Viral Host Research Articles

Immunogenetics of longevity and its association with human endogenous retrovirus K

IntroductionThe human immune system is equipped to neutralize and eliminate viruses and other foreign antigens via binding of human leukocyte antigen (HLA) molecules with foreign antigen epitopes and presenting them to T cells. HLA is highly polymorphic, resulting in subtle differences in the binding groove that influence foreign antigen binding and elimination. Here we tested the hypothesis that certain HLA alleles may promote longevity by enhanced ability to counter virus antigens that may otherwise contribute to morbidity and mortality.MethodsWe utilized high-resolution genotyping to characterize HLA and apolipoprotein E in a large sample (N = 986) of participants (469 men, 517 women) ranging in age from 24 to 90+ years old (mean age: 58.10 years) and identified 244 HLA alleles that occurred in the sample. Since each individual carries 12 classical HLA alleles (6 alleles of each Class I and Class II), we determined in silico the median predicted binding affinity for each individual (across the 12 HLA alleles) and each of 13 common viruses (Human Herpes Virus 1 [HHV1], HHV2, HHV3, HHV4, HHV5, HHV6A, HHV6B, HHV7, HHV8, human papilloma virus [HPV], human polyoma virus [JCV], human endogenous retrovirus K [HERVK], and HERVW). Next, we performed a stepwise multiple linear regression where the age of the participant was the dependent variable and the 13 median predicted HLA-virus binding affinities were the independent variables.ResultsThe analyses yielded only one statistically significant effect–namely, a positive association between age and HERVK (P = 0.005). Furthermore, we identified 13 HLA alleles (9 HLA-I and 4 HLA-II) that occurred at greater frequency in very old individuals (age ≥90 years) as compared to younger individuals. Remarkably, for those 13 alleles, the predicted binding affinities were significantly higher for HERVK than for the other viruses (P < 0.001). ApoE genotypes did not differ significantly between older and younger groups.DiscussionTaken together, the results showed that HLA-HERVK binding affinity is a robust predictor of longevity and that HLA alleles that bind with high affinity to HERVK were enriched in very old individuals. The findings of the present study highlight the influence of interactions between host immunogenetics and virus exposure on longevity and suggest that specific HLA alleles may promote longevity via enhanced immune response to specific common viruses, notably HERVK.

BV/ODV-E26 is a conserved baculoviral inhibitory factor for optimizing viral virulence in lepidopteran hosts.

Alphabaculoviruses induce abnormal behavior in lepidopteran larval hosts. A baculoviral gene, bv/odv-e26, is crucial for behavioral manipulation in Bombyx mori larvae by Bombyx mori nucleopolyhedrovirus (BmNPV). However, how bv/odv-e26 fulfills its role in this phenotype remains largely unknown. In this study, we found that the overexpression of BmNPV bv/odv-e26 delayed viral infection in cultured cells and decreased pathogenicity in B.mori larvae. We also discovered that homologs of bv/odv-e26 are conserved more widely in alphabaculoviruses than previously thought. The inhibitory activity was demonstrated in bv/odv-e26 homologs of phylogenetically close and distant baculoviruses, indicating conserved inhibitory function among alphabaculoviruses. Furthermore, locomotory analyses revealed that bv/odv-e26 increased larval locomotory activity but had little effect on the timing of abnormal behavior initiation. Collectively, our findings demonstrate that bv/odv-e26 is a baculoviral inhibitory factor that is widely conserved in the genus alphabaculovirus and may reduce viral virulence for successful host behavioral manipulation.

Zinc-finger PARP proteins ADP-ribosylate alphaviral proteins and are required for interferon-γ-mediated antiviral immunity.

Viral manipulation of posttranslational modifications (PTMs) is critical to enable control over host defenses. Evidence suggests that one such PTM, adenosine 5'-diphosphate (ADP)-ribosylation, is important for viral replication, but the host and viral components involved are poorly understood. Here, we demonstrate that several human poly(ADP-ribose) polymerase (PARP) proteins, including the zinc-finger domain containing PARP7 (TiPARP) and PARP12, directly ADP-ribosylate the alphaviral nonstructural proteins (nsPs), nsP3 and nsP4. These same human PARP proteins inhibit alphavirus replication in a manner that can be antagonized by the ADP-ribosylhydrolase activity of the virally encoded macrodomain. Last, we find that knockdown of any of the three CCCH zinc-finger domain containing PARPs, PARP7, PARP12, or the enzymatically inactive PARP13 (ZAP/ZC3HAV1), attenuates the antiviral effects of interferon-γ on alphavirus replication. Combined with evolutionary analyses, these data suggest that zinc-finger PARPs share an ancestral antiviral function that can be antagonized by the activity of viral macrodomains, indicative of an ongoing evolutionary conflict between host ADP-ribosylation and viruses.

The Japanese encephalitis virus NS1' protein facilitates virus infection in mosquitoes.

The Japanese encephalitis virus (JEV), a mosquito-borne flavivirus, is known for its capacity to cause severe neurological disease in Asia. Neurotropic flaviviruses within the Japanese encephalitis (JE) serogroup possess the distinctive feature of expressing a unique nonstructural protein, NS1'. The NS1' protein consists of the full NS1 protein with an additional 52 amino acid extension at the C-terminus and has been demonstrated to exhibit virulence in mammalian hosts upon infection. However, the precise role of the NS1' protein in the mosquito vectors has yet to be elucidated. In this study, an NS1'-defective virus (rG66A) was engineered, and its effect on the infection of mosquito cells was investigated. The results demonstrated a significant reduction in the infectivity of the rG66A virus in mosquito cells by RT-qPCR, indicating that the absence of the NS1' protein impedes JEV replication in Culex mosquitoes. Additionally, this research elucidated the underlying mechanism by which the NS1' protein enhances viral infection in mosquitoes by RNA-Seq analysis. Specifically, the NS1' protein was found to facilitate infection through the suppression of antimicrobial peptides (AMPs) regulated by the Toll pathway. Our research demonstrated that the JEV NS1' protein contributes to immune escape, thereby enhancing viral infection in mosquitoes. This finding offers new insights into the transmission mechanisms of JEV, elucidating novel aspects of viral propagation.

A Novel HTNV Budding Inhibitor Interferes the Interaction Between Viral Glycoprotein and Host ESCRT Accessory Protein ALIX.

Virus budding is a critical step in the replication cycle of enveloped viruses, closely linked to viral spread, disease progression, and clinical outcomes. The budding of many enveloped RNA viruses is facilitated by the hijacking of the host endosomal sorting complex required for transport (ESCRT) proteins through viral late domains. These late domains are essential for progeny virus production and are highly conserved, making the interaction between late domains and host ESCRT proteins a potential target for the development of antiviral therapeutics. In this study, we elucidated the functional role of the conserved YRTL motif within the glycoprotein Gn cytoplasmic tail of Orthohantavirus hantanense (Hantaan virus, HTNV), demonstrating that HTNV production is regulated by the interaction between YRTL and the ESCRT accessory protein ALIX (ALG-2 interacting protein X). Through virtual molecule docking screening, followed by in vitro and in vivo assays, we discovered a novel compound, AN-329, which disrupts the YRTL-ALIX interaction and effectively inhibits infectious HTNV production, as well as Crimean-Congo hemorrhagic fever virus (CCHFV) and Rift Valley fever virus (RVFV) VLP release. This makes AN-329 a promising therapeutic candidate for reducing viral dissemination. Given that YRTL is conserved across many hantaviruses, our findings may serve as a prototype for the development of broad-spectrum antiviral drugs.

Review of the mechanisms of virus-induced immunosuppression in poultry

A healthy immune system is the basis of successfully poultry farming. The industry suffers economic losses due to prolonged immunosuppression mediated by several viruses: Marek's disease virus (MDV), chicken infectious anemia virus (CIAV), infectious bursal disease virus (IBDV), reoviruses, some retroviruses as well as their associations. Two categories of causes of viral immunosuppression have been identified: apoptosis and/or necrosis of lymphoid cells and changes caused by the virus in the regulation of the immune response due to disruption of the cytokine profile. In many cases, the actual molecular interactions between virus proteins and host cells are poorly understood. Future research should focus on better understanding the interplay between viruses, immunocompetent cells, and cytokine regulation. Recent developments in the understanding of the immunotoxic and immunosuppressive effects of viruses may potentially offer a way to prevent these conditions.

Proliferative and viability effects of two cyanophages on freshwater bloom-forming species Microcystis aeruginosa and Raphidiopsis raciborskii vary between strains

Viruses that infect cyanobacteria are an integral part of aquatic food webs, influencing nutrient cycling and ecosystem health. However, the significance of virus host range, replication efficiency, and host compatibility on cyanobacterial dynamics, growth, and toxicity remains poorly understood. In this study, we examined the effects of cyanophage additions on the dynamics and activity of optimal, sub-optimal, and non-permissive cyanobacterial hosts in cultures of Microcystis aeruginosa and Raphidiopsis raciborskii. Our findings reveal that cross-infectivity can substantially reduce the proliferative success of the cyanophage under conditions of high-density of sub-optimal hosts which suggests phage dispersal limitation as a result of shared infections, in turn impairing their top-down control over the host community. Furthermore, we found that cyanophage addition triggers host strain-specific responses in photosynthetic performance, population size and toxin production, even among non-permissive hosts. These non-lytic effects suggest indirect impacts on co-existing cyanobacteria, increasing the overall complexity and variance in many ecologically relevant cyanobacterial traits. The high variability in responses observed with a limited subset of cyanophage-cyanobacteria combinations not only highlights the intricate role of viral infections in microbial ecosystems but also underscores the significant challenges in predicting the composition, toxicity, and dynamics of cyanobacterial blooms.

Immunological findings of West Caucasian bat virus in an accidental host.

Although all lyssaviruses cause fatal encephalomyelitis in mammals, they display a different host tropism and pathogenicity, with the ecology of Rabies virus (RABV) continually evolving and adapting to new host species. In 2020, West Caucasian bat virus (WCBV) was identified as the causative agent of rabies in a domestic cat in Italy. This event raised concerns about its public health consequences, due to the absence of biologicals against the infection. Our study investigates the host immune response triggered by WCBV in comparison with a pathogenic strain of RABV and the low pathogenic Duvenhage lyssavirus (DUVV), as a proxy to understand the mechanisms leading to lyssavirus spillover and pathogenicity. We overall confirm that previous evidence indicating an inverse relationship between lyssavirus pathogenicity and immune response is applicable for WCBV as well. Importantly, this work represents the first transcriptomic analysis of the WCBV interaction in the central nervous system with an accidental host.

Visualizing Tomato Spotted Wilt Virus Protein Localization: Cross-Kingdom Comparisons of Protein-Protein Interactions.

Tomato spotted wilt virus (TSWV) is an orthotospovirus that infects both plants and insect vectors, and understanding its protein localization and interactions is crucial for unraveling the infection cycle and host-virus interactions. We investigated and compared the localization of TSWV proteins. The localization between plant and insect cells was overall consistent, indicating a similar mechanism is utilized by the virus in both types of cells. However, a change in localization over time was associated with the viral proteins that did not contain signal peptides and transmembrane domains such as N, NSs, and NSm, which only occurred in the plant cells and not in the insect cells. We also tested the localization of the proteins during an active plant infection using free red fluorescent protein (RFP) as a marker to highlight the nucleus and cytoplasm. Voids in the cytoplasm were shown only during infection, and N, NSs, NSm, and to a lesser extent GN and GC were surrounding these areas, suggesting it may be a site of replication or morphogenesis. Furthermore, we tested the interactions of viral proteins using both bimolecular fluorescence complementation (BiFC) and membrane-based yeast two-hybrid (MbY2H) assays. These revealed self-interactions of NSm, N, GN, GC, and NSs. We also identified interactions between different TSWV proteins, indicating their possible roles, such as between NSs and GC and N and GC, which may be necessary during the replication and assembly processes, respectively. This research expands our knowledge of TSWV infection and elaborates on the intricate relationships between viral proteins, cellular dynamics, and host responses. [Formula: see text] Copyright © 2025 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Strategies for the Viral Exploitation of Nuclear Pore Transport Pathways

Viruses frequently exploit the host’s nucleocytoplasmic trafficking machinery to facilitate their replication and evade immune defenses. By encoding specialized proteins and other components, they strategically target host nuclear transport receptors (NTRs) and nucleoporins within the spiderweb-like inner channel of the nuclear pore complex (NPC), enabling efficient access to the host nucleus. This review explores the intricate mechanisms governing the nuclear import and export of viral components, with a focus on the interplay between viral factors and host determinants that are essential for these processes. Given the pivotal role of nucleocytoplasmic shuttling in the viral life cycle, we also examine therapeutic strategies aimed at disrupting the host’s nuclear transport pathways. This includes evaluating the efficacy of pharmacological inhibitors in impairing viral replication and assessing their potential as antiviral treatments. Furthermore, we emphasize the need for continued research to develop targeted therapies that leverage vulnerabilities in nucleocytoplasmic trafficking. Emerging high-resolution techniques, such as advanced imaging and computational modeling, are transforming our understanding of the dynamic interactions between viruses and the NPC. These cutting-edge tools are driving progress in identifying novel therapeutic opportunities and uncovering deeper insights into viral pathogenesis. This review highlights the importance of these advancements in paving the way for innovative antiviral strategies.

Acinetobacter baumannii clinical isolates resist complement-mediated lysis by inhibiting the complement cascade and improperly depositing MAC.

Acinetobacter baumannii is a gram-negative opportunistic bacterium that causes life-threatening infections in immunocompromised hosts. The World Health Organization (WHO) recognizes the high mortality and increasing antimicrobial resistance of A. baumannii and calls for new treatment options. The complement system is a critical mechanism of innate immunity that protects the human body from bacterial infections. Complement activation leads to the deposition of the membrane attack complex (MAC), which can directly lyse gram-negative bacteria. However, A. baumannii has developed evasion mechanisms to protect itself from complement. Here, we examined clinical isolates of A. baumannii and found 11 isolates with MAC deposition and 5 isolates without deposition. Trypsinization of MAC-positive isolates significantly reduced MAC, indicating incorrect insertion, consistent with a lack of lysis of these strains. MAC-negative isolates inhibited alternative pathway activation and were significantly more serum-resistant. These strains were also more virulent in a Galleria mellonella infection model. Whole genome sequencing revealed that MAC-negative isolates carried more virulence genes, and both MAC-negative and MAC-positive A. baumannii significantly differed in capsule type. Importantly, a correlation was observed between complement inhibition and capsule type (e.g., capsule locus KL171) of MAC-negative bacteria, while the capsule type (e.g., KL230) of MAC-positive A. baumannii was associated with increased sensitivity to MAC-mediated lysis. Thus, our findings suggest a relationship between capsule type, complement resistance, and host virulence in A. baumannii.

The cytoplasmic tail of IBV spike mediates intracellular retention via interaction with COPI-coated vesicles in retrograde trafficking.

Coronaviruses are characterized by their progeny assembly and budding in the endoplasmic reticulum-Golgi intermediate compartment (ERGIC). Our previous studies demonstrated that truncation of 9 amino acids in the cytoplasmic tail (CT) of the infectious bronchitis virus (IBV) spike (S) protein impairs its localization to the ERGIC, resulting in increased expression at the plasma membrane. However, the precise mechanism underlying this phenomenon remained elusive. In this study, we provide evidence that the IBV S protein could utilize coatomer protein-I (COPI)-coated vesicles for retrograde transport from the Golgi to the endoplasmic reticulum (ER). We identified the KKSV motif as the critical binding site within the CT domain of IBV S protein for COPI interaction. Further analysis reveals that IBV infection does not modulate host COPI expression. However, when COPI expression is disrupted, a higher proportion of S protein escapes to the plasma membrane. Moreover, inhibition of COPI-mediated transport during viral infection severely impairs progeny virion production and leads to increased S protein accumulation at the plasma membrane, inducing cell-cell fusion and syncytia formation. Our findings contribute to a deeper understanding of S protein intracellular trafficking during coronavirus infection, and offer valuable insights into the molecular mechanisms of viral replication and host cell biology.IMPORTANCEViruses hijack or modify host cellular machinery and associated pathways to facilitate their own replication. Here, we demonstrate that the infectious bronchitis virus (IBV) S protein directly interacts with coatomer protein-I (COPI)-coated vesicles through the KKSV motif in its cytoplasmic tail. COPI-coated vesicles mediate the retrograde transport of S protein from the Golgi apparatus to the endoplasmic reticulum-Golgi intermediate compartment, where viral particle assembly occurs. Our findings not only advance our understanding of IBV S protein trafficking mechanisms but also provide valuable insights for developing more effective vaccine strategies.

Correction to: First report of Solanum elaeagnifolium as a natural host of chickpea chlorotic dwarf virus in Tunisia

Correction to: First report of Solanum elaeagnifolium as a natural host of chickpea chlorotic dwarf virus in Tunisia

Viremic non-progression in HIV/SIV infection: A tied game between virus and host.

High-efficacy antiretroviral treatment (ART) has been a game-changer for HIV/AIDS pandemic, but incomplete CD4+ Tcell recovery and persistent chronic immune activation still affect HIV-suppressed people. Exceptional cases of HIV infection that naturally exhibit delayed disease progression provide invaluable insights into protective biological mechanisms with potential clinical application. Viremic non-progressors (VNPs) represent an extremely rare population of individuals with HIV, characterized by preservation of the CD4+ Tcell compartment despite persistent high levels of viral load (>10,000 copies/mL). While only a few studies have investigated the immunovirological characteristics of adult and pediatric VNPs, most of our knowledge about this phenotype stems from its non-human-primate counterpart, the natural simian immunodeficiency virus (SIV) hosts. In this review, we synthesize the insights gained from recent studies of natural SIV hosts and VNPs and evaluate the potential similarities and differences in the mechanisms that underlie the absence of pathogenesis, with special focus on the control of immune activation.

Variants and vaccines impact nasal immunity over three waves of SARS-CoV-2.

Viral variant and host vaccination status impact infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), yet how these factors shift cellular responses in the human nasal mucosa remains uncharacterized. We performed single-cell RNA sequencing (scRNA-seq) on nasopharyngeal swabs from vaccinated and unvaccinated adults with acute Delta and Omicron SARS-CoV-2 infections and integrated with data from acute infections with ancestral SARS-CoV-2. Patients with Delta and Omicron exhibited greater similarity in nasal cell composition driven by myeloid, T cell and SARS-CoV-2hi cell subsets, which was distinct from that of ancestral cases. Delta-infected samples had a marked increase in viral RNA, and a subset of PER2+EGR1+GDF15+ epithelial cells was enriched in SARS-CoV-2 RNA+ cells in all variants. Prior vaccination was associated with increased frequency and activation of nasal macrophages. Expression of interferon-stimulated genes negatively correlated with coronavirus disease 2019 (COVID-19) severity in patients with ancestral and Delta but not Omicron variants. Our study defines nasal cell responses and signatures of disease severity across SARS-CoV-2 variants and vaccination.

Nicking Activity of M13 Bacteriophage Protein 2.

Gene II Protein (Gp2/P2) is a nicking enzyme of the M13 bacteriophage that plays a role in the DNA replication of the viral genome. P2 recognizes a specific sequence at the f1 replication origin and nicks one of the strands and starts replication. This study was conducted to address the limitations of previous experiments, improve methodologies, and precisely determine the biochemical activity conditions of the P2 enzyme in vitro. For these purposes, the gene encoding P2 was cloned in Escherichia coli and expressed as a hybrid protein together with a green fluorescent protein (P2-GFP). P2-GFP was purified via metal affinity chromatography, and its nicking activity was determined by conversion of supercoiled DNA to open circular or linear forms. We discovered that, among the two loops of the f1 origin defined previously, P2 can recognize just the A1 loop. When a supercoiled plasmid containing the f1 origin was treated with P2-GFP, the plasmid was present in an open circular form, indicating that a nick was created on only one of the strands. However, when the A1 loop sequence was inserted into the 3' ends of both strands by cloning a PCR product obtained by primers with the A1 loop sequence, the plasmid was linearized by treatment with P2-GFP, indicating that nicks were created on both strands. Certain infectious diseases are caused by single-stranded DNA viruses, and some of them have specific nicking enzymes that enable strand displacement and free 3' end of a single strand that works as a primer for their replication mechanisms like M13 bacteriophages, such as parvovirus B19. Despite there being different host viruses such as bacteria and humans, their DNA replication mechanisms are very similar in this concept. Investigating the features of the P2-nicking enzyme may deepen the understanding of human pathogenic single-stranded viruses and facilitate the development of drugs that inhibit viral replication.

Rapid and comprehensive detection of viral antibodies and nucleic acids via an acoustofluidic integrated molecular diagnostics chip: AIMDx.

Precise and rapid disease detection is critical for controlling infectious diseases like COVID-19. Current technologies struggle to simultaneously identify viral RNAs and host immune antibodies due to limited integration of sample preparation and detection. Here, we present acoustofluidic integrated molecular diagnostics (AIMDx) on a chip, a platform enabling high-speed, sensitive detection of viral immunoglobulins [immunoglobulin A (IgA), IgG, and IgM] and nucleic acids. AIMDx uses acoustic vortexes and Gor'kov potential wells at a 1/10,000 subwavelength scale for concurrent isolation of viruses and antibodies while excluding cells, bacteria, and large (>200 nanometers) vesicles from saliva samples. The chip facilitates on-chip viral RNA enrichment, lysis in 2 minutes, and detection via transcription loop-mediated isothermal amplification, alongside electrochemical sensing of antibodies, including mucin-masked IgA. AIMDx achieved nearly 100% recovery of viruses and antibodies, a 32-fold RNA detection improvement, and an immunity marker sensitivity of 15.6 picograms per milliliter. This breakthrough provides a transformative tool for multiplex diagnostics, enhancing early infectious disease detection.

Nucleolin a Central Player in Host Virus Interactions and its Role in Viral Progeny Production.

Nucleolin (NCL) is a prevalent and widely distributed nucleolar protein in cells. While primarily located in the nucleolus, NCL is also found within the nucleoplasm, cytoplasm, and even on the cell surface. NCL's unique nature arises from its multifaceted roles and extensive interactions with various proteins. The structural stability of NCL is reliant on protease inhibitors, particularly in proliferating cells, indicating its essential role in cellular maintenance. This review is centered on elucidating the structure of NCL, its significance in host-viral interactions, and its various contributions to viral progeny production. This work is to enhance the scientific community's understanding of NCL functionality and its implications for viral infection processes. NCL is highlighted as a crucial host protein that viruses frequently target, exploiting it to support their own life cycles and establish infections. Understanding these interactions is key to identifying NCL's role in viral pathogenesis and its potential as a therapeutic target. Our current knowledge, alongside extensive scientific literature, underscores the critical role of host proteins like NCL in both viral infections and other diseases. As a target for viral exploitation, NCL supports viral replication and survival, making it a promising candidate for therapeutic intervention. By delving deeper into the intricacies of NCL-viral protein interactions, researchers may uncover effective antiviral mechanisms. This review aspires to inspire further research into NCL's role in viral infections and promote advancements in antiviral therapeutic development.

Immune responses to avian influenza viruses in chickens.

Chickens are a key species in both the manifestation of avian influenza and the potential for zoonotic transmission. Avian influenza virus (AIV) infection in chickens can range from asymptomatic or mild disease with low pathogenic AIVs (LPAIVs) to systemic fatal disease with high pathogenic AIVs (HPAIVs). During AIV infection in chickens, Toll-like receptor 7 and melanoma differentiation-associated gene 5 are upregulated to detect the single-stranded ribonucleic acid genomes of AIV, triggering a signaling cascade that produces interferons (IFNs) and pro-inflammatory cytokines. These inflammatory mediators induce the expression of antiviral proteins and recruit immune system cells, such as macrophages and dendritic cells, to the infection site. AIV evades these antiviral responses primarily through its non-structural protein 1, which suppresses type I IFNs, influencing viral pathogenicity. The uncontrolled release of pro-inflammatory cytokines may contribute to the pathogenicity and high mortality associated with HPAIV infections. AIV modulates apoptosis in chicken cells to enhance its replication, with variations in apoptosis pathways influenced by viral strain and host cell type. The presentation of AIV antigens to T and B cells leads to the production of neutralizing antibodies and the targeted destruction of infected cells by CD8+ T cells, respectively, which enhances protection and establishes immunological memory. This review explores the diverse innate and adaptive immune responses in chickens to different AIVs, focusing on the dynamics of these responses relative to protection, susceptibility, and potential immunopathology. By understanding these immune mechanisms, informed strategies for controlling AIV infection and improving chicken health can be developed.

A novel broad-spectrum bacteriophage cocktail against methicillin-resistant Staphylococcus aureus: Isolation, characterization, and therapeutic potential in a mastitis mouse model.

Bovine mastitis is a considerable challenge within the dairy industry, causing significant financial losses and threatening public health. The increased occurrence of methicillin-resistant Staphylococcus aureus (MRSA) has provoked difficulties in managing bovine mastitis. Bacteriophage therapy presents a novel treatment strategy to combat MRSA infections, emerging as a possible substitute for antibiotics. This study evaluated the therapeutic potency of a novel bacteriophage cocktail against MRSA mastitis. Two new bacteriophages (vB_SauR_SW21 and vB_SauR_SW25) with potent lytic activity against MRSA were isolated and characterized. The one-step growth curve displayed a rapid latent period (20-35 min) and substantial burst size (418 and 316 PFU/ cell). In silico analyses have confirmed the absence of antimicrobial resistance or virulence factor-encoding genes within their genomes. According to the results, combining these phages augmented their host range and virulence. The phage cocktail significantly reduced bacterial burden in a BALB/c mastitis model, demonstrating efficacy comparable to antibiotic treatment. Moreover, its administration led to decreased concentrations of IL-1β and TNF-α compared to the negative control group. The bacteriophage cocktail (SW21-SW25) exhibits a promising profile for therapeutic applications and may represent a novel substitute to antibiotics for managing MRSA bovine mastitis.