB_VpaM_XM1 in the evolutionary tree in Figure 5.\u003c/p\u003e\n\u003cp\u003e(B) Genome organization and comparisons of phage vB_VpaM_XM1 with Vibrio phage Rostov 7, Vibrio phage X29, and Vibrio phage phi2. ORFs are depicted by leftward or rightward oriented arrows according to the direction of transcription. Each color indicates a putative function, including host lysis (red), DNA packaging (blue), DNA replication and metabolism (green), structure protein (purple), other protein (orange), or hypothetical protein (gray).\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-4560493/v1/ac837fb383b9332acc25ec6d.png"},{"id":59173466,"identity":"f85d75ba-79e6-4a65-b11c-4dc19967a075","added_by":"auto","created_at":"2024-06-27 08:58:15","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":893838,"visible":true,"origin":"","legend":"\u003cp\u003eLocal details of the Genome BLAST distance phylogeny (GBDP) tree constructed for \u003cem\u003eVibrio\u003c/em\u003ephages XM1, Rostov 7, X29, and phi 2 and 154 other viruses representing all the 66 known families in realm Duplodnaviria. The truncated phylogenetic tree shows that vibrio phages XM1, Rostov 7, X29, and phi 2 are phylogenetically grouped and form a unique viral cluster unaffiliated with any known viral families in Duplodnaviria. The new phage family is tentatively named as Weiviridae. The complete GBDP tree is shown in Supplementary Figure 1. Numbers at the nodes are GBDP pseudo-bootstrap values (100 replications and values \u0026gt; 50%).\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-4560493/v1/a5609210701402683bb4d4b0.png"},{"id":59173465,"identity":"4f67395f-ae62-4e38-bb5f-607ebe7de4cf","added_by":"auto","created_at":"2024-06-27 08:58:15","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":601223,"visible":true,"origin":"","legend":"\u003cp\u003eNeighbour-joining phylogenetic trees of phage vB_VpaM_XM1.\u003c/p\u003e\n\u003cp\u003e(A) Phylogenetic tree based on the amino acid sequence of the terminase large subunit.\u003c/p\u003e\n\u003cp\u003e(B) Phylogenetic tree based on the major capsid protein, showing the relationships between phage vB_VpaM_XM1 and other nearest phages.\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-4560493/v1/9dd1a3fcfd6b590a8aca7dd1.png"},{"id":59241671,"identity":"d9226ab7-81cd-4502-b8d4-c8cfa991d723","added_by":"auto","created_at":"2024-06-28 05:32:49","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7152893,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4560493/v1/84003139-e086-486a-983b-ad1dbccd8903.pdf"},{"id":59174014,"identity":"7a942241-6a21-45e7-93e6-7d82e612bf59","added_by":"auto","created_at":"2024-06-27 09:06:14","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":404873,"visible":true,"origin":"","legend":"","description":"","filename":"supplementary.docx","url":"https://assets-eu.researchsquare.com/files/rs-4560493/v1/ef771d491f969d7614cded00.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Characterization and Genomic Analyses of dsDNA Vibriophage vB_VpaM_XM1, Representing a New Viral Family","fulltext":[{"header":"Background","content":"\u003cp\u003e \u003cem\u003eVibrio parahaemolyticus\u003c/em\u003e is a Gram-negative halophilic bacterium widely distributed in estuarine, coastal, and marine environments [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. This bacterium is predominantly associated with various sea animal food, including fish and shellfish such as shrimps, lobsters, crabs, and oysters[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. \u003cem\u003eV. parahaemolyticus\u003c/em\u003e has also been found as a causative agent of acute gastroenteritis in humans resulting from the consumption of undercooked or raw seafood [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. In China, \u003cem\u003eV. parahaemolyticus\u003c/em\u003e has been a leading cause of foodborne disease outbreaks and infectious diarrhea cases in coastal areas [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBacteriophages (phages) are viruses that specifically infect bacteria and represent the most abundant biological entity on the planet. They can kill nearly half of the bacterial population every two days and play a critical role in bacterial control in the natural environment [\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Bacteriophages are highly diverse morphologically and genetically, and they can facilitate horizontal gene transfer and, therefore, are crucial to bacterial diversity and evolution [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Furthermore, phage genomes contain many new genes with unknown functions, so they may be one of the largest unexplored gene pools [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAntibiotic resistance has increased significantly due to widespread antibiotic abuse, leading to the emergence of multidrug-resistant bacteria in marine aquaculture and natural environments, causing the spread and therapeutic difficulty of bacterial pathogens [\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Phage therapy has been considered a promising method to control antibiotic-resistant bacterial pathogens [\u003cspan additionalcitationids=\"CR18 CR19\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Bacteriophages with high host specificity, high lytic activity, and eco-friendly properties are beneficial candidates as bio-control agents [\u003cspan additionalcitationids=\"CR22\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Indeed, specific phages have been successfully applied as biocontrol agents to control foodborne pathogens [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. However, the phage resources are still very minimal. As of 30 June 2023, only 122 Vibrio and 26 \u003cem\u003eV. parahaemolyticus\u003c/em\u003e phage genomes were recorded in the NCBI RefSeq database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.ncbi.nlm.nih.gov/genome\u003c/span\u003e\u003cspan address=\"http://www.ncbi.nlm.nih.gov/genome\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). It is necessary to isolate and characterize new phages to broaden our understanding of the ecology, evolution, and diversity of both phages and their bacterial hosts further.\u003c/p\u003e \u003cp\u003eBacteriophage classification and genetic backgrounds are critical for the application of phage therapy [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. In earlier studies, phages were classified mainly according to their morphological similarity and nucleic acid composition [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Specific conserved genes of phages are also used for phage taxonomic analyses, such as those encoding the large subunit of terminases and the major capsid proteins [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Sequencing technology is getting more advanced, and phage taxonomic classifications based on genomes, transcription mechanisms, and gene contents are becoming more accurate [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Based on virus taxonomic classification by the International Committee on Taxonomy of Viruses (ICTV), Duplodnaviria currently contains one kingdom (Heunggongvirae), two phyla (Peploviricota and Uroviricota), two classes (Herviviricetes and Caudoviricetes), eight orders, and 66 families including those that do not belong to any defined orders. Tailed phages all belong to class Caudoviricetes according to the latest nomenclature rules of ICTV. In contrast, they had previously been classified as Myoviridae, Podoviridae, and Siphoviridae based on their tail morphology. With the development of genome sequencing and phylogenetic analysis, phage taxonomy has changed [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. At the time of writing, the ICTV has abolished the phage nomenclature of Myoviridae, Podoviridae, and Siphoviridae.\u003c/p\u003e \u003cp\u003eThe present study reported a new vibriophage isolated from \u003cem\u003eV. parahaemolyticus\u003c/em\u003e (\u003cem\u003ei.e.\u003c/em\u003e, vB_VpaM_XM1). The morphology, host range, one-step growth curve, stabilities against pH, salinity, and thermal changes of vB_VpaM_XM1 were evaluated. Based on genomic annotation and comparative genomic and phylogenetic characterizations, vB_VpaM_XM1 and three other phages represent a new viral family.\u003c/p\u003e"},{"header":"Results and discussion","content":"\u003cp\u003e\u003cstrong\u003eBiological characterization of XM1.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe phage vB_VpaM_XM1 (XM1 in short) was isolated from temporary maintenance water of marketed marine fish using \u003cem\u003eV. parahaemolyticus\u003c/em\u003e as the host bacteria. It was able to form clear, circular, and boundary-smooth plaques (Figure 1A). As shown in Figure 1B, transmission electron microscope (TEM) analysis reveals that XM1 carries an icosahedral head (76.92\u0026plusmn;2.65 nm long and 64.10\u0026plusmn;1.36 nm wide) and a long contractile tail (130.77\u0026plusmn;5.68 nm).\u003c/p\u003e\n\u003cp\u003eThe lytic cycle of XM1 was determined with a one-step growth curve at 0.01 multiplicity of infection (MOI). The latent period of phage XM1 was about 40 min, and its burst size was approximately 264 PFU/cell (Figure 2A). Previously, phage vB_VPAP_DE10 infecting \u003cem\u003eV. parahaemolyticus\u003c/em\u003e has been shown to have a latent period of approximately 0-25 min, with a burst size of 19 PFU/cell [32]. Phages F23s2 and H256D1 showed a latent period of 0-20 min and 0-5 min, respectively, with a burst size of 12 PFU/cell and 131 PFU/cell, respectively [33]. Vibrio phage VP06 has a latent period of 30 min and a burst size of 60 PFU/cell [34]. Compared with these previously studied \u003cem\u003eV. parahaemolyticus\u003c/em\u003e-infecting phages, phage XM1 exhibited a longer latent period and a much larger burst size.\u003c/p\u003e\n\u003cp\u003eTo examine the host range of phage XM1, a spotting test was performed against 58 bacterial strains isolated from various environments (Table S1). The results showed that XM1 only lyses \u003cem\u003eV. xuii\u003c/em\u003e LMG 21346 (T) JL2919, \u003cem\u003eV.\u003c/em\u003e \u003cem\u003eparahaemolyticus\u003c/em\u003e 1.1997, and \u003cem\u003eV.\u003c/em\u003e \u003cem\u003eparahaemolyticus\u003c/em\u003e MCCC 1H00029, suggesting that XM1 has a narrow range of and high specificity to its hosts.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFurthermore, the effects of pH, temperature, and salinity on the stability of phage XM1 were tested. XM1 maintained activity from pH 4 to 10, with the highest lytic activity at pH 9 (Figure 2B). The optimal pH for \u003cem\u003eV. parahaemolyticus\u003c/em\u003e phages CA8 and BA3 was from pH 5 to 7 and pH 6 to 7, respectively [12], while \u003cem\u003eV. parahaemolyticus\u003c/em\u003e phage R18L exhibited stability from pH 6 to 11 [35]. Thermal stability tests showed that phage XM1 was stable at 4 ℃ to 60 ℃ for 3 h with a decreasing stability trend with increasing temperature and a total loss of activity at 70 ℃ (Figure 2C). Phages CA8 and BA3 were stable only at temperatures ranging from 20 ℃ to 40 ℃ [12], while phage R18L was stable from 4 ℃ to 40 ℃ [35]. Phage XM1 in SM buffer at 3% salinity showed the most significant activity but could not grow at 0% salinity (Figure 2D), indicating coastal and marine environments as XM1\u0026rsquo;s habitat. \u003cem\u003eV. parahaemolyticus\u003c/em\u003e phage VB_VpP_BT-1011 can survive at 0% to 3% NaCl [36], indicating this phage also includes estuarine environments as its habitat. Results from the current study show that phage XM1 is stable at broad pH, temperature, and salinity ranges, indicating its tolerance and adaptation to various environmental stresses.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eV. parahaemolyticus\u003c/em\u003e was infected with XM1 at different MOIs to investigate its effect on bacterial growth. The killing curve (Figure 3) indicated that increasing the MOI increased bacterial growth inhibition. There was an initial increase in the concentration of host bacteria. However, with the release of phages, the concentration of host bacteria began to decrease, with no observable difference in the XM1 inhibitory effects observed after 11 h of infection, regardless of different MOIs. The intense bactericidal activity indicates that XM1 is a potent candidate for use in phage therapy.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGenome sequence of vibrio phage XM1.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGenome analyses revealed that Phage vB_VpaM_XM1 is a double-stranded DNA virus belonging to the Duplodnaviria realm in the ICTV. Its genome size is 46,056 bp with a total G + C content of 42.51%, containing 69 predicted open reading frames (ORFs). The size of XM1 protein-coding sequences (CDSs) ranges from 51 to 825 amino acid residues (207.74 on average). Among the 69 ORFs, 66 are transcribed in the forward direction, while the other 3 (\u003cem\u003ei.e.\u003c/em\u003e, ORF4, ORF35, and ORF36) are transcribed in the reverse direction. The 69 predicted genes primarily encode viral structure proteins and proteins for DNA packaging, DNA metabolism and replication, and host lysis (Figure 4). Nearly two-thirds of the predicted genes (45/69) can be assigned functions according to their homology to known sequences of other phages. Concretely speaking, 19 predicted genes (ORF5, ORF8, ORF9, ORF11, ORF12, ORF13, ORF15, ORF16, ORF18, ORF19, ORF20, ORF21, ORF22, ORF23, ORF24, ORF26, ORF27, ORF29, and ORF30) are related to viral structure proteins, 3 predicted genes (ORF2, ORF3, and ORF6) are associated with DNA packaging, and 14 predicted genes (ORF1, ORF7, ORF10, ORF37, ORF40, ORF41, ORF42, ORF44, ORF46, ORF48, ORF56, ORF58, ORF59, and ORF64) are connected with DNA replication and metabolism.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOn the other hand, the remaining predicted genes (24 ORFs) encode hypothetical proteins with unknown functions. Specifically, protein sequences encoded by ORF4, ORF51, ORF60, and ORF66 show no homology to any known protein sequences in the database, potentially indicating their sequence and functional novelties. The predicted terminase large subunit protein (ORF3) plays an important role in the late stage of viral DNA packaging\u0026nbsp;[37]. The predicted portal protein (ORF6) is involved in dsDNA viral genome packaging and release\u0026nbsp;[38]. Three ORFs are predicted to encode host lysis proteins, including a lysozyme (encoded by ORF25), a sporulation-specific N-acetylmuramoyl-L-alanine amidase (encoded by ORF32) that belongs to one of the four families of peptidoglycan hydrolases\u0026nbsp;[39], and a Rz-like spanin (encoded by ORF36) that interacts with bacterial outer membrane and plays a role in the final step of host lysis\u0026nbsp;[40]. Additionally, ORF45 encodes a site-specific integrase that may be a lysogen-related protein\u0026nbsp;[41]. Moreover, no virulence gene or factor was found in the genome of phage XM1. Therefore, phage XM1 may be safe for use in phage therapy.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePhylogenetic and comparative genomic analyses of phage XM1.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo determine the phylogenetic taxonomy of phage XM1, a proteomic tree based on viral whole genomes was generated using VipTree (\u003ca href=\"https://www.genome.jp/viptree\"\u003ehttps://www.genome.jp/viptree\u003c/a\u003e, accessed on 10 Jun 2023)[42]. The result shows that XM1 is closely clustered with Vibrio phages Rostov 7 (accession: MK575466.1)[43], X29 (accession: NC_024369.2)[44], and phi2 (accession: KJ545483.2)[45] (Figure 5A and 5B), indicating that these four phages may form a new taxonomic group of viruses.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThrough BLASTN in the NCBI database, phage XM1 has the highest genomic sequence identity to \u003cem\u003eVibrio\u003c/em\u003e phages Rostov 7, X29, and phi2, with a 77.76%, 77.60%, and 77.54% identity scores and 33%, 25%, and 25% query coverage, respectively (accessed on 10 Jun 2023). The ANI value of the XM1 genome with the viral genome of Rostov 7, X29, and phi 2 was 73.35%, 72.22%, and 72.23%, respectively (Figure 6A). However, phage XM1 showed no genomic match with other NCBI viral genomes. Phage XM1 shares a similar overall genomic organization with Vibrio phages Rostov 7, X29, and phi 2 (Figure 6B). The predicted genes of XM1 showed 66% to 100% sequence identities to genes of the other three Vibrio phages, except several genes that had no sequence match.\u003c/p\u003e\n\u003cp\u003eTo further confirm the taxonomic novelties of phage XM1 and its closest relatives (\u003cem\u003ei.e.\u003c/em\u003e, \u003cem\u003eVibrio\u003c/em\u003e phages Rostov 7, X29, and phi 2), a whole-genome phylogenetic tree (Supplementary Figure 1, Figure 7) was constructed with 154 representative viral genomes selected from all the 66 families of Duplodnaviria currently defined by ICTV (Accessed on 9 Jun 2023). The phylogenetic tree showed that the phages Rostov 7, X29, and phi 2 belong to Duplodnaviria, Heunggongvirae, Uroviricota, Caudoviricetes. In addition, the analytic result indicated that phages XM1, Rostov 7, X29, and phi 2 are phylogenetically grouped and form a unique viral cluster not affiliated with any known viral families. Therefore, we tentatively name Weiviridae as the new family name for these four novel \u003cem\u003eVibrio\u003c/em\u003e-lysing phages.\u003c/p\u003e\n\u003cp\u003eThe terminase large subunit is a relatively conserved protein used as a marker for establishing phage phylogenetic relationships [46]. Similarly, the major capsid protein, the primary component of phage capsid, is conserved among phylogenetically related phages and is frequently used in phage classification [47]. In this study, a phylogenetic tree was constructed based on the protein sequences of phage terminase large subunit and phage capsid protein, respectively (Figure 8). Our analyses show that phage XM1 is grouped with \u003cem\u003eVibrio\u003c/em\u003e pahges Rostov 7, X29, and phi 2 and phylogenetically distant from other phage families. The genome phylogenetic tree also showed similar results (Figure S2).\u003c/p\u003e\n\u003cp\u003eThe gene sequences selected in these two phylogenetic trees were based on Blast in NCBI (Accessed on 10 Jun 2023). Values at the nodes indicate the bootstrap support calculated from 1000 replicates.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAccording to phylogenetic trees (Figures 5, 7, and 8), the \u003cem\u003eVibrio\u003c/em\u003e phages Rostov 7, X29, and phi 2 belong to class Caudoviricetes. However, none of them are classified into any known phage families. Phage XM1 and these phages are grouped as a new clade and different from previously described phages. These results indicate that phages XM1, Rostov 7, X29, and phi 2 can be classified as a new phage family.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThis study isolated and fully characterized a new phage, vB_VpaM_XM1, infecting \u003cem\u003eV. parahaemolyticus\u003c/em\u003e and having a large burst size and narrow host range. XM1 has a broad range of temperature, pH, and salinity adaptability, and exhibits strong lytic activity. These indicated that XM1 has great potential as a novel antibacterial agent for the biological control of vibriosis in aquaculture. Moreover, the complete XM1 genome sequence was determined and compared with its phage relatives. Furthermore, phylogenetic analyses revealed that XM1 clusters a new clade with vibrio phages Rostov 7, X29, and phi 2, and should belong to a new viral family named Weiviridae. Our report provides an in-depth analysis of phage at the genomic, phylogenetic, and ecological levels and provides a potential antimicrobial candidate for pathogenic \u003cem\u003eV. parahaemolyticus\u003c/em\u003e.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e \u003cb\u003ePhage isolation and purification.\u003c/b\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eV. parahaemolyticus\u003c/em\u003e 1.1997 was used as the bacterial host [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. It was grown in a rich organic (RO) medium with a shaking speed of 160 rpm/min at 28 ℃. Firstly, 1 mL of water sample from seafood markets (Xiamen, China) was added into 10 mL of an exponentially growing culture of \u003cem\u003eV. parahaemolyticus\u003c/em\u003e 1.1997 and incubated for 24 h. The mixed culture was then passed through a 0.22 \u0026micro;m filter membrane ( Millipore, Bedford, MA, USA) to remove bacterial cells. The filtrate was diluted and mixed with exponential host cultures to get phage plaque using the double-layer agar method [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. After the above-mentioned steps, the well-separated plaque was removed and stored in the storage medium (SM) buffer (50 mM Tris-HCl, 0.1 M NaCl, and 8 mM MgSO\u003csub\u003e4\u003c/sub\u003e, pH 7.5) at 4 ℃ for later use.\u003c/p\u003e \u003cp\u003e \u003cb\u003ePhage enrichment.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo obtain highly concentrated phage, 1 L phage lysate was treated with DNase I and RNase A at room temperature for 1 h until the final concentration reached 1\u0026micro;g/mL, then 1 M NaCl was supplied for 30 min at 4 ℃ to promote the separation of phage particles and cell debris. Finally, the solution was mixed with 10% polyethylene glycol (PEG 8000) and stored for 3 d at 4 ℃ to precipitate virions. Viral particles were subsequently collected by centrifugation (12,000\u0026times;g, 60 min, 4 ℃) and resuspended in 6 mL of SM buffer. The phage suspensions were prepared via cesium chloride gradient centrifugation (1.3, 1.5, 1.7 g/mL) centrifuged at 200,000\u0026times;g for 24 h at 4 ℃ using an Optima L-100 XP ultracentrifuge (Beckman Coulter, CA, USA). The visible phage band was extracted and future dialyzed through 30-kD super-filters (Millipore, MA, USA) [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cb\u003eMorphology observation.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe phage morphology was observed using a JEM-2100 transmission electron microscope (JEOL, Tokyo, Japan) at an acceleration voltage of 80 kV. To prepare the samples for observation, 20 \u0026micro;L of high-titer phage concentrate was plated on 200-mesh formvar-coated copper electron microscope grids and allowed to absorb for 10 min, then negatively stained with 1% phosphotungstic acid for 1 min, followed by air drying for 10 min. The size of phage particles was measured from at least five TEM images using ImageJ software [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cb\u003eHost range.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe host range of XM1 was determined by spot testing and confirmed by the double-layer agar method [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. 1 mL of exponentially growing bacteria (10\u003csup\u003e8\u003c/sup\u003e CFU/mL) was mixed with 5 mL of the pre-warmed (50\u0026deg;C) semisolid liquid medium, then poured onto a solid agar plate immediately. After 10 min of air drying, 5 \u0026micro;L of purified phage solution was spotted on the host bacterial lawn. The plate was then incubated at 28 ℃ for 24 h. Phage infection was determined by visual examination of the plates for plaques. The used bacteria included 58 strains in the genera \u003cem\u003eVibrio\u003c/em\u003e, \u003cem\u003eIdiomarina\u003c/em\u003e, \u003cem\u003ePseudoalteromonas\u003c/em\u003e, \u003cem\u003ePhotobacterium\u003c/em\u003e, and \u003cem\u003eShewanella\u003c/em\u003e (listed in Table S1).\u003c/p\u003e \u003cp\u003e \u003cb\u003eOne-step growth curve.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe one-step growth curve of phage XM1 was determined using the previously described method [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. Briefly, 1 mL of exponentially growing bacteria (10\u003csup\u003e8\u003c/sup\u003e CFU/mL) was exposed to phages at a MOI of approximately 0.01, then placed in the dark for 10 min. Bacteria were then pelleted (6,000\u0026times;g, 5 min), and the non-adsorbed phages in the supernatant were discarded. The pellet was then washed twice and resuspended in 100 mL RO medium, and the culture was then incubated at 28 ℃ with a shaking speed of 160 rpm/min. Every 10 min, subsamples were collected, and the viral abundance was detected using the double-layer agar method. The burst size was calculated as the ratio between the number of virions at the growth plateau and the initial number of infected host cells [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cb\u003epH, temperature and salinity tolerance.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eA series of 3 experiments were designed to determine the influence of pH, temperature, and salinity on the stability of phage XM1. In all experiments, the double-layer agar method was applied to estimate the infection activity of the phage. In the pH experiment, the pH of the SM buffer was adjusted from 2 to 12 with HCl or NaOH solution. The phage concentrate was added to the SM buffer so that the final concentration was 10\u003csup\u003e14\u003c/sup\u003e PFU/mL, and then all treatments were incubated at 4 ℃ for 3 h, 24 h, and 48 h. For the experiment that investigated the thermal stability of the phage, phage in all treatments was incubated for 3 h, with incubation temperatures set at 4 ℃, 24 ℃, 37 ℃, 50 ℃, 60 ℃, and 70 ℃, respectively. As for the salinity tolerance experiment, solutions with salinity ranging from 0 to 5% were used for phage incubation (incubation time: 12 h).\u003c/p\u003e \u003cp\u003e \u003cb\u003eGrowth curve experiment.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe phage XM1 was mixed with the host \u003cem\u003eV. parahaemolyticus\u003c/em\u003e at different MOIs (0.001, 0.01, 0.1, 1, 10) and incubated at 28 \u0026ordm;C. Meanwhile, \u003cem\u003eV. parahaemolyticus\u003c/em\u003e at the same MOI level but without phage was used as a positive control. The growth curves were monitored over 12 h, and optical density (OD\u003csub\u003e600\u003c/sub\u003e) measurements were recorded every 1 h. Three independent assays were carried out for each assay.\u003c/p\u003e \u003cp\u003e \u003cb\u003eDNA extraction and sequencing.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eViral genomic DNA was extracted using the Takara MiniBEST Viral RNA/DNA Extraction Kit according to the manufacturer's protocol. In brief, 200 \u0026micro;L viral concentrate was mixed with 200 \u0026micro;L Buffer VGB, 20 \u0026micro;L Proteinase K, and 1 \u0026micro;L Carrier RNA, then incubated at 56 ℃ for 10 min. After that, 200 \u0026micro;L of ethanol was added to the mixture before a 2-min centrifugation (12,000\u0026times;g). Next, 500 \u0026micro;L RWA was added, and the solution was centrifuged at 12,000\u0026times;g for 1 min. Following that, 700 \u0026micro;L RWB was added and the mixture was centrifuged at 12,000\u0026times;g for 1 min, and this step was repeated twice. Finally, 30 \u0026micro;L RNase-free dH\u003csub\u003e2\u003c/sub\u003eO was added into the centrifuge tube and incubated for 5 min at room temperature before the final centrifugation was conducted (12,000\u0026times;g for 2 min). The extracted DNA was stored at -20 ℃. Phage genome sequencing was performed using the Illumina Nova platform by the Shanghai Hanyu Bio-Tech Co., Ltd (Shanghai, China).\u003c/p\u003e \u003cp\u003e \u003cb\u003eGenome annotation and phylogenetic analysis.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe opening reading frames (ORFs) of the XM1 genome were predicted by the Glimmer3/GeneMarkS/Prodigal online server and annotated by BLASTp search against the National Center for Biotechnology Information (NCBI) nonredundant (nr) protein sequences (Accessed on 27 Feb 2023) [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e, \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. Gene map was created based on the genome annotations using CGView-Circular Genome Viewer (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://proksee.ca\u003c/span\u003e\u003cspan address=\"https://proksee.ca\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. Genomic structures and comparison maps of phages belonging to the same categories were made using EasyFig [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eA phylogenetic tree based on genome sequence similarities computed by tBLASTx was constructed using the Viral Proteomic Tree server (VipTree, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.genome.jp/viptree/\u003c/span\u003e\u003cspan address=\"https://www.genome.jp/viptree/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e, accessed on 10 Jun 2023) [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. OrthoFinder was used to compare the genomic similarity by orthology (OrthoANI), which was calculated using the BLASTp analysis [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. To explore the phage taxonomic status, the complete nucleotide sequence of phage XM1 and its related viral genomic sequences were submitted to the virus classification and tree building online resource (VICTOR) (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://ggdc.dsmz.de/victor.php\u003c/span\u003e\u003cspan address=\"http://ggdc.dsmz.de/victor.php\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e, accessed on 9 Jun 2023) for phylogenetic analysis, with the recommended settings of genome BLAST distance phylogeny (GBDP) method being used [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. The terminase large subunit protein and capsid protein sequences of XM1 were used to construct phylogenetic trees to analyze its evolutionary relationships, and a Neighbor-joining method in the MEGA 6.0 software package with 1000 bootstrap replicates was used to construct the phylogenetic tree (Accessed on 10 Jun 2023) [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e, \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e].\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Jie Wu and Lanfeng Dai for helpful guidance on the experimental methods. We also thank Jan Meier-Kolthoff of the Leibniz Institute DSMZ scientific community for his help in the construction of the GBDP tree.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eZ.W., C.A., and H.D. developed ideas and organized the study; Z.W. performed the experiments; Z.W. and X.L. analyzed the data; Z.W. wrote the manuscript. H.D., X.L. and C.A. made revisions to the manuscript. All authors have read and agreed to the published version of the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Key Research and Development Program of China (grant No. 2020YFA0608302) and the National Natural Science Foundation of China (grant Nos. 42076111, 42141003, 42188102, and 41861144018).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSu YC, Liu C. 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Mol Biol Evol. 1987;4(4):406\u0026ndash;25.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-4560493/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4560493/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003e \u003cem\u003eVibrio parahaemolyticus\u003c/em\u003e has been a leading cause of foodborne disease outbreaks and infectious diarrhea cases in coastal areas, antibiotic resistance has increased significantly due to widespread antibiotic abuse, bacteriophages (phages) are viruses that specifically infect bacteria, it is necessary to isolate and characterize new phages to broaden our understanding of the ecology, evolution, and diversity of both phages and their bacterial hosts further.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eA novel vibriophage vB_VpaM_XM1 (XM1) was described in the present study. The morphological analysis revealed that phage XM1 had \u003cem\u003eMyoviridae\u003c/em\u003e-like morphology, with an oblate icosahedral head and a long contractile tail. The genome size of XM1 is 46,056 bp, with a G\u0026thinsp;+\u0026thinsp;C content of 42.51%, encoding 69 open reading frames (ORFs). Moreover, XM1 showed a narrow host range only lysing \u003cem\u003eVibrio xuii\u003c/em\u003e LMG 21346 (T) JL2919, \u003cem\u003eVibrio parahaemolyticus\u003c/em\u003e 1.1997, and \u003cem\u003eVibrio parahaemolyticus\u003c/em\u003e MCCC 1H00029 among the tested bacteria. One-step growth curves showed that XM1 has a 40-minute latent period and 264 plaque-forming units (PFU)/cell burst size. In addition, XM1 exhibited broad pH, thermal, and salinity stability, as well as strong lytic activity, even at a multiplicity of infection (MOI) of 0.001. Multiple genome comparisons and phylogenetic analyses showed that phage XM1 is grouped in a clade with three other phages, including \u003cem\u003eVibrio\u003c/em\u003e phages Rostov 7, X29, and phi 2, and is distinct from all known viral families that have ratified by the standard genomic analysis of the International Committee on Taxonomy of Viruses (ICTV).\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eTherefore, the above four phages might represent a new viral family, tentatively named Weiviridae. The broad physiological adaptability of phage XM1 and its high lytic activity and host specificity indicated that this novel phage is a good candidate for being used as a therapeutic bioagent against infections caused by certain \u003cem\u003eVibrio parahaemolyticus\u003c/em\u003e strains.\u003c/p\u003e","manuscriptTitle":"Characterization and Genomic Analyses of dsDNA Vibriophage vB_VpaM_XM1, Representing a New Viral Family","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-27 08:58:09","doi":"10.21203/rs.3.rs-4560493/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a93ed0f1-2c9e-499a-8a66-951cc3cc9af5","owner":[],"postedDate":"June 27th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-07-01T09:06:58+00:00","versionOfRecord":[],"versionCreatedAt":"2024-06-27 08:58:09","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4560493","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4560493","identity":"rs-4560493","version":["v1"]},"buildId":"FbvkV6FR0MCFSLy54lSbu","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}