Isolation and characterization of a novel lytic bacteriophage Ecolivirus Myo-P293 targeting avian pathogenic Escherichia coli

Isolation and characterization of a novel lytic bacteriophage Ecolivirus Myo-P293 targeting avian pathogenic Escherichia coli | 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 Isolation and characterization of a novel lytic bacteriophage Ecolivirus Myo-P293 targeting avian pathogenic Escherichia coli Hai Xu, Qiurong Qi, Yalu Zhu, Erzhong Wu, Guiqin Yan, Yu Lu, Yaming Feng This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7281919/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 29 Mar, 2026 Read the published version in Archives of Virology → Version 1 posted 5 You are reading this latest preprint version Abstract A novel lytic bacteriophage, Ecolivirus Myo‑P293 (P293), targeting avian pathogenic Escherichia coli (APEC), was isolated from duck farm sewage in Jiangsu, China. P293 formed clear plaques approximately 2 mm in diameter and displayed the characteristic morphology of Myoviridae family, with an icosahedral head (~ 70 nm) and a contractile tail (~ 100 nm), as observed under transmission electron microscopy. Adsorption assays showed that over 70% of phages adsorbed to host cells within 5 min. One-step growth analysis revealed a latent period of approximately 30 min and a burst size of 284 ± 45 PFU per infected cell. P293 exhibited stability across a pH range of 5–9 and at temperature between 20–40°C, but its infectivity was significantly reduced when exposed to temperature ≥ 60°C or 60% ethanol. In vitro bacteriolytic assays demonstrated strong antibacterial activity, which was dependent on the multiplicity of infection (MOI). Notably, P293 achieved approximately 45% clearance of mature E. coli biofilms after 24 h treatment. Whole-genome sequencing of P293 revealed an 89.5 kb double-stranded DNA genome encoding 95 open reading frames (ORFs), including modules related to structure, replication, lysis, and host interaction. Approximately 40% of the encoded genes are annotated as hypothetical proteins. Phylogenetic and comparative genomic analyses placed P293 within the unclassified Felixounavirus clade, closely related to Escherichia phage wV8 and Salmonella phage Felix O1, while displaying distinct tail fiber gene signatures associated with host specificity. These findings support the potential of P293 as a candidate for phage-based biocontrol strategies against APEC in poultry production. Avian pathogenic Escherichia coli Lytic bacteriophage Whole-genome sequencing Biological characteristics Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Avian pathogenic Escherichia coli (APEC), a member of the extraintestinal pathogenic E. coli (ExPEC) group, specifically infects avian hosts and induces both respiratory and systemic disease [ 1 ]. Traditionally, APEC has been viewed as a secondary or opportunistic pathogen that emerges under conditions of intensive farming, inadequate sanitation or other stress factors such [ 2 ]as viral respiratory infections or poor husbandry practices [ 3 ]. However, it is now clear that APEC can also act as a primary pathogen in immunocompetent avian hosts [ 4 ]. Moreover, its tight linkage to commercial poultry production systems renders APEC a significant reservoir of antimicrobial-resistance determinants. While antibiotics have long been the cornerstone for preventing and treating APEC infections, their widespread misuse and overuse have driven the emergence of multidrug-resistant (MDR) strains [ 5 , 6 ]. These MDR APEC isolates frequently carry plasmids encoding multiple antibiotic resistance genes (ARGs), which facilitate horizontal dissemination across bacterial populations [ 7 ]. To mitigate the acceleration of bacterial antimicrobial resistance, the World Health Organization (WHO), the Food and Agriculture Organization of the United Nations (FAO), and the World Organization for Animal Health (OIE) have launched global initiatives to regulate and reduce the misuse of antibiotics [ 8 ]. Consequently, effective control of APEC necessitates a multifaceted approach; the pathogen’s documented epidemiological expansion following restrictions on antimicrobial use, together with mounting regulatory pressures, highlights the urgent need for non-antibiotic intervention strategies. Bacteriophages are viruses capable of lysing bacteria and are ubiquitous in natural environments. Compared with conventional antibiotics, phage-based antibacterial agents exhibit several notable characteristics: they display high host specificity, targeting only the intended bacterial cells without affecting host tissues or the normal microbiota; they exist in vast numbers in the environment, making nature itself a rich reservoir for therapeutic phages [ 9 ]; and they possess unique pharmacokinetic properties in vivo , whereby lytic phages self-amplify at the site of infection as they kill their bacterial hosts [ 10 , 11 ]. In addressing the challenge of MDR bacteria, phages can exert their distinctive bactericidal effects through multiple mechanisms; for example, by encoding enolase-inhibitory proteins that specifically bind key enzymes in carbohydrate metabolism, thereby disrupting ATP synthesis and the supply of cell-wall precursors, ultimately resulting in bacterial lysis and death [ 12 ]. Consequently, the isolation and characterization of novel bacteriophages targeting APEC are critically important for expanding phage libraries and improving strategies to prevent and treat APEC infections. In 2022, the International Committee on Taxonomy of Viruses (ICTV) formally abolished the morphology-based family-level taxa Podoviridae , Siphoviridae and Myoviridae , along with the order Caudovirales , and introduced a unified binomial species nomenclature [ 13 ]. Within the newly established class Caudoviricetes , twenty-two new families were defined, of which fourteen have been assigned to four novel orders. Although the term “ myovirus ” persists as an informal morphological grouping, its taxonomy has shifted from morphology to genome-based phylogenetic classification. Myoviruses have demonstrated both safety and efficacy in the treatment of multidrug-resistant bacterial infections. For example, Yao et al. isolated the strictly lytic phage PEC9, which protects mice against infection by APEC serotypes O1 and O2 [ 14 ], and Naghizadeh et al. showed that a phage cocktail could ameliorate organ lesions in quail infected with APEC [ 15 ]. Advances in genetic engineering and synthetic biology for phage modification are poised to accelerate the clinical application of myoviruses in antibacterial therapy [ 16 ]. In this study, we isolated and characterized a lytic bacteriophage, Ecolivirus Myo-P293 (abbreviated as P293), from a poultry farm that specifically targets APEC. Transmission electron microscopy (TEM) revealed morphological features consistent with the Myoviridae family. Comprehensive characterization was conducted based on its biological properties and whole-genome sequencing. This study provides a valuable reference for the expansion of APEC-targeting phage libraries and the development of phage-based antimicrobial agents. Materials and methods Bacterial strains and phage host range determination The bacterial strains used in this study were provided by the GuoTai (Taizhou) Center of Technology Innovation for Veterinary Biologicals. A total of 15 Escherichia coli ( E. coli ) strains were included, comprising laboratory strains DH5α, BL21, and Top10, along with 12 serotyped field isolates recovered from poultry farms (Table 1 ). All strains were cultured in Luria-Bertani (LB) broth at 37°C under aerobic conditions. For long-term storage, bacterial stocks were preserved in LB broth supplemented with 30% (v/v) glycerol at -80°C. The host range of phage P293 was assessed using a spot assay [ 17 ]. Briefly, 10 µL of phage suspension (~ 1×10⁹ PFU/mL) was spotted onto the surface of LB agar plates overlaid with lawns of each bacterial strain. Plates were incubated at 37°C for 4–6 hours, and lytic activity was evaluated based on the formation of clear lysis zones. To further quantify phage infectivity, the efficiency of plating (EOP) was determined by performing plaque assays using all 15 field E. coli strains. The EOP was calculated by comparing the plaque-forming units (PFU) on each susceptible strain with those on the original host strain ( E. coli DPE 2). Table 1 Host range of bacteriophage Ecolivirus Myo-P293 and efficiency of plating (EOP) No. of host Bacterial host Strains Serotype Spot test a Plaque production ability Total no. of plaque (pfu/ml) / Efficiency of plating (EOP) Sources b 1 Escherichia coli DH5α - - - - LS 2 Escherichia coli BL21 - - - - LS 3 Escherichia coli Top10 - - - - LS 4 Escherichia coli GPE12 O1 - - - LS 5 Escherichia coli CPE30 O1 - - - LS 6 Escherichia coli CPE3 O1 - - - LS 7 Escherichia coli DPE2 O2 ++ + 1.34×10 10 LS 8 Escherichia coli DPE14 O2 ++ + 1.18×10 10 LS 9 Escherichia coli CPE5 O2 + + 2.45×10 9 LS 10 Escherichia coli CPE11 O78 + + 1.73×10 9 LS 11 Escherichia coli GPE7 O78 - - - LS 12 Escherichia coli GPE19 O78 - - - LS 13 Escherichia coli CPE22 O145 + + 8.82×10 8 LS 14 Escherichia coli CPE8 O145 - - - LS 15 Escherichia coli DPE4 O145 - - - LS a ++ = large clear lysis, + = small clear lysis, - = no lysis of plating. b LS stands for lab stock. Phage isolation, purification, and amplification Phage P293 were isolated from sewage samples collected at a duck farm, using E. coli O2 (DPE 2) as the indicator host strain. Phage isolation was performed according to previously described methods [ 18 ], based on the plaque assay technique. To obtain clonal phage populations, individual plaques were picked using sterile micropipette tips and subjected to at least five successive rounds of purification. For phage amplification, the purified phages were enriched as follows: a 50 mL culture of the indicator host (10⁷ CFU/mL) in LB broth was infected with purified phage and incubated at 37°C with shaking at 120 rpm. After complete lysis, the culture was centrifuged at 6,000 × g for 15 min at 4°C to remove residual bacterial cells and debris. The resulting supernatant was further centrifuged at 15,000 × g for 1 h at 4°C to pellet the phage particles. The phage pellets were resuspended in SM buffer (100 mM MgSO₄·7H₂O, 10 mM NaCl, 50 mM Tris-HCl, pH 7.5) and subsequently filtered through 0.22 µm syringe filters to ensure sterility. Phage titers were determined using the double-layer agar method, which also confirmed the presence of lytic phages in the filtrates [ 2 , 19 ]. Transmission electron microscopy (TEM) Approximately 5 µL of purified P293 suspension was applied onto glow-discharged, carbon-coated copper grids (300 mesh) and allowed to adsorb for 1 minute. Excess liquid was then carefully removed using filter paper. The grids were subsequently stained with 2% (w/v) uranyl acetate for contrast enhancement and air-dried at room temperature. The negatively stained phage particles were examined using a Hitachi transmission electron microscope (Hitachi, Japan) operated at an accelerating voltage of 80 kV. Phage adsorption assay To determine the adsorption kinetics of the P293 to its host, an adsorption assay was performed with minor modifications based on previously described protocols [ 20 ]. Briefly, the E. coli DPE 2 host strain was cultured in LB broth and infected with phage at a multiplicity of infection (MOI) of 0.1. The mixture was incubated at 37°C, and aliquots were collected at 0, 5, 10, 15, 20, 25, and 30 minutes post-infection. The samples were immediately centrifuged at 10,000 × g for 10 minutes to pellet the bacterial cells. The resulting supernatants were filtered through 0.22 µm syringe filters to remove residual cells, and the titers of non-adsorbed phages were determined using the double-layer agar method. All experiments were performed in triplicate to ensure reproducibility. One-step growth curve The one-step growth experiment was performed with slight modifications based on previously described methods [ 21 ]. Briefly, the P293 was mixed with E. coli DPE 2 at a MOI of 1 and allowed to adsorb at 37°C for 10 minutes. The mixture was then centrifuged at 10,000 × g for 30 seconds to remove unbound phages, and the resulting pellet was resuspended in 10 mL of LB broth. The culture was incubated at 37°C with shaking at 200 rpm. Aliquots of 100 µL were collected every 30 minutes over a 3-hour period. Each sample was centrifuged at 10,000 × g for 5 minutes to remove bacterial debris, and the phage titers in the supernatants were determined using the double-layer agar method. All experiments were conducted in triplicate. The burst size was calculated as the ratio of the final number of released phage particles to the initial number of infected host cells. Stability assay The stability of phage P293 under various physicochemical conditions was evaluated in terms of pH, temperature, and disinfectant exposure [ 22 ]. For pH stability, the phage suspension was adjusted to a final concentration of 1 × 10⁹ PFU/mL using buffers with pH values ranging from 3 to 12. Samples were incubated at room temperature for 1, 2, and 3 hours. After incubation, residual phage titers were determined using the double-layer agar method. For thermal stability, the phage at a concentration of 1 × 10⁹ PFU/mL was incubated at 20°C, 40°C, 60°C, and 80°C for 20, 40, 60, and 80 minutes. Samples were then immediately cooled on ice, and phage titers were measured using the double-layer agar method. For disinfectant stability, the phage was diluted to 1 × 10⁹ PFU/mL in ethanol solutions at final concentrations of 30% and 60% (v/v). The mixtures were incubated at room temperature for 20, 40, 60, and 80 minutes, followed by titer determination as above. All treatments were conducted in triplicate. Phage viability under each condition was assessed by plaque formation efficiency on the host lawn, with results expressed as viable phage counts relative to the untreated control. In vitro bacteriolytic activity The in vitro bacteriolytic activity of phage P293 was assessed by monitoring the optical density (OD₆₀₀) of E. coli cultures using UV-visible spectrophotometry [ 23 ]. The host strain, E. coli DPE 2, was originally isolated from the pericardial effusion of ducks diagnosed with myocarditis. Briefly, E. coli DPE 2 cultures in the logarithmic growth phase were infected with phage P293 at different multiplicities of infection (MOIs: 100, 10, 1, 0.1, 0.01, and 0.001). Infected cultures were incubated at 37°C for 270 minutes. Uninfected E. coli DPE 2 cultures served as the positive control, and LB broth served as the negative control. The OD₆₀₀ of each culture was measured every 30 minutes using a UV-Vis spectrophotometer. Bacteriolytic activity was evaluated based on changes in turbidity (OD₆₀₀) over time compared to the control. All experiments were performed in triplicate. Biofilm clearance assay To evaluate phage-mediated biofilm clearance [ 24 ], E. coli DPE 2 was adjusted to OD₆₀₀ = 1.0 and inoculated into 96-well plates containing 200 µL LB per well (1:1000 dilution). Plates were incubated statically at 37°C for 24 hours to allow biofilm formation. After removing planktonic cells, 200 µL of phage suspension (1×10⁸ PFU/mL) in SM buffer (100 mM NaCl, 50 mM Tris-HCl, pH 7.5, 10 mM MgSO₄) was added to each well and incubated for 6, 12, or 24 hours. Wells treated with SM buffer alone served as controls. After incubation, wells were washed with PBS, stained with crystal violet, and OD₅₉₅ values were measured as described above. Biofilm clearance was calculated as: Biofilm clearance (%) = [1 − (OD₅₉₅ of phage-treated well / OD₅₉₅ of control well)] × 100. Phage DNA sequencing and bioinformatic analysis Genomic DNA of phage P293 was extracted using the phenol/chloroform method, as previously described [ 25 ]. The purified DNA was diluted to a final concentration of 0.2 ng/µL for whole-genome sequencing. Whole-genome shotgun (WGS) sequencing was conducted to characterize the phage genome. Genomic libraries with varying insert sizes were constructed and subjected to paired-end (PE) sequencing on the Illumina NovaSeq platform. Raw sequencing reads were de novo assembled using SPAdes, and contigs were filtered based on sequencing depth. High-coverage contigs were aligned against the National Center of Biotechnology Information (NCBI) nucleotide (NT) database using BLASTn to identify viral genome sequences [ 26 ]. Final genome assembly was refined by base-level error correction using Pilon. Genomic similarity between Ecolivirus Myo-P293 and previously reported phages was assessed using fastANI v1.34 [ 27 ]. Gene prediction, functional annotation, and circular genome visualization were performed using Pharokka v1.7.1 [ 28 ]. Whole-genome alignments were carried out with MAFFT v7.520 [ 29 ], and amino acid sequence alignments of the major capsid and tail fiber proteins were performed using MUSCLE v5.1 [ 30 ]. Phylogenetic relationships were inferred using IQ-TREE v2.2.5 based on the aligned sequences [ 31 ]. For comparative genomic analysis, genome collinearity and structural conservation among phage P293, Escherichia phage wV8, and Salmonella phage Felix O1 were evaluated using Mauve v2.4.0 [ 32 ]. Statistical analysis Data were expressed as mean ± standard deviation (SD) and analyzed using Graph Pad Prism version 9 software. The level of significance was set at ( p ≤ 0.05). Results Isolation and morphology of phage P293 Using E. coli DPE 2 as the host, a novel lytic bacteriophage, designated Ecolivirus Myo-P293 (hereafter referred to as P293), was isolated from duck farm sewage in Jiangsu, China. Phage P293 produced clear, uniformly sized plaques with an average diameter of approximately 2 mm on a lawn of E. coli DPE 2 (Fig. 1 A). The phage was subsequently propagated and purified for morphological examination. TEM revealed that P293 exhibits typical Myoviridae morphology, characterized by an icosahedral head (~ 70 nm in diameter) and a contractile tail (~ 100 nm in length) (Fig. 1 B). The presence of a distinct tail sheath and baseplate structure is consistent with features of the Myoviridae family. Latent times and phage burst sizes Phage adsorption kinetics demonstrated that over 70% of phage particles were adsorbed onto host cells within the first 5 minutes of incubation. After 30 minutes, 17.5% of the phages remained unadsorbed in the supernatant (Fig. 2 A). To evaluate lytic activity, P293 was added to logarithmic-phase E. coli DPE 2 cultures. The OD₆₀₀ decreased from 0.65 to 0.234 within 3 hours and further dropped to 0.189 by 10 hours post-infection, accompanied by visible clearing of the culture and lysed bacterial debris, confirming the lytic nature of the phage (Fig. 2 B). A one-step growth assay was conducted at 37°C with an MOI of 1 to assess the replication dynamics of P293. The results showed a latent period of approximately 30 minutes, followed by a large burst size of 284 ± 45 PFU per infected cell (Fig. 2 C). Host range and lytic activity of phage P293 To evaluate the biological control potential of phage P293, its lytic activity against E. coli strains was assessed using the spot assay (Table 1 ). A total of 15 E. coli strains were tested, including 12 field isolates representing serotypes O1, O2, O78, and O145, and three laboratory strains (DH5α, BL21, and Top10). Phage P293 exhibited no lytic activity against the three laboratory strains or the O1 serotype isolates. In contrast, it demonstrated clear lytic activity against all three O2 serotype strains, as well as one isolate each from the O78 and O145 serotypes. The number of plaques formed on susceptible strains varied among isolates, indicating strain-specific differences in susceptibility. Stability of phage P293 under different environmental conditions The environmental stability and persistence of phages are critical factors for their potential application in biological control. To assess the stability of phage P293, its tolerance to varying pH levels, temperatures, and exposure to common disinfectants was evaluated. Phage P293 (1 × 10⁹ PFU/mL) was incubated at pH values ranging from 3 to 12 for 1, 2, and 3 hours. As shown in Fig. 3 A, the highest phage titer was observed at pH 7, indicating it to be the optimal condition for P293 stability. The phage remained stable under near-neutral and mildly acidic or alkaline conditions, but exhibited significant titer loss under extreme pH environments. Thermal stability assays revealed that P293 was stable at 20°C to 40°C, with minimal loss of infectivity. However, phage survival rate decreased significantly at temperatures above 60°C. As shown in Fig. 3 B, treatment at 60–80°C for 20 minutes reduced the survival rate from 65–24%. Prolonged exposure further compromised phage viability, and complete inactivation was observed after 80 minutes at 80°C, indicating that P293 is heat-sensitive but remains stable at moderate temperatures. To assess its sensitivity to disinfectants, P293 was treated with 30% and 60% ethanol for 20, 40, 60, and 80 minutes (Fig. 3 C). At 30% ethanol, phage survival rate dropped to 18% after 60 minutes. In contrast, 60% ethanol led to near-complete inactivation within the same timeframe. These results suggest that P293 is highly sensitive to ethanol treatment, particularly at higher concentrations and extended exposure durations. Bacteriolytic and anti-biofilm effects of phage P293 against E. coli DPE 2 The bacteriolytic activity of phage P293 against E. coli DPE 2 was evaluated at various multiplicities of infection (MOIs). As shown in Fig. 4 A, the optical density (OD₆₀₀) of the uninfected E. coli culture increased steadily over the 270-minute incubation period, indicating normal bacterial growth. In contrast, the negative control (medium only) showed no change in absorbance. For cultures treated with phage at low MOIs (0.001, 0.01, and 0.1), a gradual increase in OD₆₀₀ was observed during the first 120–150 minutes, followed by a notable decline between 150 and 270 minutes, suggesting delayed but effective lytic activity. In comparison, at higher MOIs (1, 10, and 100), little to no increase in absorbance was observed throughout the incubation period, indicating rapid and sustained suppression of bacterial growth. These results demonstrate that phage P293 effectively lysed E. coli DPE 2 in a dose-dependent manner, with stronger and earlier lytic effects observed at higher MOIs. The ability of phage P293 to degrade mature biofilms formed by E. coli DPE 2 was evaluated using a phage suspension at a concentration of 1 × 10⁸ PFU/mL. As shown in Fig. 4 B, phage treatment resulted in a significant, time-dependent reduction in biofilm biomass compared with the untreated control. After 24 hours of exposure, phage P293 exhibited a pronounced biofilm clearance effect, with a removal efficiency of approximately 45%, indicating its potential to disrupt established E. coli biofilms. Genomic features and functional annotation of phage P293 The complete genome of phage P293 was determined to be a linear double-stranded DNA molecule comprising 89,469 base pairs (Fig. 5 ) (Supplementary Material). The overall GC content was approximately 38.5%, with a distinct pattern of GC skew observed across the genome, indicating strand-specific replication and transcriptional asymmetry. A total of 95 open reading frames (ORFs) were predicted, of which 52 were functionally annotated based on sequence similarity to known phage proteins. These ORFs were classified into several functional modules, including structural proteins, lysis-related proteins, DNA metabolism and replication, and host interaction functions. Structural and morphogenesis-related genes were primarily clustered in one region and included genes encoding major head protein, tail sheath, tail fiber, baseplate wedge subunits, and portal protein, consistent with the morphological characteristics of Myoviridae phages. Notably, genes such as tail length tape measure protein and neck protein were also identified, suggesting conserved virion assembly architecture. The lysis cassette comprised genes encoding a putative endolysin, RIIA and RIIB lysis inhibitors, and an Rz-like spanin, which are typically involved in host cell lysis through coordinated degradation of the cell wall and outer membrane. Additionally, several ORFs were associated with DNA replication and nucleotide metabolism, including predicted DNA helicases, polymerase subunits, and nucleotide-modifying enzymes. Approximately 40% of the predicted ORFs were annotated as hypothetical proteins with unknown function, suggesting potential novel phage-specific genes requiring further investigation. Comparative analysis revealed high genomic synteny and homology between P293 and members of the Phage01D_k149 group, particularly in regions encoding structural components and lysis-related functions. Comparative genomic analysis of phage P293 To investigate the genomic relatedness of Ecolivirus Myo-P293 with other well-characterized myoviruses, comparative genomic analysis was performed using Mauve alignment software. The genomes of Escherichia phage wV8 and Salmonella phage Felix O1 were selected for comparison due to their phylogenetic proximity and similar genome sizes. As shown in Fig. 6 , P293 shares extensive collinear regions with Escherichia phage wV8, particularly across the structural and lysis gene clusters. Large locally collinear blocks (LCBs) indicate a high degree of sequence conservation and genome organization, suggesting that P293 and wV8 may belong to the same genus or closely related lineages. Most conserved regions span from ~ 10 kb to ~ 85 kb, and gene order remains largely syntenic. In contrast, the alignment between P293 and Salmonella phage Felix O1 revealed fewer and less extensive homologous regions. While some conserved sequences were observed in the structural modules, several genomic rearrangements and inversions were evident, especially at the terminal regions. This suggests that although P293 and Felix O1 share some common ancestry, they have diverged considerably in genome architecture. Together, these results indicate that phage P293 exhibits a high degree of genomic similarity and collinearity with E. coli phage wV8, whereas it shares limited homology with Salmonella phage Felix O1, supporting its classification as a novel Escherichia -infecting myovirus with distinct evolutionary lineage. Phylogenetic analysis of phage P293 To determine the evolutionary position of phage Ecolivirus Myo-P293, phylogenetic trees were constructed based on its complete genome sequence (Fig. 7 A), major capsid protein (Fig. 7 B), and tail fiber protein (Fig. 7 C). As shown in the genome-based phylogenetic tree (Fig. 7 A), P293 clustered within the unclassified Felixounavirus clade, and was most closely related to Escherichia phage EF202P1 and Salmonella phage Felix O1, suggesting a shared evolutionary lineage with this group of lytic Myoviridae phages. Phylogenetic analysis based on the major capsid protein (Fig. 7 B) supported this classification, with P293 again grouping closely with Felixounaviruses such as Salmonella phage Felix O1 and Escherichia phage P7, while remaining distinct from members of the T4-like and P2-like virus clades. In contrast, the tree derived from tail fiber protein sequences (Fig. 7 C) revealed a slightly divergent pattern: P293 formed a branch near the Felixounavirus cluster but displayed increased phylogenetic distance from closely related Escherichia phages, reflecting the high variability and modular evolution typically observed in tail fiber genes. Collectively, these results suggest that P293 is a member of the unclassified Felixounavirus group, exhibiting close evolutionary relationships with known Escherichia and Salmonella phages at both the genomic and structural protein levels, while also possessing unique genetic features, particularly in its tail fiber region, that may underlie its host specificity and functional diversity. Discussion In this study, we report the isolation, characterization, and genomic analysis of P293, a novel Myoviridae phage with potential as a biocontrol agent against APEC. Our results demonstrate that P293 exhibits high lytic efficiency, efficacy against biofilms, and favorable stability-features that align with the functional profile desired for phage-based interventions in poultry. Phage P293 exhibited strong lytic activity targeting O2 serotypes and select O78/O145 APEC strains, reflecting a relatively narrow yet clinically relevant host range. This specificity is consistent with other APEC-targeting phages, such as vB_EcoP_PW8 [ 33 ] and phage AG-MK-2022-Basu [ 34 ], which both effectively lyse multidrug-resistant (MDR) strains despite having a restricted host breadth. Notably, P293 achieved a rapid adsorption rate of over 70% within 5 minutes and released a burst size of approximately 284 PFU per infected cell, which is comparable to or even surpasses other APEC-targeting phages in terms of replicative fitness. Furthermore, treatment with P293 led to the removal of around 45% of mature E. coli biofilm biomass after 24 hours, a result consistent with previous phage-based biofilm disruption studies. For instance, UPWr_E phage cocktails reduced biofilms by 50–80% across surface types [ 35 ], and vB_EcoP_PW8 similarly inhibited biofilm formation and maintenance [ 33 ]. The observed biofilm clearance results support the potential of P293 for applications in farm sanitization and the protection of poultry flocks, especially considering the resistance of APEC biofilms to traditional disinfectants. The high-quality genomic sequencing revealed a ~ 89.5 kb dsDNA genome encoding 95 ORFs, which are organized into modules associated with the phage’s head, tail, lysis, and replication. About 40% of these ORFs correspond to hypothetical proteins, which are common in other myoviruses with limited functional characterization and warrant further investigation. Comparative genomic analysis with Escherichia phage wV8 and Salmonella phage Felix O1 showed strong synteny in structural and lysis genes, suggesting that P293 shares an evolutionary lineage within the unclassified Felixounavirus clade. Phylogenetic analysis further highlighted that tail fiber proteins of P293 diverge from closely related Felixounavirus phages, indicating possible adaptive evolution in receptor binding domains. As Tail fibers are critical for host specifictiy due to their interaction with bacterial surface receptors, such as LPS and O-antigen complexes [ 36 ], the variability observed in P293’s tail fiber sequences is consistent with its narrow host range. Engineering tail fiber swaps has been shown to significantly expand host range in related myovirus systems, such as T5-like phages [ 37 ], offering a potential strategy to broaden P293's applicability. Phage P293 remained stable across a physiologically relevant pH range (5–9) and temperatures (20–40°C), but was rapidly inactivated at temperatures ≥ 60°C or in the presence of 60% ethanol, which consistent with findings in phages vB_EcoP_PW8 and AG-MK-2022-Basu [ 33 , 34 ]. These finding suggest that while P293 is suitable for applications in feed water and sanitation, however, stabilizers may be needed when exposed to heat or ethanol. For the practical application of P293 in poultry biocontrol, several avenues warrant further exploration. In vivo challenge trials are critical to assess its therapeutic potential. Previous work with phage AG-MK-2022-Basu have demonstrated protection against colibacillosis in broilers, showing improved body weight gain and reduced bacterial load [ 38 ]. A similar approach with P293 could help confirm its therapeutic potential. Additionally, engineering the tail fiber gene using directed evolution or recombination strategies could widen host range without compromising infection efficiency [ 39 ]. Lastly, the development of phage cocktails that combine P293 with other phages may mitigate resistance development and broaden its therapeutic scope [ 40 ]. Conclusions This study presents the isolation and characterization of the novel lytic phage, Ecolivirus Myo‑P293, targeting avian pathogenic Escherichia coli . Phage P293 exhibits rapid adsorption, strong lytic activity, significant biofilm clearance, and desirable thermal and pH stability under physiological conditions. Genomic and phylogenetic analyses indicate that P293 belongs to the Myoviridae family and closely related to Felixounavirus phages. These promising attributes suggest that P293 is a potential candidate for the development of alternative antibacterial agents aimed at combating APEC infections in poultry. Declarations Conflicts of Interest: We declare no conflict of interest. Funding: This work was supported by the Jiangsu Agricultural Science and Technology Independent Innovation Fund (CX (24) 2005). Author Contributions: Hai Xu and Yaming Feng conceived the project, Qiurong Qi and Yalu Zhu conducted the majority of the experiments and analyses. Erzhong Wu and Guiqin Yan were responsible for the collection of clinical samples. Hai Xu and Yu Lu drafted the manuscript, and all authors reviewed and approved the final version of the manuscript. Data availability: The raw sequencing data for Ecolivirus Myo‑P293 have been deposited in the NCBI SRA under BioProject accession number PRJNA1302722. References Hu J, Afayibo DJA, Zhang B, Zhu H, Yao L, Guo W, Wang X, Wang Z, Wang D, Peng H, Tian M, Qi J, Wang S (2022) Characteristics, pathogenic mechanism, zoonotic potential, drug resistance, and prevention of avian pathogenic Escherichia coli (APEC). Front Microbiol 13:1049391. https://doi.org/10.3389/fmicb.2022.1049391 Kropinski AM, Mazzocco A, Waddell TE, Lingohr E, Johnson RP (2009) Enumeration of bacteriophages by double agar overlay plaque assay. 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Nat Commun 9:5114. https://doi.org/10.1038/s41467-018-07641-9 Bouras G, Nepal R, Houtak G, Psaltis AJ, Wormald PJ, Vreugde S (2023) Pharokka: a fast scalable bacteriophage annotation tool. Bioinformatics 39. https://doi.org/10.1093/bioinformatics/btac776 Katoh K, Kuma K, Toh H, Miyata T (2005) MAFFT version 5: improvement in accuracy of multiple sequence alignment. Nucleic Acids Res 33:511-518. https://doi.org/10.1093/nar/gki198 Edgar RC (2022) Muscle5: High-accuracy alignment ensembles enable unbiased assessments of sequence homology and phylogeny. Nat Commun 13:6968. https://doi.org/10.1038/s41467-022-34630-w Nguyen LT, Schmidt HA, von Haeseler A, Minh BQ (2015) IQ-TREE: a fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies. Mol Biol Evol 32:268-274. https://doi.org/10.1093/molbev/msu300 Darling AE, Mau B, Perna NT (2010) progressiveMauve: multiple genome alignment with gene gain, loss and rearrangement. PLoS One 5:e11147. https://doi.org/10.1371/journal.pone.0011147 Wintachai P, Thaion F, Clokie MRJ, Thomrongsuwannakij T (2024) Isolation and characterization of a novel Escherichia bacteriophage with potential to control multidrug-resistant avian pathogenic Escherichia coli and biofilms. Antibiotics (Basel) 13. https://doi.org/10.3390/antibiotics13111083 Karami M, Goudarztalejerdi A, Mohammadzadeh A, Berizi E (2024) In vitro evaluation of two novel Escherichia bacteriophages against multiple drug resistant avian pathogenic Escherichia coli . BMC Infect Dis 24:497. https://doi.org/10.1186/s12879-024-09402-0 Sliwka P, Moreno DS, Korzeniowski P, Milcarz A, Kuczkowski M, Kolenda R, Koziol S, Narajczyk M, Roesler U, Tomaszewska-Hetman L, Kuzminska-Bajor M (2025) Avian pathogenic Escherichia coli -targeting phages for biofilm biocontrol in the poultry industry. Vet Microbiol 301:110363. https://doi.org/10.1016/j.vetmic.2024.110363 Cho E, Kim J, Ha NC, Ryu S (2025) Amino acid residues in the tail fiber differentiate the host specificity of Cronobacter sakazakii bacteriophage. J Virol 99:e0028925. https://doi.org/10.1128/jvi.00289-25 Zheng X, Wang X, Li P, Zhou Y, Zhu X, Hu Z, Wang H, Chen M, Huo X, Liu Y, Zhang W (2025) The change of long tail fibers expanded the host range of a T5-like Salmonella phage and its application in milk. BMC Microbiol 25:169. https://doi.org/10.1186/s12866-025-03895-8 Eid S, Tolba HMN, Hamed RI, Al-Atfeehy NM (2022) Bacteriophage therapy as an alternative biocontrol against emerging multidrug resistant E. coli in broilers. Saudi J Biol Sci 29:3380-3389. https://doi.org/10.1016/j.sjbs.2022.02.015 Jia HJ, Jia PP, Yin S, Bu LK, Yang G, Pei DS (2023) Engineering bacteriophages for enhanced host range and efficacy: insights from bacteriophage-bacteria interactions. Front Microbiol 14:1172635. https://doi.org/10.3389/fmicb.2023.1172635 Taslem Mourosi J, Awe A, Guo W, Batra H, Ganesh H, Wu X, Zhu J (2022) Understanding bacteriophage tail fiber interaction with host surface receptor: the key "Blueprint" for reprogramming phage host range. Int J Mol Sci 23. https://doi.org/10.3390/ijms232012146 Supplementary Files SupplementaryFileS1P293assembledgenome.fa.fa Supplementary material: Draft assembled genome sequence of bacteriophage Ecolivirus Myo‑P293 (sample name: phage 01) in FASTA format. This file contains the de novo assembled genomic sequence of the phage isolated from duck farm sewage, originally labeled as phage 01 during sequencing. The sequence was obtained using Illumina MiSeq paired-end sequencing, and contigs were assembled using standard bioinformatics workflows. Cite Share Download PDF Status: Published Journal Publication published 29 Mar, 2026 Read the published version in Archives of Virology → Version 1 posted Reviewers agreed at journal 18 Aug, 2025 Reviewers invited by journal 11 Aug, 2025 Editor assigned by journal 09 Aug, 2025 First submitted to journal 07 Aug, 2025 Editorial decision: Major Revision 06 Aug, 2025 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-7281919","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":498571444,"identity":"fd708f73-59d5-46fd-9d43-77454953ab9b","order_by":0,"name":"Hai Xu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAApElEQVRIiWNgGAWjYBACAwbGBgaGCgk5fhK1nLEwlmwgXgsQMLZVJG4gWos5++EGZt55EowbGJgfPrpBjBbLnsQGxpnbJJjNGdiMjXOIctiBxAaGj9sk2CwbeNikidNy/mEDQ+IcCR6DA0RruQGypUFCghQtDxsYZxyTMJBsJtov59MfMPPU1NX3szc/fEyUFiBg/wGmmIlUPgpGwSgYBaOACAAAzxgtCMKo2TEAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0003-0419-0617","institution":"Insitute of Taizhou agricultural science, Jiangsu academy of agricultural science","correspondingAuthor":true,"prefix":"","firstName":"Hai","middleName":"","lastName":"Xu","suffix":""},{"id":498571445,"identity":"91f71a89-a14e-47a0-80f3-9c97317e18cf","order_by":1,"name":"Qiurong Qi","email":"","orcid":"","institution":"GuoTai (Taizhou) center of technology innovation for veterinary biologicals","correspondingAuthor":false,"prefix":"","firstName":"Qiurong","middleName":"","lastName":"Qi","suffix":""},{"id":498571446,"identity":"5207679b-d83d-4c9e-b4ef-62a899990a17","order_by":2,"name":"Yalu Zhu","email":"","orcid":"","institution":"GuoTai (Taizhou) center of technology innovation for veterinary biologicals","correspondingAuthor":false,"prefix":"","firstName":"Yalu","middleName":"","lastName":"Zhu","suffix":""},{"id":498571447,"identity":"b22dc9b0-204b-46ae-9bf5-ef682672a8a1","order_by":3,"name":"Erzhong Wu","email":"","orcid":"","institution":"Animal husbandry and veterinary department, Taizhou municipal bureau of agriculture and rural affairs","correspondingAuthor":false,"prefix":"","firstName":"Erzhong","middleName":"","lastName":"Wu","suffix":""},{"id":498571448,"identity":"f5253a3b-61c4-462a-acd8-b1918a37a1ed","order_by":4,"name":"Guiqin Yan","email":"","orcid":"","institution":"Yuxi Township animal husbandry and veterinary station","correspondingAuthor":false,"prefix":"","firstName":"Guiqin","middleName":"","lastName":"Yan","suffix":""},{"id":498571449,"identity":"3621956f-0bef-4cc9-a0e5-2bd7e60a6e08","order_by":5,"name":"Yu Lu","email":"","orcid":"","institution":"Insitute of veterinary immunology and engineering, Jiangsu academy of agricultural science","correspondingAuthor":false,"prefix":"","firstName":"Yu","middleName":"","lastName":"Lu","suffix":""},{"id":498571450,"identity":"5d2bed23-45a3-4635-b226-ed39adc6b301","order_by":6,"name":"Yaming Feng","email":"","orcid":"","institution":"Institue of Taizhou agricultural science, Jiangs academy of agricultural science","correspondingAuthor":false,"prefix":"","firstName":"Yaming","middleName":"","lastName":"Feng","suffix":""}],"badges":[],"createdAt":"2025-08-03 07:44:16","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7281919/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7281919/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00705-026-06602-8","type":"published","date":"2026-03-29T16:11:09+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":89280833,"identity":"3c10a085-b17d-4e91-abcf-f897e37ba882","added_by":"auto","created_at":"2025-08-18 10:29:11","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":236389,"visible":true,"origin":"","legend":"\u003cp\u003eMorphological characterization of phage \u003cem\u003eEcolivirus \u003c/em\u003eMyo‑P293 (P293)\u003c/p\u003e\n\u003cp\u003e(A) Plaque morphology of phage P293 formed on \u003cem\u003eEscherichia coli\u003c/em\u003e DPE 2 lawns after 6 h of incubation at 37 °C, showing clear and round plaques approximately 2 mm in diameter. Scale bar: 5mm. (B) Transmission electron micrograph (TEM) of negatively stained phage P293, displaying a typical \u003cem\u003eMyoviridae\u003c/em\u003emorphology with an icosahedral head (~70 nm in diameter) and a contractile tail (~100 nm in length). The image was captured at 80 kV with uranyl acetate staining. Scale bar: 100 nm.\u003c/p\u003e","description":"","filename":"Figure1.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7281919/v1/7eca6aef5fe2e307cc4f80ee.jpg"},{"id":89280835,"identity":"13ca7c01-cd7f-4e59-8ed8-a7d106e8ed30","added_by":"auto","created_at":"2025-08-18 10:29:12","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":278730,"visible":true,"origin":"","legend":"\u003cp\u003eBiological characterization of phage \u003cem\u003eEcolivirus \u003c/em\u003eMyo‑P293 (P293).\u003c/p\u003e\n\u003cp\u003e(A) Adsorption kinetics of phage P293 to \u003cem\u003eE. coli\u003c/em\u003e DPE 2 at 37 °C. Over 70% of phages adsorbed to host cells within 5 min; unadsorbed phage titers in the supernatant were measured at indicated time points. (B) \u003cem\u003eIn vitro\u003c/em\u003e bacteriolytic activity of phage P293 against \u003cem\u003eE. coli\u003c/em\u003e DPE 2 at an MOI of 1. Bacterial growth was monitored by OD₆₀₀ over 10 h. Phage infection led to a rapid decline in optical density, indicating efficient bacterial lysis. (C) One-step growth curve of phage P293 at an MOI of 1. The latent period was approximately 30 min, followed by a sharp increase in phage titer, with a calculated burst size of 284 ± 45 PFU per infected cell. Data are presented as means ± SD from three independent experiments.\u003c/p\u003e","description":"","filename":"Figure2.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7281919/v1/1e68fdb916c41c89641d9135.jpg"},{"id":89279584,"identity":"1b006c80-f645-4b82-afa2-4f8efe6478b9","added_by":"auto","created_at":"2025-08-18 10:13:11","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":261240,"visible":true,"origin":"","legend":"\u003cp\u003eEnvironmental stability of phage \u003cem\u003eEcolivirus \u003c/em\u003eMyo‑P293 (P293).\u003c/p\u003e\n\u003cp\u003e(A) pH stability assay. P293 (1×10⁹ PFU/mL) was incubated at various pH values (3-12) for 1, 2, and 3 h at room temperature. The phage titer remained stable between pH 5-9, with maximal activity at pH 7, and declined significantly outside this range. (B) Thermal stability assay. P293 was treated at different temperatures (20-80 °C) for 20, 40, 60, and 80 min. Phage infectivity was stable at 20-40 °C but decreased rapidly above 60 °C, with complete inactivation at 80 °C after 80 min. (C) Ethanol sensitivity assay. P293 was exposed to 30% and 60% ethanol for 20-80 min. Phage viability decreased significantly with increasing ethanol concentration and exposure time, with near-complete inactivation at 60% ethanol for 60 min. All data are presented as means ± SD from three independent experiments.\u003c/p\u003e","description":"","filename":"Figure3.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7281919/v1/b839d21fd5f5440537422345.jpg"},{"id":89279586,"identity":"2bd9760c-b847-4d75-9e03-978fc68fc5ad","added_by":"auto","created_at":"2025-08-18 10:13:11","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":390877,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eIn vitro\u003c/em\u003e bacteriolytic activity and biofilm clearance ability of phage \u003cem\u003eEcolivirus\u003c/em\u003e Myo‑P293 (P293).\u003c/p\u003e\n\u003cp\u003e(A) Bacteriolytic activity of phage P293 against \u003cem\u003eE. coli\u003c/em\u003e DPE 2 at different multiplicities of infection (MOIs: 0.001-100). Bacterial growth was monitored by measuring OD₆₀₀ every 30 min for 270 min. Phage treatment inhibited bacterial growth in an MOI-dependent manner, with higher MOIs showing stronger growth suppression. (B) Biofilm clearance assay. Preformed \u003cem\u003eE. coli\u003c/em\u003e DPE 2 biofilms were treated with P293 (1×10⁸ PFU/mL) for 6, 12, and 24 h. Residual biofilm biomass was quantified using crystal violet staining. P293 treatment led to a time-dependent reduction in biofilm mass, with a clearance rate of approximately 45% after 24 h. All data represent the mean ± SD of three independent experiments. Statistical differences compared to untreated controls were determined using Student’s t-test (p \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"Figure4.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7281919/v1/c4c86b2a40104806d7ef420a.jpg"},{"id":89279593,"identity":"28bb542d-5138-4b5e-b82f-f539f1fcb078","added_by":"auto","created_at":"2025-08-18 10:13:11","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":867892,"visible":true,"origin":"","legend":"\u003cp\u003eCircular genome map of phage \u003cem\u003eEcolivirus \u003c/em\u003eMyo‑P293.\u003c/p\u003e\n\u003cp\u003eThe complete genome of \u003cem\u003eEcolivirus \u003c/em\u003eMyo‑P293 is 89,518 bp of double-stranded DNA with a GC content of 38.5%. A total of 95 open reading frames (ORFs) were predicted and are displayed on the circular map. Arrows indicate the direction of transcription. ORFs are color-coded by predicted function: structural proteins (blue), DNA replication and regulation (green), host lysis (red), and hypothetical proteins (grey). Functional annotation was performed using Pharokka v1.7.1, and the map was generated based on genomic coordinates and annotation results. No antibiotic resistance or virulence genes were detected. The modular organization is consistent with that of \u003cem\u003eMyoviridae\u003c/em\u003e phages, indicating a strictly lytic lifestyle.\u003c/p\u003e","description":"","filename":"Figure5.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7281919/v1/a3f374d3ff47cc15b59f2532.jpg"},{"id":89279597,"identity":"d14a37fc-f0a8-4cfd-94b2-c3cbc6bc1782","added_by":"auto","created_at":"2025-08-18 10:13:12","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1477645,"visible":true,"origin":"","legend":"\u003cp\u003eComparative genomic analysis of phage \u003cem\u003eEcolivirus\u003c/em\u003e Myo‑P293 with related phages.\u003c/p\u003e\n\u003cp\u003eProgressive Mauve alignment of the genomes of \u003cem\u003eEcolivirus\u003c/em\u003e Myo‑P293, \u003cem\u003eEscherichia \u003c/em\u003ephage wV8, and \u003cem\u003eSalmonella\u003c/em\u003e phage Felix O1. Colored blocks represent locally collinear blocks (LCBs), indicating conserved genomic regions among the three phages. The height of each block reflects the degree of nucleotide sequence similarity. Despite overall synteny, notable variation is observed in regions encoding tail fiber and hypothetical proteins, suggesting possible divergence in host specificity and adaptation. The alignment reveals that \u003cem\u003eEcolivirus\u003c/em\u003eMyo‑P293, shares higher genome collinearity with E. coli phage wV8 than with Salmonella phage Felix O1.\u003c/p\u003e","description":"","filename":"Figure6.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7281919/v1/027c34393f9923ccaf391aea.jpg"},{"id":89279595,"identity":"42bdf28e-3e9b-4435-9b70-12a8fdf4a96d","added_by":"auto","created_at":"2025-08-18 10:13:11","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":3333578,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic analysis of phage \u003cem\u003eEcolivirus\u003c/em\u003e Myo‑P293.\u003c/p\u003e\n\u003cp\u003e(A) Whole-genome-based phylogenetic tree constructed using IQ-TREE v2.2.5, based on alignment with MAFFT v7.520. (B) Phylogenetic tree based on capsid protein amino acid sequences. (C) Phylogenetic tree based on tail fiber protein sequences.\u003c/p\u003e\n\u003cp\u003eAll trees were generated using the maximum likelihood method with 1,000 bootstrap replicates. \u003cem\u003eEcolivirus\u003c/em\u003eMyo‑P293 clusters closely with \u003cem\u003eEscherichia\u003c/em\u003e phage wV8 and \u003cem\u003eSalmonella\u003c/em\u003ephage Felix O1, forming a distinct branch within the unclassified \u003cem\u003eFelixounavirus\u003c/em\u003egroup. Variability in tail fiber protein sequence highlights divergence in host recognition potential. Bootstrap values above 70% are shown at the nodes. Scale bars indicate substitutions per site.\u003c/p\u003e","description":"","filename":"Figure7.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7281919/v1/2ddc686490dcf41e024c0451.jpg"},{"id":105755144,"identity":"392ceb90-cc30-465f-aff4-f68d49259ed2","added_by":"auto","created_at":"2026-03-30 16:25:51","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7846275,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7281919/v1/cb48cdbb-9e84-414a-81e4-63846604e623.pdf"},{"id":89279934,"identity":"4607187c-8e5f-4481-8513-7762eca71594","added_by":"auto","created_at":"2025-08-18 10:21:11","extension":"fa","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":89851,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary material: \u003c/strong\u003eDraft assembled genome sequence of bacteriophage \u003cem\u003eEcolivirus\u003c/em\u003e Myo‑P293 (sample name: phage 01) in FASTA format. This file contains the \u003cem\u003ede novo\u003c/em\u003e assembled genomic sequence of the phage isolated from duck farm sewage, originally labeled as phage 01 during sequencing. The sequence was obtained using Illumina MiSeq paired-end sequencing, and contigs were assembled using standard bioinformatics workflows.\u003c/p\u003e","description":"","filename":"SupplementaryFileS1P293assembledgenome.fa.fa","url":"https://assets-eu.researchsquare.com/files/rs-7281919/v1/ebe2b095fad36b56d60dfc90.fa"}],"financialInterests":"","formattedTitle":"Isolation and characterization of a novel lytic bacteriophage Ecolivirus Myo-P293 targeting avian pathogenic Escherichia coli","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAvian pathogenic \u003cem\u003eEscherichia coli\u003c/em\u003e (APEC), a member of the extraintestinal pathogenic \u003cem\u003eE. coli\u003c/em\u003e (ExPEC) group, specifically infects avian hosts and induces both respiratory and systemic disease [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Traditionally, APEC has been viewed as a secondary or opportunistic pathogen that emerges under conditions of intensive farming, inadequate sanitation or other stress factors such [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]as viral respiratory infections or poor husbandry practices [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. However, it is now clear that APEC can also act as a primary pathogen in immunocompetent avian hosts [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Moreover, its tight linkage to commercial poultry production systems renders APEC a significant reservoir of antimicrobial-resistance determinants. While antibiotics have long been the cornerstone for preventing and treating APEC infections, their widespread misuse and overuse have driven the emergence of multidrug-resistant (MDR) strains [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. These MDR APEC isolates frequently carry plasmids encoding multiple antibiotic resistance genes (ARGs), which facilitate horizontal dissemination across bacterial populations [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. To mitigate the acceleration of bacterial antimicrobial resistance, the World Health Organization (WHO), the Food and Agriculture Organization of the United Nations (FAO), and the World Organization for Animal Health (OIE) have launched global initiatives to regulate and reduce the misuse of antibiotics [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Consequently, effective control of APEC necessitates a multifaceted approach; the pathogen\u0026rsquo;s documented epidemiological expansion following restrictions on antimicrobial use, together with mounting regulatory pressures, highlights the urgent need for non-antibiotic intervention strategies.\u003c/p\u003e\u003cp\u003eBacteriophages are viruses capable of lysing bacteria and are ubiquitous in natural environments. Compared with conventional antibiotics, phage-based antibacterial agents exhibit several notable characteristics: they display high host specificity, targeting only the intended bacterial cells without affecting host tissues or the normal microbiota; they exist in vast numbers in the environment, making nature itself a rich reservoir for therapeutic phages [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]; and they possess unique pharmacokinetic properties \u003cem\u003ein vivo\u003c/em\u003e, whereby lytic phages self-amplify at the site of infection as they kill their bacterial hosts [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. In addressing the challenge of MDR bacteria, phages can exert their distinctive bactericidal effects through multiple mechanisms; for example, by encoding enolase-inhibitory proteins that specifically bind key enzymes in carbohydrate metabolism, thereby disrupting ATP synthesis and the supply of cell-wall precursors, ultimately resulting in bacterial lysis and death [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Consequently, the isolation and characterization of novel bacteriophages targeting APEC are critically important for expanding phage libraries and improving strategies to prevent and treat APEC infections.\u003c/p\u003e\u003cp\u003eIn 2022, the International Committee on Taxonomy of Viruses (ICTV) formally abolished the morphology-based family-level taxa \u003cem\u003ePodoviridae\u003c/em\u003e, \u003cem\u003eSiphoviridae\u003c/em\u003e and \u003cem\u003eMyoviridae\u003c/em\u003e, along with the order \u003cem\u003eCaudovirales\u003c/em\u003e, and introduced a unified binomial species nomenclature [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Within the newly established class \u003cem\u003eCaudoviricetes\u003c/em\u003e, twenty-two new families were defined, of which fourteen have been assigned to four novel orders. Although the term \u0026ldquo;\u003cem\u003emyovirus\u003c/em\u003e\u0026rdquo; persists as an informal morphological grouping, its taxonomy has shifted from morphology to genome-based phylogenetic classification. \u003cem\u003eMyoviruses\u003c/em\u003e have demonstrated both safety and efficacy in the treatment of multidrug-resistant bacterial infections. For example, Yao et al. isolated the strictly lytic phage PEC9, which protects mice against infection by APEC serotypes O1 and O2 [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], and Naghizadeh et al. showed that a phage cocktail could ameliorate organ lesions in quail infected with APEC [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Advances in genetic engineering and synthetic biology for phage modification are poised to accelerate the clinical application of \u003cem\u003emyoviruses\u003c/em\u003e in antibacterial therapy [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. In this study, we isolated and characterized a lytic bacteriophage, \u003cem\u003eEcolivirus\u003c/em\u003e Myo-P293 (abbreviated as P293), from a poultry farm that specifically targets APEC. Transmission electron microscopy (TEM) revealed morphological features consistent with the \u003cem\u003eMyoviridae\u003c/em\u003e family. Comprehensive characterization was conducted based on its biological properties and whole-genome sequencing. This study provides a valuable reference for the expansion of APEC-targeting phage libraries and the development of phage-based antimicrobial agents.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eBacterial strains and phage host range determination\u003c/h2\u003e\u003cp\u003eThe bacterial strains used in this study were provided by the GuoTai (Taizhou) Center of Technology Innovation for Veterinary Biologicals. A total of 15 \u003cem\u003eEscherichia coli\u003c/em\u003e (\u003cem\u003eE. coli\u003c/em\u003e) strains were included, comprising laboratory strains DH5α, BL21, and Top10, along with 12 serotyped field isolates recovered from poultry farms (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). All strains were cultured in Luria-Bertani (LB) broth at 37\u0026deg;C under aerobic conditions. For long-term storage, bacterial stocks were preserved in LB broth supplemented with 30% (v/v) glycerol at -80\u0026deg;C. The host range of phage P293 was assessed using a spot assay [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Briefly, 10 \u0026micro;L of phage suspension (~\u0026thinsp;1\u0026times;10⁹ PFU/mL) was spotted onto the surface of LB agar plates overlaid with lawns of each bacterial strain. Plates were incubated at 37\u0026deg;C for 4\u0026ndash;6 hours, and lytic activity was evaluated based on the formation of clear lysis zones. To further quantify phage infectivity, the efficiency of plating (EOP) was determined by performing plaque assays using all 15 field \u003cem\u003eE. coli\u003c/em\u003e strains. The EOP was calculated by comparing the plaque-forming units (PFU) on each susceptible strain with those on the original host strain (\u003cem\u003eE. coli\u003c/em\u003e DPE 2).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eHost range of bacteriophage Ecolivirus Myo-P293 and efficiency of plating (EOP)\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"8\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNo. of host\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eBacterial host\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eStrains\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eSerotype\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eSpot test \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003ePlaque production ability\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eTotal no. of plaque (pfu/ml) / Efficiency of plating (EOP)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003eSources \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eEscherichia coli\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eDH5α\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eLS\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eEscherichia coli\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eBL21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eLS\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eEscherichia coli\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eTop10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eLS\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eEscherichia coli\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eGPE12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eO1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eLS\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eEscherichia coli\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eCPE30\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eO1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eLS\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eEscherichia coli\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eCPE3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eO1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eLS\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eEscherichia coli\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eDPE2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eO2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e++\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e1.34\u0026times;10\u003csup\u003e10\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eLS\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eEscherichia coli\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eDPE14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eO2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e++\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e1.18\u0026times;10\u003csup\u003e10\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eLS\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eEscherichia coli\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eCPE5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eO2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e2.45\u0026times;10\u003csup\u003e9\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eLS\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eEscherichia coli\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eCPE11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eO78\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e1.73\u0026times;10\u003csup\u003e9\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eLS\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eEscherichia coli\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eGPE7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eO78\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eLS\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eEscherichia coli\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eGPE19\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eO78\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eLS\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eEscherichia coli\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eCPE22\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eO145\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e8.82\u0026times;10\u003csup\u003e8\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eLS\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eEscherichia coli\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eCPE8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eO145\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eLS\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eEscherichia coli\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eDPE4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eO145\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eLS\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"8\" nameend=\"c8\" namest=\"c1\"\u003e\u003cp\u003e\u003csup\u003ea\u003c/sup\u003e ++ = large clear lysis, + = small clear lysis, - = no lysis of plating.\u003c/p\u003e\u003cp\u003e\u003csup\u003eb\u003c/sup\u003e LS stands for lab stock.\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003ePhage isolation, purification, and amplification\u003c/h3\u003e\n\u003cp\u003ePhage P293 were isolated from sewage samples collected at a duck farm, using \u003cem\u003eE. coli\u003c/em\u003e O2 (DPE 2) as the indicator host strain. Phage isolation was performed according to previously described methods [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], based on the plaque assay technique. To obtain clonal phage populations, individual plaques were picked using sterile micropipette tips and subjected to at least five successive rounds of purification. For phage amplification, the purified phages were enriched as follows: a 50 mL culture of the indicator host (10⁷ CFU/mL) in LB broth was infected with purified phage and incubated at 37\u0026deg;C with shaking at 120 rpm. After complete lysis, the culture was centrifuged at 6,000 \u0026times; g for 15 min at 4\u0026deg;C to remove residual bacterial cells and debris. The resulting supernatant was further centrifuged at 15,000 \u0026times; g for 1 h at 4\u0026deg;C to pellet the phage particles. The phage pellets were resuspended in SM buffer (100 mM MgSO₄\u0026middot;7H₂O, 10 mM NaCl, 50 mM Tris-HCl, pH 7.5) and subsequently filtered through 0.22 \u0026micro;m syringe filters to ensure sterility. Phage titers were determined using the double-layer agar method, which also confirmed the presence of lytic phages in the filtrates [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eTransmission electron microscopy (TEM)\u003c/h3\u003e\n\u003cp\u003eApproximately 5 \u0026micro;L of purified P293 suspension was applied onto glow-discharged, carbon-coated copper grids (300 mesh) and allowed to adsorb for 1 minute. Excess liquid was then carefully removed using filter paper. The grids were subsequently stained with 2% (w/v) uranyl acetate for contrast enhancement and air-dried at room temperature. The negatively stained phage particles were examined using a Hitachi transmission electron microscope (Hitachi, Japan) operated at an accelerating voltage of 80 kV.\u003c/p\u003e\n\u003ch3\u003ePhage adsorption assay\u003c/h3\u003e\n\u003cp\u003eTo determine the adsorption kinetics of the P293 to its host, an adsorption assay was performed with minor modifications based on previously described protocols [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Briefly, the \u003cem\u003eE. coli\u003c/em\u003e DPE 2 host strain was cultured in LB broth and infected with phage at a multiplicity of infection (MOI) of 0.1. The mixture was incubated at 37\u0026deg;C, and aliquots were collected at 0, 5, 10, 15, 20, 25, and 30 minutes post-infection. The samples were immediately centrifuged at 10,000 \u0026times; g for 10 minutes to pellet the bacterial cells. The resulting supernatants were filtered through 0.22 \u0026micro;m syringe filters to remove residual cells, and the titers of non-adsorbed phages were determined using the double-layer agar method. All experiments were performed in triplicate to ensure reproducibility.\u003c/p\u003e\n\u003ch3\u003eOne-step growth curve\u003c/h3\u003e\n\u003cp\u003eThe one-step growth experiment was performed with slight modifications based on previously described methods [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Briefly, the P293 was mixed with \u003cem\u003eE. coli\u003c/em\u003e DPE 2 at a MOI of 1 and allowed to adsorb at 37\u0026deg;C for 10 minutes. The mixture was then centrifuged at 10,000 \u0026times; g for 30 seconds to remove unbound phages, and the resulting pellet was resuspended in 10 mL of LB broth. The culture was incubated at 37\u0026deg;C with shaking at 200 rpm. Aliquots of 100 \u0026micro;L were collected every 30 minutes over a 3-hour period. Each sample was centrifuged at 10,000 \u0026times; g for 5 minutes to remove bacterial debris, and the phage titers in the supernatants were determined using the double-layer agar method. All experiments were conducted in triplicate. The burst size was calculated as the ratio of the final number of released phage particles to the initial number of infected host cells.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eStability assay\u003c/h2\u003e\u003cp\u003eThe stability of phage P293 under various physicochemical conditions was evaluated in terms of pH, temperature, and disinfectant exposure [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. For pH stability, the phage suspension was adjusted to a final concentration of 1 \u0026times; 10⁹ PFU/mL using buffers with pH values ranging from 3 to 12. Samples were incubated at room temperature for 1, 2, and 3 hours. After incubation, residual phage titers were determined using the double-layer agar method. For thermal stability, the phage at a concentration of 1 \u0026times; 10⁹ PFU/mL was incubated at 20\u0026deg;C, 40\u0026deg;C, 60\u0026deg;C, and 80\u0026deg;C for 20, 40, 60, and 80 minutes. Samples were then immediately cooled on ice, and phage titers were measured using the double-layer agar method. For disinfectant stability, the phage was diluted to 1 \u0026times; 10⁹ PFU/mL in ethanol solutions at final concentrations of 30% and 60% (v/v). The mixtures were incubated at room temperature for 20, 40, 60, and 80 minutes, followed by titer determination as above. All treatments were conducted in triplicate. Phage viability under each condition was assessed by plaque formation efficiency on the host lawn, with results expressed as viable phage counts relative to the untreated control.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eIn vitro bacteriolytic activity\u003c/h3\u003e\n\u003cp\u003eThe \u003cem\u003ein vitro\u003c/em\u003e bacteriolytic activity of phage P293 was assessed by monitoring the optical density (OD₆₀₀) of \u003cem\u003eE. coli\u003c/em\u003e cultures using UV-visible spectrophotometry [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. The host strain, \u003cem\u003eE. coli\u003c/em\u003e DPE 2, was originally isolated from the pericardial effusion of ducks diagnosed with myocarditis. Briefly, \u003cem\u003eE. coli\u003c/em\u003e DPE 2 cultures in the logarithmic growth phase were infected with phage P293 at different multiplicities of infection (MOIs: 100, 10, 1, 0.1, 0.01, and 0.001). Infected cultures were incubated at 37\u0026deg;C for 270 minutes. Uninfected \u003cem\u003eE. coli\u003c/em\u003e DPE 2 cultures served as the positive control, and LB broth served as the negative control. The OD₆₀₀ of each culture was measured every 30 minutes using a UV-Vis spectrophotometer. Bacteriolytic activity was evaluated based on changes in turbidity (OD₆₀₀) over time compared to the control. All experiments were performed in triplicate.\u003c/p\u003e\n\u003ch3\u003eBiofilm clearance assay\u003c/h3\u003e\n\u003cp\u003eTo evaluate phage-mediated biofilm clearance [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], \u003cem\u003eE. coli\u003c/em\u003e DPE 2 was adjusted to OD₆₀₀ = 1.0 and inoculated into 96-well plates containing 200 \u0026micro;L LB per well (1:1000 dilution). Plates were incubated statically at 37\u0026deg;C for 24 hours to allow biofilm formation. After removing planktonic cells, 200 \u0026micro;L of phage suspension (1\u0026times;10⁸ PFU/mL) in SM buffer (100 mM NaCl, 50 mM Tris-HCl, pH 7.5, 10 mM MgSO₄) was added to each well and incubated for 6, 12, or 24 hours. Wells treated with SM buffer alone served as controls. After incubation, wells were washed with PBS, stained with crystal violet, and OD₅₉₅ values were measured as described above. Biofilm clearance was calculated as: Biofilm clearance (%) = [1 \u0026minus; (OD₅₉₅ of phage-treated well / OD₅₉₅ of control well)] \u0026times; 100.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003ePhage DNA sequencing and bioinformatic analysis\u003c/h2\u003e\u003cp\u003eGenomic DNA of phage P293 was extracted using the phenol/chloroform method, as previously described [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. The purified DNA was diluted to a final concentration of 0.2 ng/\u0026micro;L for whole-genome sequencing. Whole-genome shotgun (WGS) sequencing was conducted to characterize the phage genome. Genomic libraries with varying insert sizes were constructed and subjected to paired-end (PE) sequencing on the Illumina NovaSeq platform. Raw sequencing reads were de novo assembled using SPAdes, and contigs were filtered based on sequencing depth. High-coverage contigs were aligned against the National Center of Biotechnology Information (NCBI) nucleotide (NT) database using BLASTn to identify viral genome sequences [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Final genome assembly was refined by base-level error correction using Pilon. Genomic similarity between \u003cem\u003eEcolivirus\u003c/em\u003e Myo-P293 and previously reported phages was assessed using fastANI v1.34 [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Gene prediction, functional annotation, and circular genome visualization were performed using Pharokka v1.7.1 [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Whole-genome alignments were carried out with MAFFT v7.520 [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], and amino acid sequence alignments of the major capsid and tail fiber proteins were performed using MUSCLE v5.1 [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Phylogenetic relationships were inferred using IQ-TREE v2.2.5 based on the aligned sequences [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. For comparative genomic analysis, genome collinearity and structural conservation among phage P293, \u003cem\u003eEscherichia\u003c/em\u003e phage wV8, and \u003cem\u003eSalmonella\u003c/em\u003e phage Felix O1 were evaluated using Mauve v2.4.0 [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eData were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD) and analyzed using Graph Pad Prism version 9 software. The level of significance was set at (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.05).\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eIsolation and morphology of phage P293\u003c/h2\u003e\u003cp\u003eUsing \u003cem\u003eE. coli\u003c/em\u003e DPE 2 as the host, a novel lytic bacteriophage, designated \u003cem\u003eEcolivirus\u003c/em\u003e Myo-P293 (hereafter referred to as P293), was isolated from duck farm sewage in Jiangsu, China. Phage P293 produced clear, uniformly sized plaques with an average diameter of approximately 2 mm on a lawn of \u003cem\u003eE. coli\u003c/em\u003e DPE 2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). The phage was subsequently propagated and purified for morphological examination. TEM revealed that P293 exhibits typical \u003cem\u003eMyoviridae\u003c/em\u003e morphology, characterized by an icosahedral head (~\u0026thinsp;70 nm in diameter) and a contractile tail (~\u0026thinsp;100 nm in length) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). The presence of a distinct tail sheath and baseplate structure is consistent with features of the \u003cem\u003eMyoviridae\u003c/em\u003e family.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eLatent times and phage burst sizes\u003c/h2\u003e\u003cp\u003ePhage adsorption kinetics demonstrated that over 70% of phage particles were adsorbed onto host cells within the first 5 minutes of incubation. After 30 minutes, 17.5% of the phages remained unadsorbed in the supernatant (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). To evaluate lytic activity, P293 was added to logarithmic-phase \u003cem\u003eE. coli\u003c/em\u003e DPE 2 cultures. The OD₆₀₀ decreased from 0.65 to 0.234 within 3 hours and further dropped to 0.189 by 10 hours post-infection, accompanied by visible clearing of the culture and lysed bacterial debris, confirming the lytic nature of the phage (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). A one-step growth assay was conducted at 37\u0026deg;C with an MOI of 1 to assess the replication dynamics of P293. The results showed a latent period of approximately 30 minutes, followed by a large burst size of 284\u0026thinsp;\u0026plusmn;\u0026thinsp;45 PFU per infected cell (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eHost range and lytic activity of phage P293\u003c/h2\u003e\u003cp\u003eTo evaluate the biological control potential of phage P293, its lytic activity against \u003cem\u003eE. coli\u003c/em\u003e strains was assessed using the spot assay (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). A total of 15 \u003cem\u003eE. coli\u003c/em\u003e strains were tested, including 12 field isolates representing serotypes O1, O2, O78, and O145, and three laboratory strains (DH5α, BL21, and Top10). Phage P293 exhibited no lytic activity against the three laboratory strains or the O1 serotype isolates. In contrast, it demonstrated clear lytic activity against all three O2 serotype strains, as well as one isolate each from the O78 and O145 serotypes. The number of plaques formed on susceptible strains varied among isolates, indicating strain-specific differences in susceptibility.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eStability of phage P293 under different environmental conditions\u003c/h2\u003e\u003cp\u003eThe environmental stability and persistence of phages are critical factors for their potential application in biological control. To assess the stability of phage P293, its tolerance to varying pH levels, temperatures, and exposure to common disinfectants was evaluated. Phage P293 (1 \u0026times; 10⁹ PFU/mL) was incubated at pH values ranging from 3 to 12 for 1, 2, and 3 hours. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, the highest phage titer was observed at pH 7, indicating it to be the optimal condition for P293 stability. The phage remained stable under near-neutral and mildly acidic or alkaline conditions, but exhibited significant titer loss under extreme pH environments. Thermal stability assays revealed that P293 was stable at 20\u0026deg;C to 40\u0026deg;C, with minimal loss of infectivity. However, phage survival rate decreased significantly at temperatures above 60\u0026deg;C. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, treatment at 60\u0026ndash;80\u0026deg;C for 20 minutes reduced the survival rate from 65\u0026ndash;24%. Prolonged exposure further compromised phage viability, and complete inactivation was observed after 80 minutes at 80\u0026deg;C, indicating that P293 is heat-sensitive but remains stable at moderate temperatures. To assess its sensitivity to disinfectants, P293 was treated with 30% and 60% ethanol for 20, 40, 60, and 80 minutes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). At 30% ethanol, phage survival rate dropped to 18% after 60 minutes. In contrast, 60% ethanol led to near-complete inactivation within the same timeframe. These results suggest that P293 is highly sensitive to ethanol treatment, particularly at higher concentrations and extended exposure durations.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eBacteriolytic and anti-biofilm effects of phage P293 against\u003c/b\u003e \u003cb\u003eE. coli\u003c/b\u003e \u003cb\u003eDPE 2\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe bacteriolytic activity of phage P293 against \u003cem\u003eE. coli\u003c/em\u003e DPE 2 was evaluated at various multiplicities of infection (MOIs). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, the optical density (OD₆₀₀) of the uninfected \u003cem\u003eE. coli\u003c/em\u003e culture increased steadily over the 270-minute incubation period, indicating normal bacterial growth. In contrast, the negative control (medium only) showed no change in absorbance. For cultures treated with phage at low MOIs (0.001, 0.01, and 0.1), a gradual increase in OD₆₀₀ was observed during the first 120\u0026ndash;150 minutes, followed by a notable decline between 150 and 270 minutes, suggesting delayed but effective lytic activity. In comparison, at higher MOIs (1, 10, and 100), little to no increase in absorbance was observed throughout the incubation period, indicating rapid and sustained suppression of bacterial growth. These results demonstrate that phage P293 effectively lysed \u003cem\u003eE. coli\u003c/em\u003e DPE 2 in a dose-dependent manner, with stronger and earlier lytic effects observed at higher MOIs.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe ability of phage P293 to degrade mature biofilms formed by \u003cem\u003eE. coli\u003c/em\u003e DPE 2 was evaluated using a phage suspension at a concentration of 1 \u0026times; 10⁸ PFU/mL. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, phage treatment resulted in a significant, time-dependent reduction in biofilm biomass compared with the untreated control. After 24 hours of exposure, phage P293 exhibited a pronounced biofilm clearance effect, with a removal efficiency of approximately 45%, indicating its potential to disrupt established \u003cem\u003eE. coli\u003c/em\u003e biofilms.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003eGenomic features and functional annotation of phage P293\u003c/h2\u003e\u003cp\u003eThe complete genome of phage P293 was determined to be a linear double-stranded DNA molecule comprising 89,469 base pairs (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e) (Supplementary Material). The overall GC content was approximately 38.5%, with a distinct pattern of GC skew observed across the genome, indicating strand-specific replication and transcriptional asymmetry. A total of 95 open reading frames (ORFs) were predicted, of which 52 were functionally annotated based on sequence similarity to known phage proteins. These ORFs were classified into several functional modules, including structural proteins, lysis-related proteins, DNA metabolism and replication, and host interaction functions. Structural and morphogenesis-related genes were primarily clustered in one region and included genes encoding major head protein, tail sheath, tail fiber, baseplate wedge subunits, and portal protein, consistent with the morphological characteristics of Myoviridae phages. Notably, genes such as tail length tape measure protein and neck protein were also identified, suggesting conserved virion assembly architecture. The lysis cassette comprised genes encoding a putative endolysin, RIIA and RIIB lysis inhibitors, and an Rz-like spanin, which are typically involved in host cell lysis through coordinated degradation of the cell wall and outer membrane. Additionally, several ORFs were associated with DNA replication and nucleotide metabolism, including predicted DNA helicases, polymerase subunits, and nucleotide-modifying enzymes. Approximately 40% of the predicted ORFs were annotated as hypothetical proteins with unknown function, suggesting potential novel phage-specific genes requiring further investigation. Comparative analysis revealed high genomic synteny and homology between P293 and members of the Phage01D_k149 group, particularly in regions encoding structural components and lysis-related functions.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003eComparative genomic analysis of phage P293\u003c/h2\u003e\u003cp\u003eTo investigate the genomic relatedness of Ecolivirus Myo-P293 with other well-characterized myoviruses, comparative genomic analysis was performed using Mauve alignment software. The genomes of \u003cem\u003eEscherichia\u003c/em\u003e phage wV8 and \u003cem\u003eSalmonella\u003c/em\u003e phage Felix O1 were selected for comparison due to their phylogenetic proximity and similar genome sizes. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, P293 shares extensive collinear regions with Escherichia phage wV8, particularly across the structural and lysis gene clusters. Large locally collinear blocks (LCBs) indicate a high degree of sequence conservation and genome organization, suggesting that P293 and wV8 may belong to the same genus or closely related lineages. Most conserved regions span from ~\u0026thinsp;10 kb to ~\u0026thinsp;85 kb, and gene order remains largely syntenic. In contrast, the alignment between P293 and \u003cem\u003eSalmonella\u003c/em\u003e phage Felix O1 revealed fewer and less extensive homologous regions. While some conserved sequences were observed in the structural modules, several genomic rearrangements and inversions were evident, especially at the terminal regions. This suggests that although P293 and Felix O1 share some common ancestry, they have diverged considerably in genome architecture. Together, these results indicate that phage P293 exhibits a high degree of genomic similarity and collinearity with \u003cem\u003eE. coli\u003c/em\u003e phage wV8, whereas it shares limited homology with \u003cem\u003eSalmonella\u003c/em\u003e phage Felix O1, supporting its classification as a novel \u003cem\u003eEscherichia\u003c/em\u003e-infecting myovirus with distinct evolutionary lineage.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003ePhylogenetic analysis of phage P293\u003c/h2\u003e\u003cp\u003eTo determine the evolutionary position of phage Ecolivirus Myo-P293, phylogenetic trees were constructed based on its complete genome sequence (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA), major capsid protein (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB), and tail fiber protein (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC). As shown in the genome-based phylogenetic tree (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA), P293 clustered within the unclassified \u003cem\u003eFelixounavirus\u003c/em\u003e clade, and was most closely related to \u003cem\u003eEscherichia\u003c/em\u003e phage EF202P1 and \u003cem\u003eSalmonella\u003c/em\u003e phage Felix O1, suggesting a shared evolutionary lineage with this group of lytic \u003cem\u003eMyoviridae\u003c/em\u003e phages. Phylogenetic analysis based on the major capsid protein (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB) supported this classification, with P293 again grouping closely with \u003cem\u003eFelixounaviruses\u003c/em\u003e such as \u003cem\u003eSalmonella\u003c/em\u003e phage Felix O1 and \u003cem\u003eEscherichia\u003c/em\u003e phage P7, while remaining distinct from members of the T4-like and P2-like virus clades. In contrast, the tree derived from tail fiber protein sequences (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC) revealed a slightly divergent pattern: P293 formed a branch near the \u003cem\u003eFelixounavirus\u003c/em\u003e cluster but displayed increased phylogenetic distance from closely related \u003cem\u003eEscherichia\u003c/em\u003e phages, reflecting the high variability and modular evolution typically observed in tail fiber genes. Collectively, these results suggest that P293 is a member of the unclassified \u003cem\u003eFelixounavirus\u003c/em\u003e group, exhibiting close evolutionary relationships with known \u003cem\u003eEscherichia\u003c/em\u003e and \u003cem\u003eSalmonella\u003c/em\u003e phages at both the genomic and structural protein levels, while also possessing unique genetic features, particularly in its tail fiber region, that may underlie its host specificity and functional diversity.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we report the isolation, characterization, and genomic analysis of P293, a novel \u003cem\u003eMyoviridae\u003c/em\u003e phage with potential as a biocontrol agent against APEC. Our results demonstrate that P293 exhibits high lytic efficiency, efficacy against biofilms, and favorable stability-features that align with the functional profile desired for phage-based interventions in poultry.\u003c/p\u003e\u003cp\u003ePhage P293 exhibited strong lytic activity targeting O2 serotypes and select O78/O145 APEC strains, reflecting a relatively narrow yet clinically relevant host range. This specificity is consistent with other APEC-targeting phages, such as vB_EcoP_PW8 [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e] and phage AG-MK-2022-Basu [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e], which both effectively lyse multidrug-resistant (MDR) strains despite having a restricted host breadth. Notably, P293 achieved a rapid adsorption rate of over 70% within 5 minutes and released a burst size of approximately 284 PFU per infected cell, which is comparable to or even surpasses other APEC-targeting phages in terms of replicative fitness.\u003c/p\u003e\u003cp\u003eFurthermore, treatment with P293 led to the removal of around 45% of mature E. coli biofilm biomass after 24 hours, a result consistent with previous phage-based biofilm disruption studies. For instance, UPWr_E phage cocktails reduced biofilms by 50\u0026ndash;80% across surface types [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e], and vB_EcoP_PW8 similarly inhibited biofilm formation and maintenance [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. The observed biofilm clearance results support the potential of P293 for applications in farm sanitization and the protection of poultry flocks, especially considering the resistance of APEC biofilms to traditional disinfectants.\u003c/p\u003e\u003cp\u003eThe high-quality genomic sequencing revealed a\u0026thinsp;~\u0026thinsp;89.5 kb dsDNA genome encoding 95 ORFs, which are organized into modules associated with the phage\u0026rsquo;s head, tail, lysis, and replication. About 40% of these ORFs correspond to hypothetical proteins, which are common in other \u003cem\u003emyoviruses\u003c/em\u003e with limited functional characterization and warrant further investigation. Comparative genomic analysis with \u003cem\u003eEscherichia\u003c/em\u003e phage wV8 and \u003cem\u003eSalmonella\u003c/em\u003e phage Felix O1 showed strong synteny in structural and lysis genes, suggesting that P293 shares an evolutionary lineage within the unclassified \u003cem\u003eFelixounavirus\u003c/em\u003e clade.\u003c/p\u003e\u003cp\u003ePhylogenetic analysis further highlighted that tail fiber proteins of P293 diverge from closely related \u003cem\u003eFelixounavirus\u003c/em\u003e phages, indicating possible adaptive evolution in receptor binding domains. As Tail fibers are critical for host specifictiy due to their interaction with bacterial surface receptors, such as LPS and O-antigen complexes [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e], the variability observed in P293\u0026rsquo;s tail fiber sequences is consistent with its narrow host range. Engineering tail fiber swaps has been shown to significantly expand host range in related \u003cem\u003emyovirus\u003c/em\u003e systems, such as T5-like phages [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e], offering a potential strategy to broaden P293's applicability.\u003c/p\u003e\u003cp\u003ePhage P293 remained stable across a physiologically relevant pH range (5\u0026ndash;9) and temperatures (20\u0026ndash;40\u0026deg;C), but was rapidly inactivated at temperatures\u0026thinsp;\u0026ge;\u0026thinsp;60\u0026deg;C or in the presence of 60% ethanol, which consistent with findings in phages vB_EcoP_PW8 and AG-MK-2022-Basu [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. These finding suggest that while P293 is suitable for applications in feed water and sanitation, however, stabilizers may be needed when exposed to heat or ethanol.\u003c/p\u003e\u003cp\u003eFor the practical application of P293 in poultry biocontrol, several avenues warrant further exploration. \u003cem\u003eIn vivo\u003c/em\u003e challenge trials are critical to assess its therapeutic potential. Previous work with phage AG-MK-2022-Basu have demonstrated protection against colibacillosis in broilers, showing improved body weight gain and reduced bacterial load [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. A similar approach with P293 could help confirm its therapeutic potential. Additionally, engineering the tail fiber gene using directed evolution or recombination strategies could widen host range without compromising infection efficiency [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Lastly, the development of phage cocktails that combine P293 with other phages may mitigate resistance development and broaden its therapeutic scope [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e].\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThis study presents the isolation and characterization of the novel lytic phage, \u003cem\u003eEcolivirus\u003c/em\u003e Myo‑P293, targeting avian pathogenic \u003cem\u003eEscherichia coli\u003c/em\u003e. Phage P293 exhibits rapid adsorption, strong lytic activity, significant biofilm clearance, and desirable thermal and pH stability under physiological conditions. Genomic and phylogenetic analyses indicate that P293 belongs to the \u003cem\u003eMyoviridae\u003c/em\u003e family and closely related to \u003cem\u003eFelixounavirus\u003c/em\u003e phages. These promising attributes suggest that P293 is a potential candidate for the development of alternative antibacterial agents aimed at combating APEC infections in poultry.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eConflicts of Interest:\u003c/h2\u003e\u003cp\u003eWe declare no conflict of interest.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding:\u003c/h2\u003e\u003cp\u003eThis work was supported by the Jiangsu Agricultural Science and Technology Independent Innovation Fund (CX (24) 2005).\u003c/p\u003e\u003ch2\u003eAuthor Contributions:\u003c/h2\u003e\u003cp\u003eHai Xu and Yaming Feng conceived the project, Qiurong Qi and Yalu Zhu conducted the majority of the experiments and analyses. Erzhong Wu and Guiqin Yan were responsible for the collection of clinical samples. Hai Xu and Yu Lu drafted the manuscript, and all authors reviewed and approved the final version of the manuscript.\u003c/p\u003e\u003ch2\u003eData availability:\u003c/h2\u003e\u003cp\u003eThe raw sequencing data for Ecolivirus Myo‑P293 have been deposited in the NCBI SRA under BioProject accession number PRJNA1302722.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eHu J, Afayibo DJA, Zhang B, Zhu H, Yao L, Guo W, Wang X, Wang Z, Wang D, Peng H, Tian M, Qi J, Wang S (2022) Characteristics, pathogenic mechanism, zoonotic potential, drug resistance, and prevention of avian pathogenic \u003cem\u003eEscherichia coli\u003c/em\u003e (APEC). Front Microbiol 13:1049391. https://doi.org/10.3389/fmicb.2022.1049391\u003c/li\u003e\n\u003cli\u003eKropinski AM, Mazzocco A, Waddell TE, Lingohr E, Johnson RP (2009) Enumeration of bacteriophages by double agar overlay plaque assay. 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Saudi J Biol Sci 29:3380-3389. https://doi.org/10.1016/j.sjbs.2022.02.015\u003c/li\u003e\n\u003cli\u003eJia HJ, Jia PP, Yin S, Bu LK, Yang G, Pei DS (2023) Engineering bacteriophages for enhanced host range and efficacy: insights from bacteriophage-bacteria interactions. Front Microbiol 14:1172635. https://doi.org/10.3389/fmicb.2023.1172635\u003c/li\u003e\n\u003cli\u003eTaslem Mourosi J, Awe A, Guo W, Batra H, Ganesh H, Wu X, Zhu J (2022) Understanding bacteriophage tail fiber interaction with host surface receptor: the key \u0026quot;Blueprint\u0026quot; for reprogramming phage host range. Int J Mol Sci 23. https://doi.org/10.3390/ijms232012146\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"archives-of-virology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"arvi","sideBox":"Learn more about [Archives of Virology](https://www.springer.com/journal/705)","snPcode":"705","submissionUrl":"https://submission.nature.com/new-submission/705/3","title":"Archives of Virology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Avian pathogenic Escherichia coli, Lytic bacteriophage, Whole-genome sequencing, Biological characteristics","lastPublishedDoi":"10.21203/rs.3.rs-7281919/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7281919/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eA novel lytic bacteriophage, \u003cem\u003eEcolivirus\u003c/em\u003e Myo‑P293 (P293), targeting avian pathogenic \u003cem\u003eEscherichia coli\u003c/em\u003e (APEC), was isolated from duck farm sewage in Jiangsu, China. P293 formed clear plaques approximately 2 mm in diameter and displayed the characteristic morphology of \u003cem\u003eMyoviridae\u003c/em\u003e family, with an icosahedral head (~\u0026thinsp;70 nm) and a contractile tail (~\u0026thinsp;100 nm), as observed under transmission electron microscopy. Adsorption assays showed that over 70% of phages adsorbed to host cells within 5 min. One-step growth analysis revealed a latent period of approximately 30 min and a burst size of 284\u0026thinsp;\u0026plusmn;\u0026thinsp;45 PFU per infected cell. P293 exhibited stability across a pH range of 5\u0026ndash;9 and at temperature between 20\u0026ndash;40\u0026deg;C, but its infectivity was significantly reduced when exposed to temperature\u0026thinsp;\u0026ge;\u0026thinsp;60\u0026deg;C or 60% ethanol. \u003cem\u003eIn vitro\u003c/em\u003e bacteriolytic assays demonstrated strong antibacterial activity, which was dependent on the multiplicity of infection (MOI). Notably, P293 achieved approximately 45% clearance of mature \u003cem\u003eE. coli\u003c/em\u003e biofilms after 24 h treatment. Whole-genome sequencing of P293 revealed an 89.5 kb double-stranded DNA genome encoding 95 open reading frames (ORFs), including modules related to structure, replication, lysis, and host interaction. Approximately 40% of the encoded genes are annotated as hypothetical proteins. Phylogenetic and comparative genomic analyses placed P293 within the unclassified \u003cem\u003eFelixounavirus\u003c/em\u003e clade, closely related to \u003cem\u003eEscherichia\u003c/em\u003e phage wV8 and \u003cem\u003eSalmonella\u003c/em\u003e phage Felix O1, while displaying distinct tail fiber gene signatures associated with host specificity. These findings support the potential of P293 as a candidate for phage-based biocontrol strategies against APEC in poultry production.\u003c/p\u003e","manuscriptTitle":"Isolation and characterization of a novel lytic bacteriophage Ecolivirus Myo-P293 targeting avian pathogenic Escherichia coli","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-18 10:13:07","doi":"10.21203/rs.3.rs-7281919/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2025-08-18T10:16:24+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-08-11T07:19:34+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-08-09T12:10:45+00:00","index":"","fulltext":""},{"type":"submitted","content":"Archives of Virology","date":"2025-08-07T21:07:41+00:00","index":"","fulltext":""},{"type":"decision","content":"Major Revision","date":"2025-08-06T05:29:46+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"archives-of-virology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"arvi","sideBox":"Learn more about [Archives of Virology](https://www.springer.com/journal/705)","snPcode":"705","submissionUrl":"https://submission.nature.com/new-submission/705/3","title":"Archives of Virology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"232e553d-62c0-498e-8328-fe276c3a54a9","owner":[],"postedDate":"August 18th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-03-30T16:21:22+00:00","versionOfRecord":{"articleIdentity":"rs-7281919","link":"https://doi.org/10.1007/s00705-026-06602-8","journal":{"identity":"archives-of-virology","isVorOnly":false,"title":"Archives of Virology"},"publishedOn":"2026-03-29 16:11:09","publishedOnDateReadable":"March 29th, 2026"},"versionCreatedAt":"2025-08-18 10:13:07","video":"","vorDoi":"10.1007/s00705-026-06602-8","vorDoiUrl":"https://doi.org/10.1007/s00705-026-06602-8","workflowStages":[]},"version":"v1","identity":"rs-7281919","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7281919","identity":"rs-7281919","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}