Versatile Platinum Nanoparticles-Decorated Phage Nanozyme Integrating Recognition, Bacteriolysis, and Catalysis Capabilities for On-Site Detection of Foodborne Pathogenic Strains Vitality Based on Bioluminescence/Pressure Dual-Mode Bioassay.

Sensitive and on-site discrimination of live and dead foodborne pathogenic strains remains a significant challenge due to the lack of appropriate assay and signal probes. In this work, a versatile platinum nanoparticle-decorated phage nanozyme (P2@PtNPs) that integrated recognition, bacteriolysis, and catalysis was designed to establish the bioluminescence/pressure dual-mode bioassay for on-site determination of the vitality of foodborne pathogenic strains. Benefiting from the bacterial strain-level specificity of phage, the target Salmonella typhimurium (S.T) was specially captured to form sandwich complexes with P2@PtNPs on another phage-modified glass microbead (GM@P1). As the other part of the P2@PtNPs nanozyme, the introduced PtNPs could not only catalyze the decomposition of hydrogen peroxide to generate a significant oxygen pressure signal but also produce hydroxyl radicals around the target bacteria to enhance the bacteriolysis of phage and adenosine triphosphate release. It significantly improved the bioluminescence signal. The two signals corresponded to the total and live target bacteria counts, so the dead target could be easily calculated from the difference between the total and live target bacteria counts. Meanwhile, the vitality of S.T was realized according to the ratio of live and total S.T. Under optimal conditions, the application range of this proposed bioassay for bacterial vitality was 102-107 CFU/mL, with a limit of detections for total and live S.T of 30 CFU/mL and 40 CFU/mL, respectively. This work provides an innovative and versatile nanozyme signal probe for the on-site determination of bacterial vitality for food safety.

Magnetically modified bacteriophage-triggered ATP release activated EXPAR-CRISPR/Cas14a system for visual detection of Burkholderia pseudomallei.

Burkholderia pseudomallei, widely distributed in tropical and subtropical ecosystems, is capable of causing the fatal zoonotic disease melioidosis and exhibiting a global trend of dissemination. Rapid and sensitive detection of B. pseudomallei is essential for environmental monitoring as well as infection control. Here, we developed an innovative biosensor for quantitatively detecting B. pseudomallei relies on ATP released triggered by bacteriophage-induced bacteria lysis. The lytic bacteriophage vB_BpP_HN01, with high specificity, is employed alongside magnetic nanoparticles assembly to create a biological receptor, facilitating the capture and enrichment of viable target bacteria. Following a brief extraction and incubation process, the captured target undergoes rapid lysis to release contents including ATP. The EXPAR-CRISPR cascade reaction provides an efficient signal transduction and dual amplification module that allowing the generated ATP to guide the signal output as an activator, ultimately converting the target bacterial amount into a detectable fluorescence signal. The proposed bacteriophage affinity strategy exhibited superior performance for B. pseudomallei detection with a dynamic range from 10^2 to 10^7 CFU mL-1, and a LOD of 45 CFU mL-1 within 80 min. Moreover, with the output signal compatible across various monitoring methods, this work offers a robust assurance for rapid diagnosis and on-site environmental monitoring of B. pseudomallei.

Activation of Thoeris antiviral system via SIR2 effector filament assembly.

To survive bacteriophage (phage) infections, bacteria developed numerous anti-phage defence systems1-7. Some of them (for example, type III CRISPR-Cas, CBASS, Pycsar and Thoeris) consist of two modules: a sensor responsible for infection recognition and an effector that stops viral replication by destroying key cellular components8-12. In the Thoeris system, a Toll/interleukin-1 receptor (TIR)-domain protein, ThsB, acts as a sensor that synthesizes an isomer of cyclic ADP ribose, 1''-3' glycocyclic ADP ribose (gcADPR), which is bound in the Smf/DprA-LOG (SLOG) domain of the ThsA effector and activates the silent information regulator 2 (SIR2)-domain-mediated hydrolysis of a key cell metabolite, NAD+ (refs. 12-14). Although the structure of ThsA has been solved15, the ThsA activation mechanism remained incompletely understood. Here we show that 1''-3' gcADPR, synthesized in vitro by the dimeric ThsB' protein, binds to the ThsA SLOG domain, thereby activating ThsA by triggering helical filament assembly of ThsA tetramers. The cryogenic electron microscopy (cryo-EM) structure of activated ThsA revealed that filament assembly stabilizes the active conformation of the ThsA SIR2 domain, enabling rapid NAD+ depletion. Furthermore, we demonstrate that filament formation enables a switch-like response of ThsA to the 1''-3' gcADPR signal.

New role of bacteriophages in medical oncology.

Targeted treatment of cancer is one of the most paramount approaches in cancer treatment. Despite significant advances in cancer diagnosis and treatment methods, there are still significant limitations and disadvantages in the field, including high costs, toxicity, and unwanted damage to healthy cells. The phage display technique is an innovative method for designing carriers containing exogenic peptides with cancer diagnostic and therapeutic properties. Bacteriophages possess unique properties making them effective in cancer treatment. These characteristics include the small size enabling them to penetrate vessels; having no pathogenicity to mammals; easy manipulation of their genetic information and surface proteins to introduce vaccines and drugs to cancer tissues; lower cost of large-scale production; and greater stimulation of the immune system. Bacteriophages will certainly play a more effective role in the future of medical oncology; however, studies are in the early stages of conception and require more extensive research. We aimed in this review to provide some related examples and bring insights into the potential of phages as targeted vectors for use in cancer diagnosis and treatment, especially regarding their capability in gene and drug delivery to cancer target cells, determination of tumor markers, and vaccine design to stimulate anticancer immunity.

Viral Small Terminase: A Divergent Structural Framework for a Conserved Biological Function.

The genome packaging motor of bacteriophages and herpesviruses is built by two terminase subunits, known as large (TerL) and small (TerS), both essential for viral genome packaging. TerL structure, composition, and assembly to an empty capsid, as well as the mechanisms of ATP-dependent DNA packaging, have been studied in depth, shedding light on the chemo-mechanical coupling between ATP hydrolysis and DNA translocation. Instead, significantly less is known about the small terminase subunit, TerS, which is dispensable or even inhibitory in vitro, but essential in vivo. By taking advantage of the recent revolution in cryo-electron microscopy (cryo-EM) and building upon a wealth of crystallographic structures of phage TerSs, in this review, we take an inventory of known TerSs studied to date. Our analysis suggests that TerS evolved and diversified into a flexible molecular framework that can conserve biological function with minimal sequence and quaternary structure conservation to fit different packaging strategies and environmental conditions.

Modification of a Tumor-Targeting Bacteriophage for Potential Diagnostic Applications.

BACKGROUND: Tumor-targeting bacteriophages can be used as a versatile new platform for the delivery of diagnostic imaging agents and therapeutic cargo. This became possible due to the development of viral capsid modification method. Earlier in our laboratory and using phage display technology, phages to malignant breast cancer cells MDA-MB 231 were obtained. The goal of this study was the optimization of phage modification and the assessment of the effect of the latter on the efficiency of phage particle penetration into MDA-MB 231 cells. METHODS: In this work, we used several methods, such as chemical phage modification using FAM-NHS ester, spectrophotometry, phage amplification, sequencing, phage titration, flow cytometry, and confocal microscopy. RESULTS: We performed chemical phage modification using different concentrations of FAM-NHS dye (0.5 mM, 1 mM, 2 mM, 4 mM, 8 mM). It was shown that with an increase of the modification degree, the phage titer decreases. The maximum modification coefficient of the phage envelope with the FAM-NHS dye was observed with 4 mM modifying agent and had approximately 804,2 FAM molecules per phage. Through the immunofluorescence staining and flow cytometry methods, it was shown that the modified bacteriophage retains the ability to internalize into MDA-MB-231 cells. The estimation of the number of phages that could have penetrated into one tumor cell was conducted. CONCLUSIONS: Optimizing the conditions for phage modification can be an effective strategy for producing tumor-targeting diagnostic and therapeutic agents, i.e., theranostic drugs.

Identification of a peptide binding to cancer antigen Kita-kyushu lung cancer antigen 1 from a phage-display library.

Kita-kyushu lung cancer antigen 1 (KK-LC-1) is a kind of cancer-testis antigen with anti-tumor potential for clinical application. As a class of small-molecule antigen conjugate, tumor-targeting peptides have broad application prospects in gastric cancer diagnosis, imaging, and biological treatment. Here, we screened specific cyclic nonapeptides from a phage-display library. The targeting peptide with the best affinity was selected and further verified in ex vivo tissue sections. Finally, enrichment of targeting peptides in tumor tissues was observed in vivo, and the dynamic biodistribution process was also observed with micro-positron emission tomography (micro-PET)/computed tomography (CT) imaging. Studies showed that the specific cyclic nonapeptide had a high binding capacity for KK-LC-1 protein. It has a strong affinity and specificity for KK-LC-1-expressing positive tumor cells. Targeting peptides were significantly enriched at tumor sites in vivo, with very low normal tissue background. These findings demonstrated that the KK-LC-1 targeting peptide has high clinical potential.

RNA and Sugars, Unique Properties of Bacteriophages Infecting Multidrug Resistant Acinetobacter radioresistens Strain LH6.

Bacteriophages (phages) are predicted to be the most ubiquitous biological entity on earth, and yet, there are still vast knowledge gaps in our understanding of phage diversity and phage-host interactions. Approximately one hundred Acinetobacter-infecting DNA viruses have been identified, and in this report, we describe eight more. We isolated two typical dsDNA lytic podoviruses (CAP1-2), five unique dsRNA lytic cystoviruses (CAP3-7), and one dsDNA lysogenic siphovirus (SLAP1), all capable of infecting the multidrug resistant isolate Acinetobacter radioresistens LH6. Using transmission electron microscopy, bacterial mutagenesis, phage infectivity assays, carbohydrate staining, mass-spectrometry, genomic sequencing, and comparative studies, we further characterized these phages. Mutation of the LH6 initiating glycosyltransferase homolog, PglC, necessary for both O-linked glycoprotein and capsular polysaccharide (CPS) biosynthesis, prevented infection by the lytic podovirus CAP1, while mutation of the pilin protein, PilA, prevented infection by CAP3, representing the lytic cystoviruses. Genome sequencing of the three dsRNA segments of the isolated cystoviruses revealed low levels of homology, but conserved synteny with the only other reported cystoviruses that infect Pseudomonas species. In Pseudomonas, the cystoviruses are known to be enveloped phages surrounding their capsids with the inner membrane from the infected host. To characterize any membrane-associated glycoconjugates in the CAP3 cystovirus, carbohydrate staining was used to identify a low molecular weight lipid-linked glycoconjugate subsequently identified by mutagenesis and mass-spectrometry as bacterial lipooligosaccharide. Together, this study demonstrates the isolation of new Acinetobacter-infecting phages and the determination of their cell receptors. Further, we describe the genomes of a new genus of Cystoviruses and perform an initial characterization of membrane-associated glycoconjugates.

Protective efficacy of phage PVN02 against haemorrhagic septicaemia in striped catfish Pangasianodon hypophthalmus via oral administration.

Haemorrhagic septicaemia caused by Aeromonas hydrophila in striped catfish (Pangasianodon hypophthalmus) is one of the most important aquatic diseases in the Mekong Delta, Vietnam. However, antibiotic-resistant A. hydrophila strains have become popular and resulted in inadequate control of the disease in striped catfish farms. This study investigates the protective efficacy of bacteriophage PVN02 against haemorrhagic septicaemia in striped catfish via oral administration. The phage-containing pellets were prepared by spraying the phage solution on food pellets at 20 ml/kg. The rate of phage desorption from the food pellets into the water was very low; the phage titres in the water were approximately log 1.0 PFU/ml or undetectable. The in vivo experiment evaluating the protective efficacy of PVN02 against haemorrhagic septicaemia in striped catfish was conducted using 21 groups of 1,260 fish in 50-L plastic tanks in triplicate. The catfish were fed twice daily with phage-sprayed pellets. Different densities of bacterial suspensions were added into the tanks for 24 hr. Without the existence of the phage, the highest mortality rate was 68.3 ± 2.9% at the highest density of bacterial suspension. In contrast, the mortality rate at the highest density of bacterial suspension was significantly reduced to 8.33 ± 2.9% or 16.67 ± 2.9% at the phage dose of log 6.2 ± 0.09 or log 4.2 ± 0.09 PFU/g. This study provides a very practical manner of applying phage therapy to prevent disease in large-scale striped catfish farms.

Bacteriophage Capsid Modification by Genetic and Chemical Methods.

Bacteriophages are viruses whose ubiquity in nature and remarkable specificity to their host bacteria enable an impressive and growing field of tunable biotechnologies in agriculture and public health. Bacteriophage capsids, which house and protect their nucleic acids, have been modified with a range of functionalities (e.g., fluorophores, nanoparticles, antigens, drugs) to suit their final application. Functional groups naturally present on bacteriophage capsids can be used for electrostatic adsorption or bioconjugation, but their impermanence and poor specificity can lead to inconsistencies in coverage and function. To overcome these limitations, researchers have explored both genetic and chemical modifications to enable strong, specific bonds between phage capsids and their target conjugates. Genetic modification methods involve introducing genes for alternative amino acids, peptides, or protein sequences into either the bacteriophage genomes or capsid genes on host plasmids to facilitate recombinant phage generation. Chemical modification methods rely on reacting functional groups present on the capsid with activated conjugates under the appropriate solution pH and salt conditions. This review surveys the current state-of-the-art in both genetic and chemical bacteriophage capsid modification methodologies, identifies major strengths and weaknesses of methods, and discusses areas of research needed to propel bacteriophage technology in development of biosensors, vaccines, therapeutics, and nanocarriers.

Insertion of channel of phi29 DNA packaging motor into polymer membrane for high-throughput sensing.

The connector channel of bacteriophage phi29 DNA packaging motor has been inserted into the lipid bilayer membrane and has shown potential for the sensing of DNA, RNA, chemicals, peptides, and antibodies. Properties such as high solubility and large channel size have made phi29 channel an advantageous system for those applications; however, previously studied lipid membranes have short lifetimes, and they are frangible and unstable under voltages higher than 200 mV. Thus, the application of this lipid membrane platform for clinical applications is challenging. Here we report the insertion of the connector into the stable polymer membrane in MinION flow cell that contains 2048 wells for high-throughput sensing by the liposome-polymer fusion process. The successful insertion of phi29 connector was confirmed by a unique gating phenomenon. Peptide translocation through the inserted phi29 connector was also observed, revealing the potential of applying phi29 connector for high-throughput peptide sensing.

Bio-hybrid gold nanoparticles as SERS probe for rapid bacteria cell identification.

This study reports the utilization of engineered molecular networks between bacteriophage (or phage) and gold nanoparticles (AuNPs) prepared ablating a high purity gold target in water by nanosecond laser source. Gold colloids are assembled with P9b phage clone, displaying the specific peptide (QRKLAAKLT), able to bind P. aeruginosa. The single components and assembled systems were characterized by spectroscopic and electronic techniques, such as the conventional optical absorption and micro-Raman spectroscopies as well as the Dynamic Light Scattering (DLS) and Scanning Transmission Electron Microscopy (STEM) techniques. The performance of the AuNPs-phage assembly as substrate for Surface-Enhanced Raman Spectroscopy (SERS) was tested against the detection of the characteristics Raman vibrational features of the Pseudomonas aeruginosa bacteria.

Use of anti-CRISPR protein AcrIIA4 as a capture ligand for CRISPR/Cas9 detection.

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) ribonucleoprotein (RNP) complex is an RNA-guided DNA-nuclease that is part of the bacterial adaptive immune system. CRISPR/Cas9 RNP has been adapted for targeted genome editing within cells and whole organisms with new applications vastly outpacing detection and quantification of gene-editing reagents. Detection of the CRISPR/Cas9 RNP within biological samples is critical for assessing gene-editing reagent delivery efficiency, retention, persistence, and distribution within living organisms. Conventional detection methods are effective, yet the expense and lack of scalability for antibody-based affinity reagents limit these techniques for clinical and/or field settings. This necessitates the development of low cost, scalable CRISPR/Cas9 RNP affinity reagents as alternatives or augments to antibodies. Herein, we report the development of the Streptococcus pyogenes anti-CRISPR/Cas9 protein, AcrIIA4, as a novel affinity reagent. An engineered cysteine linker enables covalent immobilization of AcrIIA4 onto glassy carbon electrodes functionalized via aryl diazonium chemistry for detection of CRISPR/Cas9 RNP by electrochemical, fluorescent, and colorimetric methods. Electrochemical measurements achieve a detection of 280 pM RNP in reaction buffer and 8 nM RNP in biologically representative conditions. Our results demonstrate the ability of anti-CRISPR proteins to serve as robust, specific, flexible, and economical recognition elements in biosensing/quantification devices for CRISPR/Cas9 RNP.

Crystal structures of phage NrS-1 N300-dNTPs-Mg2+ complex provide molecular mechanisms for substrate specificity.

A novel DNA polymerase from the deep-sea vent phage NrS-1, was characterized as a primase-polymerase (referred to as prim-pol), which works as a self-priming DNA polymerase to synthesize de novo long DNA strands. Functional research on the NrS-1 prim-pol illustrated that the N-terminal 300 residues (referred to as N300) have de novo synthesis activity similar to that of the full-length enzyme. Just like other prim-pols, NrS-1 prim-pol was able to initiate DNA synthesis, proficiently discriminating against ribonucleotides (NTPs), exclusively using deoxynucleotides (dNTPs). However, the structural basis for this discrimination is not well understood. Here, the three kinds of crystal structures of N300-dNTPs-Mg2+ complex were determined. These complex structures shared the identical steric architecture and hydrogen-bond interactions in the catalytic center. The results of biochemical studies indicated that R145 possibly plays an indispensable role in the primer extension. Mutagenesis and structural simulation showed that the backbone carboxyl group of Y146, as a potential sugar selector, was involved in steric clashing with the incoming 2'-OH group of NTPs. However, the mechanism of substrate discrimination probably was different from that of other prim-pols, according to the structural analyses and sequence comparison.

Strategies for purification of the bacteriophage HK97 small and large terminase subunits that yield pure and homogeneous samples that are functional.

Packaging the viral genome in the head of double-stranded DNA viruses, such as bacteriophages, requires the activity of a terminase. The bacteriophage terminase consists of a small terminase subunit (TerS), which binds the viral DNA, and a large terminase subunit (TerL) that possesses the ATPase and nuclease activities for packaging the DNA in the phage head. Some phages require additional components for DNA packaging, such as the HNH endonuclease gp74 in the bacteriophage HK97. Gp74 enhances the activity of terminase-mediated digestion of the cohesive (cos) site that connects individual genomes in phage concatemeric DNA, a pre-requisite to DNA packaging, and this enhancement requires an intact HNH motif in gp74. Testing of whether gp74 alters the terminase DNA binding or enzymatic activities requires obtaining isolated samples of pure TerS and TerL, which has been challenging owing to the poor solubility of these proteins. To this end, we developed methods to obtain purified TerS and TerL proteins that are active. TerS is expressed solubly in E. coli as a fusion with SUMO, which can be removed during purification to yield a TerS nonamer (TerS9). Homogenous samples of a TerL monomer are also obtained, but the homogeneity of the sample depends on the solution conditions, as seen for other terminases. DNA binding, ATPase, and nuclease assays demonstrate that our preparations of TerS9 and TerL are functional, and that they also function with gp74. Purified TerS9 and TerL enable studies into the molecular basis by which gp74 regulates terminase activity in phage maturation.

Probing Surface Membrane Receptors Using Engineered Bacteriophage Bioconjugates.

Specific recognition of ligands by surface receptors of eukaryotic cells is a fundamental process in sensing of the exogenous environment, including cell-to-cell communication. These interactions are therefore widely probed in both basic studies and drug development to enhance or interrupt them. Here, we designed a high-throughput publicly available platform for visualization and selection of eukaryotic cells according to the specificity of surface-exposed receptors by consolidation of phage display and flow cytometry techniques. Polypeptide ligands for membrane receptors are incorporated into every copy of p3 protein of M13K07 bacteriophage, which is intracellularly biotinylated to further accept PE-Cy7-labled streptavidin. Transgenic antigen-specific B-cells expressing membrane-tethered lymphoid B-cell receptor in a single-chain format interacted with engineered bacteriophages exposing the polypeptide ligand with an unprecedented selectivity of 97% and a false-positive detection value of 2.0%. Multivalent binding of the phage bioconjugates with the receptor provided significantly better specificity and sensitivity allowing application of engineered bacteriophage bioconjugates at a concentration 3 orders of magnitude lower in comparison with synthetic biotinylated peptide. We suggest that the platform described in this work may be applied either for routine staining or characterization of orphan membrane receptors exposed on the surface of living mammalian cells in their native environment.

Effect of C-terminal His-tag and purification routine on the activity and structure of the metalloenzyme, l-alanyl-d-glutamate peptidase of the bacteriophage T5.

In this work, we studied the effect of the C-terminally attached poly-histidine tag (His-tag), as well as the peculiarities of the protein purification procedure by the immobilized metal affinity chromatography (IMAC) on the activity and structure of the metalloenzyme, l-alanyl-d-glutamate peptidase of bacteriophage T5 (EndoT5), whose zinc binding site and catalytic aspartate are located near the C-terminus. By itself, His-tag did not have a significant effect on either activity or folding of the polypeptide chain, nor on the binding of zinc and calcium ions to the protein. However, the His-tagged EndoT5 samples had low shelf-life, with storage of these samples resulting in an increased propensity for protein self-association and decreased enzymatic activity of EndoT5. Furthermore, disastrous effects on the activity of the enzyme were exerted by the presence of imidazole and nickel ions accompanying metal chelate chromatography. The activity of the protein can be restored by thorough washing off of these low molecular impurities via the prolonged dialysis of the His-tagged EndoT5 samples at the specifically elaborated conditions.

Immunostimulation of Cyprinus carpio using phage lysate of Aeromonas hydrophila.

Over the last 50 years, various approaches have been established for the development of antigens for immunostimulation. We used phage lysate (PL), composed of inactivated antigens by the lytic bacteriophage pAh 6-c for Aeromonas hydrophila JUNAH strain to develop a vaccine for the prevention of A. hydrophila infection in Cyprinus carpio (common carp). We also assessed the poly D,L lactide-co-glycolic acid (PLGA) microparticles encapsulation method to increase the efficiency of the vaccine. Six groups of vaccines involving encapsulated by PLGA, formalin killed cells, or phage lysate at low or high concentration were prepared for intraperitoneal injection in C. carpio. Blood specimens and head kidney samples were collected at various time points for bacterial agglutination assay and to assess relative expression of immune-related genes interleukin-1 beta (IL-1ß), tumor necrosis factor alpha (TNF-α), lysozyme C, and serum amyloid A (SAA). The vaccine groups using high dose phage lysate antigen showed significantly higher agglutination titers than all other groups at 4- and 6-weeks post vaccination (wpv), with the titer of the PLGA encapsulated vaccine group being highest from 10 wpv to the end of the experiment. The survival rate of fish immunized with the phage lysate vaccines were higher than that of fish immunized with the formailin killed cells vaccine in the challenge experiment conducted 6 wpv. Additionally, the PLGA-encapsulated high dose phage lysate antigen vaccinated groups showed the best protective efficacy in the challenge experiment 12 wpv. Vaccines using the phage lysate antigen also showed higher IL-1ß and lysozyme C gene expression at 7 days post vaccination (dpv) and 2 wpv, and higher TNF-α gene expression was seen at 7 dpv. Higher SAA gene expression was seen in these groups at 1 dpv. These results suggest that phage lysate antigen has the potential to induce robust immune responses than formalin killed cells-based vaccines, and could be more effective as a novel inactivated antigen in preventing A. hydrophila infection in C. carpio.

Intracellular RNA-tracking methods.

RNA tracking allows researchers to visualize RNA molecules in cells and tissues, providing important spatio-temporal information regarding RNA dynamics and function. Methods such as fluorescent in situ hybridization (FISH) and molecular beacons rely on complementary oligonucleotides to label and view endogenous transcripts. Other methods create artificial chimeric transcripts coupled with bacteriophage-derived coat proteins (e.g. MS2, λN) to tag molecules in live cells. In other approaches, endogenous RNAs are recognized by complementary RNAs complexed with noncatalytic Cas proteins. Each technique has its own set of strengths and limitations that must be considered when planning an experiment. Here, we discuss the mechanisms, advantages, and weaknesses of in situ hybridization, molecular beacons, MS2 tagging and Cas-derived systems, as well as how RNA tracking can be employed to study various aspects of molecular biology.

Phages bearing specific peptides with affinity for porcine reproductive and respiratory syndrome virus GP4 protein prevent cell penetration of the virus.

Porcine reproductive and respiratory syndrome (PRRS) has caused significant economic losses to the pig industry worldwide over the last 30 years. GP4 is a minor highly glycosylated structural protein composed of 187 and 183 amino acids in types I and II porcine reproductive and respiratory syndrome virus (PRRSV), respectively. The GP4 protein co-localizes with cluster of differentiation 163 (CD163), the major receptor on the target cell membrane, to mediate PRRSV internalization and disassembly. However, it remains to be established whether blocking interactions between GP4 and host cells can inhibit viral proliferation. In the present study, recombinant GP4 protein prepared and purified using the Escherichia coli system effectively recognized PRRSV-positive serum. Phage display biopanning on GP4 protein showed that the specific phages obtained could distinguish PRRSV from the other viruses. The exogenous peptide WHEYPLVWLSGY displayed on one of the candidate phages showed high affinity for GP4 protein and exerted a significant inhibitory effect on PRRSV penetration in vitro. Moreover, the N-terminus of GP4 was predicted as the critical receptor binding site and the beginning of the fifth scavenger receptor cysteine-rich domain of CD163 as the critical ligand recognition site based on sequence alignment and model prediction analyses. The current study expands our understanding of PRRSV GP4 and its receptor CD163 and provides a fresh perspective for the development of novel peptide-based viral inhibition reagents.