Phage Particles Research Articles

Free DNA partially clarifies discrepancies between qPCR and the conventional phage quantification method.

To use phages in a personalized therapy and industrial applications, an accurate quantification is needed. The gold standard method, namely the culture-based double agar overlay (DAO) method, provides an accurate estimate of the number of infectious phages but is laborious and time-intensive. Quantitative polymerase chain reaction (qPCR) can be used as a fast alternative but tends to overestimate the number of infectious phage particles. Here we describe the use of a DNase treatment before quantification of the Staphylococcus aureus phage ISP with qPCR to obtain a more accurate estimate of the number of infectious phage particles. We showed that DNase treatment results in a significant decrease of the concentration when measured with qPCR although for two out of three tested ISP phage stocks, there was still a significant difference with the DAO method. We also showed that the discrepancy between quantification with qPCR and the DAO method is dependent on the storage period of the phage stock, with a larger discrepancy for older stocks. Additionally, we used negative contrast immune electron microscopy to confirm the presence of DNA in the medium of the phage stock and the impact of the DNase treatment on the free DNA.

Electrochemical immunosensor based on phages/CdSe nanoparticles as tracers to quantify clomazone in water

Clomazone, an herbicide used worldwide, contaminates river and lakes, as well as surface and ground water. Therefore, there is great interest in developing new analytical tools for detecting environmental pollutants that are simple, highly sensitive and provide an extended analytical range. This work describes a disposable, highly sensitive and reproducible electrochemical immunosensor based on a non-competitive and enzyme-free immunoassay to quantify clomazone in water samples. A specific clone of recombinant phage particles labelled with CdSe nanoparticles, which form a trivalent complex with the clomazone/monoclonal antibody anti clomazone immuno-complex, was used as tracer. Clomazone was determined indirectly by square wave anodic stripping voltammetry through the anodic oxidation of Cd previously deposited in an accumulation step from the reduction of Cd2+ ions released from CdSe nanoparticles. An excellent analytical performance, with a limit of detection and a midpoint sensitivity of 0.038 ng mL−1 and 1.34 ng mL−1, respectively, were found. The limit of detection was 60-fold lower than the limit of detection obtained using the same antibody and un-label phage in an optimized non-competitive ELISA. The developed electrochemical immunosensor was successively used to assay undiluted river water samples, where very good recoveries percentages were reached. The very low limit of detection achieved with the electrochemical immunosensor allows it to be used as an important environmental tool for the determination of clomazone.

Selection of LRP1 ligand phage-displayed single domain antibody that transmigrates BBB

Effective drug delivery to the central nervous system (CNS) remains a challenge due to the blood–brain barrier (BBB). Macromolecules such as proteins and peptides are unable to cross BBB and have poor therapeutic efficacy due to little or no drug distribution. A promising alternative is the conjugation of a drug to a shuttle molecule that can reach the CNS via receptor-mediated transcytosis (RMT). Several receptors have been described for RMT, such as low-density lipoprotein receptor-related protein 1 (LRP1). We used phage display technology combined with an in vitro BBB model to identify LRP1 ligands. A single domain antibody (dAb) library was used to enrich for species that selectively bind to immobilised LRP1 ligand. We obtained a novel nanobody, dAb D11, that selectively binds to LRP1 receptor and mediates in vitro internalisation of phage particles in brain endothelial cells, with a dissociation constant Kd of 183.1 ± 85.8 nM. The high permeability of D11 was demonstrated by an in vivo biodistribution assay in mice. We discovered D11, the first LRP1 binding dAb with BBB permeability. Our findings will contribute to the development of RMT-based drugs for the treatment of CNS diseases.

The role of bacteriophages in facilitating the horizontal transfer of antibiotic resistance genes in municipal wastewater treatment plants

Bacteriophages play integral roles in the ecosystem; however, their precise involvement in horizontal gene transfer and the spread of antibiotic resistance genes (ARGs) are not fully understood. In this study, a coculture system involving consortia of bacteriophages and multidrug-resistant bacteria from an aerobic tank in a municipal wastewater treatment plant (WWTP) was established to investigate the functions of bacteriophages in ARG transfer and spread. The results of the cocultivation of the MRB and bacteriophage consortia indicated that the bacterial community remained stable throughout the whole process, but the addition of bacteriophages significantly increased ARG abundance, especially in bacteriophage DNA. Nine out of the 11 identified ARGs significantly increased, indicating that more bacteriophage particles carried ARGs in the system after cocultivation. In addition, 686 plasmids were detected during cocultivation, of which only 3.36 % were identified as conjugative plasmids, which is significantly lower than the proportion found among previously published plasmids (25.2 %, totaling 14,029 plasmids). Our findings revealed that bacteriophages may play important roles in the horizontal transfer of ARGs through both bacteriophage-mediated conduction and an increase in extracellular ARGs; however, conjugative transfer may not be the main mechanism by which multidrug-resistant bacteria acquire and spread ARGs. Unlike in most previous reports, a coculture system of diverse bacteria and bacteriophages was established in this study to assess bacteriophage functions in ARG transfer and dissemination in the environment, overcoming the limitations associated with the isolation of bacteria and bacteriophages, as well as the specificity of bacteriophage hosts.

Generation of Antibody Libraries for Phage Display: Preparation of Helper Phage.

The generation and selection of antibody libraries by phagemid-based phage display requires three components; namely, phagemid library, host bacterial cells, and helper phage. The use of helper phage is necessary for the selection of phagemid libraries by phage display because it provides all genes needed for production of infectious phage particles. Here, we describe the generation of high-titer helper phage preparations suitable for phagemid-based phage display. The approach is based on helper phage VCSM13, which includes a gene for kanamycin resistance and a mutated packaging signal that, in the presence of a phagemid with an unmutated packaging signal, favors the production of infectious phage particles with phagemid phenotype and genotype.

Ultrasensitive Electrochemical Detection of Salmonella typhimurium in Food Matrices Using Surface-Modified Bacterial Cellulose with Immobilized Phage Particles.

The rapid and sensitive detection of Salmonella typhimurium in food matrices is crucial for ensuring food safety. This study presents the development of an ultrasensitive electrochemical biosensor using surface-modified bacterial cellulose (BC) integrated with polypyrrole (Ppy) and reduced graphene oxide (RGO), further functionalized with immobilized S. typhimurium-specific phage particles. The BC substrate, with its ultra-fibrous and porous structure, was modified through in situ oxidative polymerization of Ppy and RGO, resulting in a highly conductive and flexible biointerface. The immobilization of phages onto this composite was facilitated by electrostatic interactions between the polycationic Ppy and the negatively charged phage capsid heads, optimizing phage orientation and enhancing bacterial capture efficiency. Morphological and chemical characterization confirmed the successful fabrication and phage immobilization. The biosensor demonstrated a detection limit of 1 CFU/mL for S. typhimurium in phosphate-buffered saline (PBS), with a linear detection range spanning 100 to 107 CFU/mL. In real samples, the sensor achieved detection limits of 5 CFU/mL in milk and 3 CFU/mL in chicken, with a linear detection range spanning 100 to 106 CFU/mL, maintaining high accuracy and reproducibility. The biosensor also effectively discriminated between live and dead bacterial cells, demonstrating its potential in real-world food safety applications. The biosensor performed excellently over a wide pH range (4-10) and remained stable for up to six weeks. Overall, the developed BC/Ppy/RGO-phage biosensor offers a promising tool for the rapid, sensitive, and selective detection of S. typhimurium, with robust performance across different food matrices.

Characterisation of a new virulent phage isolated from Hainan Island with potential against multidrug-resistant Pseudomonas aeruginosa infections

Multidrug-resistant (MDR) Pseudomonas aeruginosa is a serious life-threatening pathogen. The rise in P. aeruginosa resistance rates has renewed interest in phages as an alternative therapeutic approach for treating bacterial infections. In this study, we investigated the characteristics of the first Pseudomonas phage, vB_PaP_HN01, isolated from Hainan, the only tropical island in China. The lytic rate of this phage against P. aeruginosa reached 64.3 % (27/42). Under the optimal multiplicity of infection (MOI) of 0.1, more than 90 % of phage particles absorb onto the host cell within 10 min, with an eclipse period of around 15 min, and a high titer phage production (1011 PFU/ml) within 90 min was demonstrated. vB_PaP_HN01 maintains a robust titer after 1 h exposure to pH values and temperatures (up to 50 °C). Genome annotation revealed that vB_PaP_HN01 did not contain drug-resistance or lysogeny-associated genes. It can effectively inhibit the formation of biofilms of MDR P. aeruginosa and eliminated aggressive biofilms (removal rate about 70 %). In the in vivo infection models, it was demonstrated that the survival rate and lifespan of Galleria mellonella larvae were increased alongside the injection of vB_PaP_HN01. These data revealed the potential of vB_PaP_HN01 against P. aeruginosa in clinic.

Single particle tracking reveals through-membrane diffusion of bacteriophage during process disruption of virus filtration

In the biopharmaceutical industry, virus filters are crucial for ensuring the removal of endogenous and adventitious viruses as part of the viral clearance strategy. Although traditionally described as a size-exclusion mechanism, virus retention has a process-dependent nature where challenging conditions, such as process disruptions, may compromise membrane retention and significantly increase virus filtrate concentrations. The detailed mechanisms underlying this loss of retention are challenging to determine using traditional breakthrough experiments. In this work, single particle tracking and kinetic simulations were employed to connect individual particle behavior to the observed macroscopic losses in virus retention. Our experiments, using fluorescently labeled ΦX174 bacteriophage as a model parvovirus, replicated conditions representative of process disruptions within the Pegasus SV4, a homogeneous polymeric virus filtration membrane. During flow, phage particles retained were trapped within relatively large cavity spaces that had downstream constrictions aligned with the flow direction; the trapped particles were dynamic and exhibited significant intra-cavity motion. Upon flow stoppage, particles escaped from these retention locations rapidly, with approximately 90 % of previously trapped particles being remobilized for process disruption times ranging from 2 to 10 min, suggesting that local cavity escape had reached saturation at these timescales. Diffusion experiments within the membrane revealed isotropic and Fickian motion, hindered by more than an order of magnitude compared to diffusion in unconfined liquid. Despite the reduced mobility within the membrane, the substantial diffusion coefficient of 4.19 ± 0.06 μm2/s indicated that virus particles could travel tortuous but non-retentive pathways through the membrane on length scales equal to or greater than the membrane thickness during a disruption event. A 1D kinetic Monte-Carlo simulation successfully connected single-particle behavior to macroscopically observed virus release, indicating that significant diffusive release into the filtrate can occur even without the resumption of flow. This work provides crucial insights into the retention behavior of homogeneous membranes during periods of disruption, enabling the design of more robust mitigation strategies and filter designs.

Abundance measurements reveal the balance between lysis and lysogeny in the human gut microbiome.

The human gut contains diverse communities of bacteriophage, whose interactions with the broader microbiome and potential roles in human health are only beginning to be uncovered. Here, we combine multiple types of data to quantitatively estimate gut phage population dynamics and lifestyle characteristics in human subjects. Unifying results from previous studies, we show that an average human gut contains a low ratio of phage particles to bacterial cells (~1:100), but a much larger ratio of phage genomes to bacterial genomes (~4:1), implying that most gut phage are effectively temperate (e.g., integrated prophage, phage-plasmids, etc.). By integrating imaging and sequencing data with a generalized model of temperate phage dynamics, we estimate that phage induction and lysis occurs at a low average rate (~0.001-0.01 per bacterium per day), imposing only a modest fitness burden on their bacterial hosts. Consistent with these estimates, we find that the phage composition of a diverse synthetic community in gnotobiotic mice can be quantitatively predicted from bacterial abundances alone, while still exhibiting phage diversity comparable to native human microbiomes. These results provide a foundation for interpreting existing and future studies on links between the gut virome and human health.

Isolation of phage-antibodies against Eimeria species that infect chickens

Eimeria is one of the most economically important pathogens in poultry production. Diagnosis of infection has the potential to inform treatment and prevention strategies. Here, phage display technology was used to isolate single chain antibodies (scFvs) that had a broad specificity against oocysts from the seven pathogenic species of Eimeria found in poultry. Three such scFvs, representing 2 scFv HCDR3 motifs, were isolated by random picks of clones isolated after five rounds of iterative enrichment (panning) of phage against the seven Eimeria species. Phage-antibody binding to Eimeria oocysts was also interrogated using next generation sequencing of the HCDR3 region of scFv genes contained with phage particles. This analysis demonstrated that the most abundant scFv found after 5 rounds of panning accounted for over >90 % of scFvs. Furthermore, the three scFvs isolated from random picks of clones were the only antibodies that were enriched through each round of panning. They were also seen to be enriched through the stages of phage panning that included binding to the Eimeria oocysts (selection phase) and to be selected against during the stages that consisted solely of phage propagation (growth only phase). The NGS data was further analysed to identify an additional scFv that demonstrated specific enrichment against 3 Eimeria species at the third round of panning and had the same pattern of enrichment during the selection and growth phases of panning. Rescue and analysis of this phage-scFv demonstrated a binder with broad specificity for Eimeria species. The four antibodies with broad specificity detected all seven Eimeria species in immunoassays. The binding of one such scFv that recognised all species was further validated by fluorescent microscopy.

Aerosolic Application of Phages Against S. infantis on Plates and Chicken Skin.

Phages are known as a promising method to combat antimicrobial resistance (AMR) in the human and veterinary sector. Use of phage aerosols enormously increases the application field, although the impact on the infectivity of phages during nebulization needs to be evaluated. In this study S. infantis was treated on plates and chicken skin with nebulized phage particles of the Myoviridae type, identified by transmission electron microscopy, using a commercial nebulizer primarily used for H2O2 disinfection. The reduction of bacterial number by aerosol applied phage particles was evaluated. It could clearly be shown that the phage particles were able to infect Salmonella after being nebulized using ultrasound technology. Further studies on other types of phages as well as other conditions must be performed to standardize the aerosolic application of phages.

In vitro antimicrobial and antibiofilm activity of phage cocktail against Mammaliicoccus sciuri, a causative agent of bovine mastitis.

In this research paper the in vitro antimicrobial and antibiofilm activity of phage cocktail against the coagulase negative Mammaliicoccus sciuri was investigated. Three M. sciuri isolates obtained from clinical bovine mastitis samples were characterized and identified by 16S rRNA gene sequencing. Bacteriophages with lytic activity against M. sciuri isolates were isolated from dairy farm effluents. Two typical phages were isolated using standard enrichment and plaque assay techniques, purified by polyethylene glycol precipitation, and morphologically characterized based on shape and size using transmission electron microscopy. This was followed by determination of host range using spot tests and stability to varying temperature, pH and UV treatment. The phage cocktail suppressed bacterial activity within 30 min of exposure. Crystal violet assay showed that the tested phages and their cocktail significantly reduced the biofilm biomass of all three M. sciuri strains compared to the untreated control in vitro within 24 h with a single dosing. Transmission electron micrography of the purified phage particle revealed an icosahedral head and a rigid contractile tail, characteristic of the class Caudoviricetes. The findings open new avenues in phage-based antimicrobial approaches for controlling contagious and teat skin opportunistic bacteria causing bovine mastitis.

Isolation and Characterization of a Novel Phage against Vibrio alginolyticus Belonging to a New Genus.

Vibrio alginolyticus causes substantial economic losses in the aquaculture industry. With the rise of multidrug-resistant Vibrio strains, phages present a promising solution. Here, a novel lytic Vibrio phage, vB_ValC_RH2G (RH2G), that efficiently infects the pathogenic strain V. alginolyticus ATCC 17749T, was isolated from mixed wastewater from an aquatic market in Xiamen, China. Transmission electron microscopy revealed that RH2G has the morphology of Siphoviruses, featuring an icosahedral head (73 ± 2 nm diameter) and long noncontractile tail (142 ± 4 nm). A one-step growth experiment showed that RH2G had a short latent period (10 min) and a burst size of 48 phage particles per infected cell. Additionally, RH2G was highly species-specific and was relatively stable at 4-55 °C and pH 4-10. A genomic analysis showed that RH2G has a 116,749 bp double-stranded DNA genome with 43.76% GC content. The intergenomic similarity between the genome sequence of RH2G and other phages recorded in the GenBank database was below 38.8%, suggesting that RH2G represents a new genus. RH2G did not exhibit any virulence or resistance genes. Its rapid lysis capacity, lytic activity, environmental resilience, and genetic safety suggested that RH2G may be a safe candidate for phage therapy in combatting vibriosis in aquaculture settings.

An electrochemical biosensor for sensitive detection of live Salmonella in food via MXene amplified methylene blue signals and electrostatic immobilization of bacteriophages.

Anovel bacteriophage-targeted electrochemical biosensor designed for accurate and quantitative detection of live Salmonella in food samplesis presented. The biosensor is simply constructed by electrostatic immobilizing bacteriophages on MXene-nanostructured electrodes. MXene, renowned for its high surface area, biocompatibility, and conductivity, serves as an ideal platform for bacteriophage immobilization. This allows for a high-density immobilization of bacteriophage particles, achieving approximately 71 pcs μm-2. Remarkably, the bacteriophages immobilized MXene nanostructured electrodes still maintain their viability and functionality, ensuring their effectiveness in pathogen detection. Therefore, the proposed biosensor exhibited enhanced sensitivity with a low limit of detection (LOD) of 5CFUmL-1. Notably, the biosensor shows excellent specificity in the presence of other bacteria that commonly contaminate food and can distinguish live Salmonella from a mixed population. Furthermore, it is applicable in detecting live Salmonella in food samples, which highlights its potential in food safety monitoring. This biosensor offers simplicity, convenience, and suitability for resource-limited environments, making it a promising tool for on-site monitoring of foodborne pathogenic bacteria.

Nanomechanical resilience and thermal stability of RSJ2 phage

As the world moves toward a green economy and sustainable agriculture, bacterial viruses or bacteriophages (phages) become attractive biocontrol agents for controlling crop diseases. Effective utilization of phages in farms requires integrated knowledge of crops, pathogens, phages, and surroundings. Phages must encounter environmental fluctuations, including temperature, and must remain infectious for successful bacteria lysis. This work studied a soilborne RSJ2 phage discovered in Thailand, which can eliminate Ralstonia solanacearum, causing bacterial wilt disease in chili. We investigated how phage infectivity and nanomechanics responded to thermal changes. The plaque-based assay showed that the infectivity of the RSJ2 phage was stable within 24–40 °C, an average temperature fluctuation in tropical regions. The structural examination also showed that the phage remained intact. The nanomechanical property of the phage was inspected by the atomic force microscopy-based nanoindentation. The result revealed that the phage stiffness within 24–40 °C was statistically similar (0.05–0.06 N/m). Upon heating at 40 °C for 1, 5, and 10 h and resting at 25 °C, the stiffness of the phage particles increased to 0.09–0.11 N/m (54–83% increase). The stiffness results suggest structural adaptation of the protein subunits as a response to thermal alteration. The study exhibits that the phage structure is highly dynamic and can nanomechanically respond to varying temperatures. The phage stiffness may reveal insight into phage adaptation to environmental factors. Equipped with the knowledge of phage infectivity, structure, and nanomechanics, we can design practical guidelines for effective phage usage in farming and propelling green and safe agriculture.

Preparation of VCSM13 Helper Phage for Display Library Reamplification and Bio-Panning.

Phage-displayed antibody libraries can be constructed using any species that is easily immunized. The pComb3XSS phagemid vector is commonly used for library cloning and phage display. This phagemid encodes the origin of replication of the filamentous bacteriophage f1 but lacks all the genes required for replication and assembly of phage particles. The replication and the assembly of phage from these phagemids thus requires a "helper" phage that provides the genes essential for those steps during library production and bio-panning. One of those helper phages is VCSM13. In this protocol, we describe the preparation of VCSM13 helper phage. Users should prepare VCSM13 helper phage for library reamplification and for bio-panning.

CrAss-like phages are suitable indicators of antibiotic resistance genes found in abundance in fecally polluted samples

Antibiotic resistance genes (ARGs) have been extensively observed in bacterial DNA, and more recently, in phage particles from various water sources and food items. The pivotal role played by ARG transmission in the proliferation of antibiotic resistance and emergence of new resistant strains calls for a thorough understanding of the underlying mechanisms. The aim of this study was to assess the suitability of the prototypical p-crAssphage, a proposed indicator of human fecal contamination, and the recently isolated crAssBcn phages, both belonging to the Crassvirales group, as potential indicators of ARGs. These crAss-like phages were evaluated alongside specific ARGs (blaTEM, blaCTX-M-1, blaCTX-M-9, blaVIM, blaOXA-48, qnrA, qnrS, tetW and sul1) within the total DNA and phage DNA fractions in water and food samples containing different levels of fecal pollution. In samples with high fecal load (>103 CFU/g or ml of E. coli or somatic coliphages), such as wastewater and sludge, positive correlations were found between both types of crAss-like phages and ARGs in both DNA fractions. The strongest correlation was observed between sul1 and crAssBcn phages (rho = 0.90) in sludge samples, followed by blaCTX-M-9 and p-crAssphage (rho = 0.86) in sewage samples, both in the phage DNA fraction. The use of crAssphage and crAssBcn as indicators of ARGs, considered to be emerging environmental contaminants of anthropogenic origin, is supported by their close association with the human gut. Monitoring ARGs can help to mitigate their dissemination and prevent the emergence of new resistant bacterial strains, thus safeguarding public health.

Synergistic removal of Staphylococcus aureus biofilms by using a combination of phage Kayvirus rodi with the exopolysaccharide depolymerase Dpo7.

Bacteriophages have been shown to penetrate biofilms and replicate if they find suitable host cells. Therefore, these viruses appear to be a good option to tackle the biofilm problem and complement or even substitute more conventional antimicrobials. However, in order to successfully remove biofilms, in particular mature biofilms, phages may need to be administered along with other compounds. Phage-derived proteins, such as endolysins or depolymerases, offer a safer alternative to other compounds in the era of antibiotic resistance. This study examined the interactions between phage Kayvirus rodi with a polysaccharide depolymerase (Dpo7) from another phage (Rockefellervirus IPLA7) against biofilms formed by different Staphylococcus aureus strains, as determined by crystal violet staining, viable cell counts and microscopy analysis. Our results demonstrated that there was synergy between the two antimicrobials, with a more significant decreased in biomass and viable cell number with the combination treatment compared to the phage and enzyme alone. This observation was confirmed by microscopy analysis, which also showed that polysaccharide depolymerase treatment reduced, but did not eliminate extracellular matrix polysaccharides. Activity assays on mutant strains did not identify teichoic acids or PNAG/PIA as the exclusive target of Dpo7, suggesting that may be both are degraded by this enzyme. Phage adsorption to S. aureus cells was not significantly altered by incubation with Dpo7, indicating that the mechanism of the observed synergistic interaction is likely through loosening of the biofilm structure. This would allow easier access of the phage particles to their host cells and facilitate infection progression within the bacterial population.

Prophage induction by non-antibiotic compounds promotes transformation of released antibiotic resistance genes from cell lysis

Prophages are prevalent among bacterial species, including strains carrying antibiotic resistance genes (ARGs). Prophage induction can be triggered by the SOS response to stressors, leading to cell lysis. In environments polluted by chemical stressors, ARGs and prophage co-harboring strains might pose an unknown risk of spreading ARGs through chemical pollutant-mediated prophage induction and subsequent cell lysis. In this study, we investigated the effects of common non-antibiotic water pollutants, triclosan and silver nanoparticles, on triggering prophage induction in clinical isolates carrying ARGs and the subsequent uptake of released ARGs by the naturally competent bacterium Acinetobacter baylyi. Our results demonstrate that both triclosan and silver nanoparticles, at environmentally relevant concentrations and those found in commercial products, significantly enhance prophage induction among various clinical isolates. Transmission electron microscopy imaging and plaque assays confirmed the production of infectious phage particles under non-antibiotic pollutants-mediated prophage induction. In addition, the rate of ARG transformation to A. baylyi significantly increased after the release of extracellular ARGs from prophage induction-mediated cell lysis. The mechanism of non-antibiotic pollutants-mediated prophage induction is primarily associated with excessive oxidative stress, which provokes the SOS response. Our findings offer insights into the role of non-antibiotic pollutants in promoting the dissemination of ARGs by triggering prophage induction.

Globoside Is an Essential Intracellular Factor Required for Parvovirus B19 Endosomal Escape.

Human parvovirus B19 (B19V), like most parvoviruses, possesses phospholipase A2 (PLA2) activity, which is thought to mediate endosomal escape by membrane disruption. Here, we challenge this model and find evidence for a mechanism of B19V entry mediated by the glycosphingolipid globoside without endosome disruption and retrograde transport to the Golgi. We show that B19V PLA2 activity requires specific calcium levels and pH conditions that are not optimal in endosomes. Accordingly, endosomal membrane integrity was maintained during B19V entry. Furthermore, endosomes remained intact when loaded with MS2 bacteriophage particles pseudotyped with multiple B19V PLA2 subunits, providing superior enzymatic potential compared to native B19V. In globoside knockout cells, incoming viruses are arrested in the endosomal compartment and the infection is blocked. Infection can be rescued by promoting endosomal leakage with polyethyleneimine (PEI), demonstrating the essential role of globoside in facilitating endosomal escape. Incoming virus colocalizes with Golgi markers and interfering with Golgi function blocks infection, suggesting that globoside-mediated entry involves the Golgi compartment, which provides conditions favorable for the lipolytic PLA2. Our study challenges the current model of B19V entry and identifies globoside as an essential intracellular receptor required for endosomal escape.