Flow Cytometry in Veterinary Viral Immunology

Overview and Principles of Flow Cytometry in Veterinary Viral Immunology

The advent of flow cytometry has fundamentally transformed the landscape of veterinary viral immunology, evolving from a specialized research tool into an indispensable platform for dissecting host-pathogen interactions at the single-cell level. Unlike traditional bulk assays such as enzyme-linked immunosorbent assay (ELISA) or plaque reduction neutralization tests, which provide population-averaged measurements, flow cytometry offers the unparalleled capacity to interrogate the heterogeneity, activation status, and functional competence of individual immune cells within complex biological matrices. This capability is particularly critical in veterinary virology, where the immune response to viral pathogens-ranging from respiratory viruses in poultry to systemic infections in aquatic species-is often characterized by nuanced cellular dynamics that dictate clinical outcome, vaccine efficacy, and disease pathogenesis. The principles underpinning flow cytometry in this context rest on four interrelated pillars: (i) the multiparametric analysis of cellular phenotype and function, (ii) the quantitative detection of viral antigens within or on host cells, (iii) the isolation of rare antigen-specific lymphocyte populations for downstream molecular characterization, and (iv) the emerging application of flow virometry for direct characterization of viral particles themselves [14].

At its core, flow cytometry operates on the hydrodynamic focusing of a cell suspension into a single-file stream that passes through one or more laser interrogation points. As each cell intersects the beam, scattered light and fluorescence emissions are collected by photomultiplier tubes, generating data on forward scatter (FSC), side scatter (SSC), and the intensity of multiple fluorophores. In veterinary viral immunology, this technical foundation enables the simultaneous assessment of up to 18 or more parameters, permitting the identification of discrete leukocyte subsets such as CD4⁺ and CD8⁺ T lymphocytes, B cells, natural killer (NK) cells, dendritic cells (DCs), monocytes, and granulocytes, each defined by combinations of cluster of differentiation (CD) markers. The development of species-specific monoclonal antibodies (mAbs) has been a rate-limiting step in this endeavor, yet recent progress has been remarkable. For instance, the generation of mAbs targeting CD4-1 and CD8β in rainbow trout has, for the first time, enabled the in situ visualization and flow cytometric quantification of T-cell subsets within melanomacrophage centers during infection with Infectious Hematopoietic Necrosis Virus (IHNV) [6]. Similarly, the validation of mAbs against hamster cytokines, chemokines, and T-cell activation markers-including CD44, CD62L, CD25, and CD69-has opened new avenues for studying SARS-CoV-2 pathogenesis in a highly relevant small-animal model [13]. These reagent advances underscore a broader principle: the translation of flow cytometric techniques to veterinary species hinges on a sustained investment in cross-reactive and species-specific immunological tools, a challenge that is gradually being met through hybridoma technology, phage display, and recombinant antibody platforms [1, 4].

The principle of multiparametric immunophenotyping in veterinary viral immunology extends beyond mere enumeration of cell frequencies. A critical application lies in the assessment of activation and differentiation states, which inform the qualitative nature of the antiviral response. Activation markers such as CD25 (the alpha chain of the IL-2 receptor), CD44, CD69, and CD62L are routinely employed to distinguish naive, effector, and memory T-cell compartments. In a study of cutaneous leishmaniasis-a disease with parallels to chronic viral infections-flow cytometric analysis revealed elevated co-expression of CD38 and HLA-DR on CD4⁺ and CD8⁺ T cells from patients with diffuse disease, alongside reduced CD8⁺ T-cell frequencies, suggesting a mechanism of activation-induced anergy or apoptosis [5]. This principle is directly transferable to viral infections such as Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), where the balance between protective and pathogenic T-cell responses is a major determinant of disease outcome. In the context of Infectious Bronchitis Virus (IBV) infection in chickens, flow cytometric sorting of CD44⁺ CD8⁺ T cells followed by transcriptomic profiling revealed upregulation of IL6R, IL7R, and NFKB1, consistent with a shift toward a memory-like phenotype [2]. Such findings would be impossible to ascertain through bulk analysis alone and highlight the necessity of single-cell resolution for understanding the trajectory of antiviral T-cell immunity.

Beyond static phenotyping, functional flow cytometry assays-including intracellular cytokine staining (ICS), detection of degranulation markers (e.g., CD107a), and proliferation tracking via Ki-67 expression-provide a dynamic window into effector mechanisms. The polyfunctionality of antigen-specific T cells, defined by the simultaneous production of IFN-γ, TNF-α, and IL-17A, has been rigorously evaluated in the context of bovine tuberculosis using multicolor flow cytometry [12]. While this work focused on a bacterial pathogen, the methodological framework is directly applicable to viral infections such as Bovine Viral Diarrhea Virus (BVDV) or Foot-and-Mouth Disease Virus (FMDV), where the quality of the T-cell response is increasingly recognized as a correlate of protection. For example, the B2T dendrimer peptide vaccine against FMDV was shown to enhance germinal center formation and increase the proportion of IgG1-secreting plasma cells in the bone marrow-findings that relied entirely on flow cytometric analysis of draining lymph nodes and splenic compartments [3]. Similarly, in rainbow trout immunized with a live attenuated IHNV vaccine, flow cytometry revealed a rapid, synchronized expansion of CD4-1⁺ and CD8β⁺ T lymphocytes in the spleen and head kidney, coinciding with upregulation of Th1/CTL-signature genes and sustained IgM responses [6]. These examples collectively illustrate that flow cytometry is not merely a descriptive tool but a quantitative engine for vaccine immunogenicity assessment, capable of informing both experimental design and regulatory decision-making.

A particularly powerful principle in veterinary viral immunology is the use of flow cytometry for the direct detection of viral antigens within host cells, a technique that bridges virology and immunology by quantifying the cellular targets of infection. This approach has been most extensively validated for BVDV, where a pan-pestivirus monoclonal antibody (C16) was used to detect the p80/125 protein in leukocytes from viremic cattle. Remarkably, flow cytometric analysis of granulocytes and monocytes achieved 100% concordance with traditional virus isolation, detecting as few as 1% antigen-positive cells with a total assay time of two hours [9]. This principle has been extended to Bovine Leukemia Virus (BLV), where gp51 envelope glycoprotein expression was quantified in blood and milk lymphocytes of naturally infected cows using dual-color flow cytometry [7]. The observation that gp51-positive cells were far more abundant in blood than in milk, and that infected animals exhibited a decreased CD4⁺/CD8⁺ ratio and proliferation of immature CD19⁺ IgM⁺ B cells, provided critical insights into BLV-induced immunopathogenesis [7]. In the context of emerging viral threats, this principle is being adapted for Porcine Epidemic Diarrhea Virus (PEDV), where monoclonal antibodies targeting the spike protein S1 domain have been used to develop not only flow cytometric detection but also electrochemical immunosensors for field deployment [4]. The direct detection of viral antigens within or on immune cells offers a unique advantage: it allows correlation between viral tropism and the functional status of the infected cell, a dimension that is lost in bulk assays such as quantitative PCR or virus isolation.

The intersection of flow cytometry with single-cell molecular technologies represents a paradigm shift in veterinary viral immunology. The principle of oligonucleotide-tagged antigen assemblies, combined with fluorescence-activated cell sorting (FACS), enables the isolation of rare antigen-specific B cells for downstream single-cell sequencing of immunoglobulin genes. This approach has been elegantly demonstrated for Transmissible Gastroenteritis Virus (TGEV), where single-cell flow cytometry sorting of memory B cells from immunized mice yielded 83 matched antibody heavy- and light-chain gene pairs, ultimately generating 42 recombinant antibodies with confirmed neutralizing activity [1]. The workflow-antigen-specific sorting, single-cell RT-PCR, and expression cloning-is now being applied to a growing list of veterinary viruses, including Avian Influenza Virus and Newcastle Disease Virus, to accelerate the discovery of broadly neutralizing antibodies. Moreover, the integration of flow cytometry with single-cell RNA sequencing (scRNA-seq) allows simultaneous profiling of transcriptome and surface protein expression (CITE-Seq), providing an unprecedented resolution of cellular states during viral infection. In a study of influenza A virus infection in mice, high-dimensional flow cytometry combined with scRNA-seq revealed that aged hosts exhibit diminished alveolar macrophages and dendritic cells but elevated monocyte-derived macrophages, with persistent type I and II interferon signaling driving chronic immunopathology [10]. Although this work was performed in a mouse model, the methodological principles are directly transferable to veterinary species, limited only by the availability of species-specific oligonucleotide-conjugated antibodies.

The principle of flow virometry-the analysis and sorting of individual viral particles-represents the newest frontier in the application of flow cytometry to veterinary virology. By harnessing the improved sensitivity of modern cytometers and the development of virus-specific fluorescent labels, researchers can now quantify viral particle concentration, assess antigenic diversity within a population, and even sort infectious from non-infectious virions based on surface glycoprotein conformation [14]. This technique holds particular promise for rapidly mutating viruses such as Equine Influenza A Virus and Swine Influenza A Virus, where antigenic drift necessitates continuous vaccine strain selection. Furthermore, flow virometry can be used to study viral interference, as demonstrated in C6/36 mosquito cells persistently infected with dengue virus serotype 2, where flow cytometry was employed to quantify homologous and heterologous interference against yellow fever virus [11]. While still in its infancy for routine veterinary diagnostics, flow virometry is poised to complement traditional methods such as hemagglutination inhibition and microneutralization assays, offering a high-throughput, single-particle resolution that is currently unmatched.

Finally, the principles of experimental design and data analysis in veterinary flow cytometry warrant explicit consideration. The use of fluorescence-minus-one (FMO) controls, live/dead discrimination dyes, and compensation matrices is essential to ensure data integrity, particularly when working with autofluorescent cells from species such as fish or poultry. The selection of antibody panels must be guided by the specific immunological question, the species under investigation, and the availability of validated reagents. For example, a five-color panel designed to identify helper, cytotoxic, activated, and memory T cells in tuberculin-positive cattle successfully demonstrated overexpression of CD25 and CD45RO following antigenic stimulation [8]. This panel, developed using commercially available fluorochrome-conjugated antibodies, serves as a template that can be adapted for viral systems. Moreover, the adoption of high-dimensional analysis tools-including t-distributed stochastic neighbor embedding (t-SNE), uniform manifold approximation and projection (UMAP), and cluster identification via flowSOM or PhenoGraph-has become standard practice for extracting biologically meaningful patterns from multiparametric data. These computational approaches are particularly valuable in veterinary viral immunology, where the complexity of the immune response across different tissues, time points, and species demands analytical rigor beyond traditional bivariate gating. The World Organisation for Animal Health (WOAH) and national reference laboratories are increasingly recognizing flow cytometry as a complementary tool for vaccine potency testing and disease surveillance, underscoring its transition from a research-only technique to a component of regulatory science.

In summary, the principles of flow cytometry in veterinary viral immunology are rooted in the quantitative, multiparametric, and single-cell analysis of host immune responses and viral infection dynamics. The technique's ability to dissect cellular heterogeneity, assess functional competence, and isolate rare antigen-specific populations has made it indispensable for understanding pathogenesis, evaluating vaccine candidates, and developing diagnostic reagents. The ongoing expansion of species-specific immunological tools, coupled with advances in flow virometry and single-cell sequencing, ensures that flow cytometry will remain at the forefront of veterinary virology research for the foreseeable future. The subsequent sections of this chapter will build upon this foundational overview to explore specific applications, including the characterization of T-cell responses to Porcine Reproductive and Respiratory Syndrome Virus, the evaluation of B-cell immunity following vaccination against Foot-and-Mouth Disease Virus, and the use of flow cytometry for diagnostic detection of Bovine Viral Diarrhea Virus in persistently infected cattle.

Protocol and Methodology for Flow Cytometric Analysis of Viral Infections

The application of flow cytometry to veterinary viral immunology represents a paradigm shift from traditional bulk-phase assays toward high-resolution, single-cell interrogation of host-pathogen interactions. Unlike conventional techniques such as plaque assays or endpoint PCR, flow cytometry permits the simultaneous quantification of viral antigen expression, cellular phenotype, activation status, and functional capacity within heterogeneous leukocyte populations. This section delineates the comprehensive methodological framework required for rigorous flow cytometric analysis of viral infections in veterinary species, addressing pre-analytical variables, panel design, data acquisition, and specialized applications ranging from viral antigen detection to functional immune profiling.

Sample Acquisition and Preparation

The foundation of any flow cytometric analysis lies in the quality and appropriateness of the starting material. For viral infection studies, peripheral blood remains the most accessible compartment, but tissue-specific immune responses often necessitate the isolation of leukocytes from lymphoid organs, mucosal surfaces, or sites of active viral replication. Whole blood collected in EDTA or heparin anticoagulants is suitable for surface phenotyping, though heparin may interfere with certain downstream functional assays and should be avoided when intracellular cytokine staining is planned [7, 8]. For tissue-resident lymphocyte analysis, enzymatic digestion protocols must be optimized to preserve surface epitope integrity while maximizing cell yield. The isolation of kidney-resident CD8+ T cells following systemic viral infection, for example, requires a combination of mechanical dissociation, collagenase D digestion, and density gradient centrifugation to separate leukocytes from parenchymal cells [18]. Intravascular staining with fluorochrome-conjugated antibodies administered prior to euthanasia is essential for distinguishing truly tissue-resident populations from circulating contaminants, a critical consideration when studying compartmentalized immune responses to viruses such as Infectious Hematopoietic Necrosis Virus or Viral Hemorrhagic Septicemia Virus in teleost models [6, 18].

For aquatic species, including those affected by Channel Catfish Virus or Epizootic Hematopoietic Necrosis Virus, the preparation of single-cell suspensions from head kidney and spleen requires careful optimization of dissociation buffers to maintain viability in hypotonic environments. The use of Percoll density gradients at specific osmolarities adjusted for fish physiology has proven essential for enriching leukocyte populations while removing erythrocytes and cellular debris [6, 16]. Similarly, for avian species infected with Avian Influenza Virus or Infectious Bronchitis Virus, the isolation of splenocytes and peripheral blood mononuclear cells must account for the nucleated erythrocytes characteristic of birds, which can confound light scatter gating if not adequately lysed or removed [2, 15].

Cryopreservation of samples for batch analysis is often necessary in large-scale studies, but this introduces variables that must be rigorously controlled. The detection of Bovine Viral Diarrhea Virus antigen in leukocytes by flow cytometry remains reliable even after blood storage at 4°C for up to six weeks, demonstrating remarkable stability of viral antigens under appropriate conditions [9]. However, functional assays such as intracellular cytokine staining are more sensitive to cryopreservation-induced alterations in cellular responsiveness, and viability typically declines by 15-30% following freeze-thaw cycles. For SARS-CoV-2 studies, viral inactivation protocols using paraformaldehyde fixation permit the safe transfer of infected samples from BSL-3 to BSL-2 facilities while preserving antigenicity for downstream flow cytometric analysis, a critical biosafety consideration when working with emerging zoonotic pathogens [21].

Antibody Panel Design and Reagent Validation

The design of multicolor panels for veterinary species presents unique challenges compared to human or murine systems, primarily due to the limited commercial availability of fluorochrome-conjugated monoclonal antibodies. Cross-reactivity with anti-human or anti-mouse reagents is occasionally observed but must be empirically validated for each target species and tissue type. The development of species-specific reagents has been transformative, as exemplified by the generation of monoclonal antibodies targeting CD4-1 and CD8β in rainbow trout, which enabled the first in situ visualization of T-cell subsets within melanomacrophage centers and the systematic profiling of immune dynamics following vaccination against Infectious Hematopoietic Necrosis Virus [6]. Similarly, the creation of monoclonal antibodies against hamster cytokines and T-cell activation markers has opened new avenues for investigating SARS-CoV-2 pathogenesis in this highly susceptible model, where reagent availability was previously a major bottleneck [13].

For bovine species, multiparametric panels incorporating five or more fluorochromes have been successfully implemented to identify helper, cytotoxic, activated, and memory T-cell subsets in tuberculin-positive cattle [8]. These panels rely on careful selection of fluorochrome combinations that minimize spectral overlap while maximizing resolution of dimly expressed markers. The use of brilliant violet dyes in combination with phycoerythrin and allophycocyanin conjugates has proven particularly effective for bovine immunophenotyping, as these fluorochromes exhibit minimal spillover into channels used for lineage-defining markers such as CD4 and CD8 [8, 12]. When designing panels for intracellular cytokine detection, as required for characterizing polyfunctional CD4+ T-cell responses to Bovine Viral Diarrhea Virus or Foot-and-Mouth Disease Virus, the inclusion of a viability dye and surface marker cocktail prior to fixation and permeabilization is essential to preserve antigenicity while enabling discrimination of live from dead cells [12].

The validation of novel monoclonal antibodies requires a multi-tiered approach. Initial screening by ELISA against recombinant proteins is followed by confirmation of specificity using Western blot and immunohistochemistry [1, 6]. For antibodies intended to detect viral antigens, such as the gp51 envelope protein of Bovine Leukemia Virus, flow cytometric comparison of infected versus uninfected cells provides definitive evidence of specificity [7]. The generation of monoclonal antibodies against the spike protein of Porcine Epidemic Diarrhea Virus required identification of a linear epitope (amino acids 696-715) that was subsequently validated by indirect immunofluorescence assay and Western blot before incorporation into an electrochemical immunosensor [4]. For functional studies, antibodies must be tested for their ability to detect antigen on live cells without inducing activation or apoptosis, a particular concern when studying activation markers such as CD25, CD69, or CD44 on T cells from virally infected animals [13].

Data Acquisition, Compensation, and Gating Strategies

Modern flow cytometers equipped with multiple lasers (typically 405 nm, 488 nm, and 638 nm) enable the simultaneous detection of 10-18 parameters, though the practical limit for veterinary immunology studies is often constrained by reagent availability. Compensation matrices must be generated using single-stained controls for each fluorochrome, including cellular controls where possible rather than compensation beads, as the autofluorescence profile of leukocytes from different species varies considerably [8, 12]. For avian samples, the high granularity of thrombocytes and heterophils can complicate light scatter gating, necessitating the use of lineage-specific markers such as CD45 or pan-leukocyte antigens to accurately identify lymphocyte populations [2, 15].

The gating strategy for viral infection studies typically begins with exclusion of doublets using FSC-A versus FSC-H and SSC-A versus SSC-H parameters, followed by viability gating using fixable amine-reactive dyes. For peripheral blood samples, the lymphocyte gate is defined by characteristic forward and side scatter properties, though this must be validated for each species. In cattle, the lymphocyte gate includes both small resting lymphocytes and larger activated blasts, which can be distinguished by increased forward scatter and granularity following viral stimulation [8, 12]. The identification of granulocytes and monocytes is particularly relevant for viruses that infect myeloid cells, such as Porcine Reproductive and Respiratory Syndrome Virus and African Swine Fever Virus, where viral antigen can be detected in CD172a+ or CD14+ populations [9, 17].

For intracellular antigen detection, such as the p80/125 protein of Bovine Viral Diarrhea Virus, fixation with paraformaldehyde followed by permeabilization with saponin or methanol-based buffers is required to allow antibody access to cytoplasmic epitopes [9]. The percentage of antigen-positive cells varies considerably depending on the virus and cell type; for BVDV, granulocytes and monocytes typically show 1-87% positivity with higher mean fluorescence intensity compared to lymphocytes, which exhibit 0-37% positivity with weaker per-cell fluorescence [9]. This heterogeneity underscores the importance of including appropriate isotype controls and fluorescence-minus-one (FMO) controls to establish accurate gates for antigen positivity.

High-dimensional data analysis using dimensionality reduction algorithms such as t-distributed stochastic neighbor embedding (t-SNE) or uniform manifold approximation and projection (UMAP) has become increasingly important for unraveling the complexity of immune responses to viral infections. These approaches have been applied to characterize natural killer cell phenotypes in HIV-infected chimpanzees, where traditional bivariate gating failed to capture the full repertoire of NK cell subsets [20]. The identification of NKG2D and NKp46 as the most stable NK cell markers across infection states was only possible through unbiased multidimensional analysis, highlighting the power of these computational approaches for veterinary immunology [20]. Similarly, factor analysis of mixed data (FAMD) has been employed to distinguish species-specific and infection-related immune response patterns in buffaloes and cattle infected with Mycobacterium bovis, revealing that IL-17A+ and IFN-γ+IL-17A+ CD4+ T-cell subsets contribute to interspecies differences that would be missed by conventional gating [12].

Specialized Applications: Flow Virometry and Viral Antigen Detection

Beyond cellular immunophenotyping, flow cytometry has been adapted for the direct detection and characterization of viral particles themselves, a technique termed flow virometry. This approach relies on the detection of fluorescently labeled virions as they pass through the laser interrogation point, enabling quantification of viral particle concentration, assessment of antigenic properties, and even sorting of individual virions for downstream characterization [14]. The successful application of flow virometry requires careful calibration using standardized beads of known size and fluorescence intensity, as viral particles are typically 50-200 nm in diameter, near the detection limit of conventional cytometers. Improvements in laser optics, photomultiplier tube sensitivity, and sheath fluid filtration have progressively enhanced the resolution of viral particle detection [14].

For veterinary applications, flow virometry has been used to study viral interference in insect cell lines persistently infected with dengue virus serotype 2, where heterotypic interference against other serotypes and homologous interference against yellow fever virus were quantified by plaque and flow cytometry assays [11]. The technique has also been applied to characterize the binding of fluorescently labeled dengue virus to monocytes and dendritic cells from dyslipidemic patients, revealing increased viral binding affinity associated with altered expression of entry receptors [19]. In the context of White Spot Syndrome Virus and Yellow Head Virus in crustaceans, flow virometry offers the potential for rapid, quantitative detection of viral particles in hemolymph samples, though the lack of species-specific antibodies remains a limitation.

The detection of viral antigens on the surface of infected cells provides complementary information to flow virometry. For Bovine Leukemia Virus, dual-color flow cytometry using monoclonal antibodies against CD markers and the gp51 envelope protein enables simultaneous identification of infected cell subsets and their phenotypic characterization [7]. This approach revealed that BLV-infected cows exhibit depleted CD4+ lymphocytes, augmented CD8+ lymphocytes, and proliferation of CD19+ IgM+ cells that are immature and show no tendency toward differentiation [7]. Such detailed immunophenotyping would be impossible with bulk assays and demonstrates the unique value of flow cytometry for understanding viral pathogenesis.

Functional Assays: Intracellular Cytokine Staining and Proliferation

The assessment of functional immune responses is critical for evaluating vaccine efficacy and understanding protective immunity against viral infections. Intracellular cytokine staining (ICS) following short-term in vitro stimulation with viral antigens enables the quantification of antigen-specific T-cell responses at the single-cell level. For veterinary species, the stimulation period must be optimized to balance cytokine induction with activation-induced cell death. Six-hour stimulation with protein antigens or peptide pools in the presence of brefeldin A or monensin is standard for detecting IFN-γ, TNF-α, and IL-2 production by CD4+ and CD8+ T cells [12]. The addition of co-stimulatory antibodies (anti-CD28 and anti-CD49d) enhances cytokine detection, particularly for weak antigens.

Polyfunctional T cells, defined by the simultaneous production of multiple cytokines, are associated with superior protective immunity against viral infections. In buffaloes and cattle exposed to M. bovis, multicolor flow cytometry enabled the identification of CD4+ T cells producing IFN-γ, TNF-α, and IL-17A simultaneously, with distinct patterns correlating with infection status [12]. For [Foot-and-Mouth Disease Virus](/knowledge

Molecular Pathogenesis and Immune Mechanisms Elucidated by Flow Cytometry

Flow cytometry has emerged as an indispensable tool for dissecting the complex interplay between viral pathogens and the host immune system at the single-cell level. In the context of veterinary viral immunology, multiparametric flow cytometric analysis enables the precise quantification and functional characterization of immune cell subsets, their activation states, and their dynamic responses to infection. This section provides an exhaustive analysis of how flow cytometry has elucidated molecular pathogenesis and immune mechanisms across a diverse spectrum of viral infections in animals, drawing upon cutting-edge studies that integrate cellular phenotyping, intracellular cytokine detection, and transcriptomic profiling.

T Cell Responses: Activation, Exhaustion, and Memory Differentiation

The characterization of T cell responses remains a cornerstone of flow cytometric analysis in viral immunology. In a seminal study on Infectious Hematopoietic Necrosis Virus (IHNV) in rainbow trout, the development and validation of monoclonal antibodies targeting CD4-1 and CD8β co-receptors enabled the first in situ visualization and quantification of T cell subsets following vaccination with a live attenuated mutant strain [6]. Flow cytometric analysis revealed that the attenuated mutant induced a rapid, robust, and synchronized expansion of both CD4-1+ and CD8β+ T lymphocytes in the spleen and head kidney, correlating with restricted viral replication and coordinated upregulation of Th1/CTL-signature genes including IL-2, IFN-γ, and perforin [6]. This study exemplifies how species-specific reagent development coupled with flow cytometry can unveil protective immune mechanisms that would otherwise remain hidden.

The phenomenon of T cell exhaustion has been extensively characterized using flow cytometry in chronic viral infections. Studies on Murine Cytomegalovirus (MCMV) infection have demonstrated that advanced age profoundly alters the antiviral T cell response, shifting immunity away from effective antigen-specific T cell responses toward inflammatory and innate pathways [24]. Using comprehensive flow cytometric panels including CD38, HLA-DR, and Ki-67, investigators demonstrated that aged mice exhibited reduced antigen-specific CD8+ T cell responses despite maintaining viral loads comparable to younger animals, while simultaneously displaying increased frequencies of non-antigen-specific bystander T cell activation [24]. This age-dependent immune imbalance was associated with heightened systemic inflammation, coagulopathy, and increased mortality, highlighting the critical importance of modeling advanced aging in preclinical viral studies.

In the context of Infectious Bronchitis Virus (IBV) infection in chickens, flow cytometric analysis of CD8+ T cells revealed that IBV infection promotes activation and differentiation toward a memory-like phenotype [2]. Splenocytes from IBV-infected chickens exhibited CD25high T cell populations with elevated expression of IL10 and IL12B, indicating T cell activation and regulatory immune responses. Transcriptomic analysis of sorted CD44+ CD8+ T cells demonstrated upregulation of IL6R, IL7R, and NFKB1 genes, consistent with a shift toward memory precursor and activated T cell status [2]. These findings provide critical insights into avian antiviral T cell immunity and support the development of T cell-based protective strategies targeting IBV variants.

Flow cytometry has also been instrumental in understanding the role of regulatory T cells (Tregs) and immune checkpoint molecules in viral pathogenesis. In a study of Infectious Bursal Disease Virus variants, investigators utilized multicolor flow cytometry to demonstrate that viral infection disrupts the balance between effector T cells and Tregs, contributing to immunopathology [5]. The co-expression of CD38 and HLA-DR on CD4+ and CD8+ T cells was significantly higher in patients with diffuse cutaneous leishmaniasis (a chronic parasitic infection with viral parallels), suggesting a mechanism of T cell activation followed by anergy or apoptosis [5]. This pattern of elevated activation markers without corresponding proliferation (Ki-67 negativity) is reminiscent of T cell exhaustion observed in chronic viral infections such as Bovine Leukemia Virus (BLV) infection.

The application of flow cytometry to study mucosal immune responses has provided unprecedented insights into tissue-resident memory T cell (TRM) biology. In a murine model of influenza A virus infection, high-dimensional flow cytometry coupled with single-cell RNA sequencing revealed that primary nasal viral infection induces coordinated stepwise changes in innate and adaptive immune cell subsets within the nasal mucosa [29]. Flow cytometric analysis identified a rare subset of Krt13+ nasal immune-interacting floor epithelial (KNIIFE) cells that concurrently increased with TRM-like cells, with the CXCL16-CXCR6 axis mediating interactions between these populations [29]. This study underscores the power of flow cytometry in mapping the spatiotemporal dynamics of immune cell interactions at mucosal surfaces.

Natural Killer Cells and Innate Lymphoid Cells in Antiviral Defense

Flow cytometry has revolutionized our understanding of natural killer (NK) cell biology in veterinary viral infections. A comprehensive study on Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) infection in pregnant gilts utilized flow cytometry to characterize uterine NK (uNK) cell subsets based on NKp46 expression levels [17]. Three distinct uNK cell populations were identified: NKp46high, NKp46int, and NKp46low. PRRSV infection did not significantly alter total NK cell counts but affected NK cell proliferation, with NKp46high subsets showing increased Ki-67 expression. Notably, an upregulation of perforin was observed in NKp46high cells, indicating potential immune responsiveness to PRRSV, while CD107a degranulation capacity was lower in fetal placenta uNK cells [17]. Critically, PRRSV infection led to suppression of IFN-γ expression in most uNK cell subsets, revealing a mechanism by which this virus compromises innate immunity at the maternal-fetal interface.

The role of NK cells in controlling orthopoxvirus infections has been elucidated through flow cytometric analysis of ectromelia virus infection in mice. The B22 family virulence factor C15, encoded by ectromelia virus, was found to target NK cell-mediated viral control early during infection [34]. Flow cytometry revealed that C15 exerts a replicative advantage as early as 32 hours post-infection, and surprisingly, significantly more IFNγ+ NK cells were detected at 48 hours post-infection with wild-type virus compared to the C15 deletion mutant [34]. This paradoxical finding suggests that C15 modulates NK cell responses in a complex manner, potentially through inhibition of cell-cell contacts between NK cells and infected cells, as confirmed by subsequent transcriptomic analyses.

In the context of SARS-CoV-2 infection, flow cytometry has been used to demonstrate that viral dose determines the magnitude and quality of NK cell responses. In K18-hACE2 transgenic mice, a high viral inoculum (1×105 PFU) induced strong host responses that did not recover until death, whereas a low inoculum (1×102 PFU) induced responses that resolved by 14 days post-infection [32]. Flow cytometric analysis showed that CD8+ T cell numbers continuously increased in low-dose infected lungs from 2 days post-infection, but this expansion was absent in high-dose infected lungs, suggesting that high viral loads impair adaptive immune activation and may contribute to severe disease outcomes [32].

Dendritic Cells and Antigen Presentation

Flow cytometric analysis of dendritic cell (DC) function has provided critical insights into the early events of viral infection. In Porcine Epidemic Diarrhea Virus (PEDV) infection, Toll-like receptor 2 (TLR2) signaling was shown to be essential for regulating DC function [26]. Using flow cytometry, TLR2 deficiency in bone marrow-derived dendritic cells (BMDCs) significantly reduced surface expression of CD40, CD86, and CD83 following PEDV infection, indicating defects in DC maturation and activation. Furthermore, IFN-γ expression was significantly downregulated in CD4+ and CD8+ T cells co-cultured with TLR2-deficient BMDCs, demonstrating impaired antigen-presenting function [26]. Transcriptomic analysis revealed that TLR2 deficiency led to aberrant activation of the non-canonical NF-κB signaling pathway, dysregulated MHC Class I/II gene expression, and enhanced apoptosis in PEDV-infected BMDCs [26]. These findings establish a critical role for TLR2 in bridging innate and adaptive immunity during coronavirus infection.

The development of subunit vaccines against PEDV has benefited from flow cytometric evaluation of DC activation. A study utilizing the Gram-positive enhancer matrix (GEM) surface-display system to present the PEDV spike protein demonstrated that particulate antigen complexes (GEM-S) significantly increased antigen-presenting cell numbers in lymph nodes and enhanced co-stimulatory molecule expression on DCs compared to soluble protein alone [25]. Flow cytometry revealed that GEM-S induced a higher IgG2a/IgG1 ratio and stronger intestinal mucosal IgA production, indicating a balanced Th1/Th2 response with mucosal homing properties [25].

B Cell Responses and Humoral Immunity

Flow cytometry has enabled detailed analysis of germinal center (GC) B cell responses following vaccination. In a study of Foot-and-Mouth Disease Virus (FMDV) dendrimer peptide vaccines, flow cytometric analysis of draining lymph nodes revealed that immunization with the B2T dendrimer (containing both B and T cell epitopes) enhanced GC formation and increased the proportion of IgG1-positive GC B cells [3]. Elevated levels of IgG1-secreting plasma cells in bone marrow were consistent with GC-derived long-lived plasma cell differentiation, highlighting the ability of this vaccine platform to potentiate antigen-specific immunity through coordinated B and T cell activation [3]. This study underscores the power of flow cytometry in tracking the differentiation trajectory of B cells from GC to plasma cell stages.

The detection of antigen-specific B cells at low frequencies has been revolutionized by oligonucleotide-tagged antigen assemblies combined with flow cytometric sorting and single-cell sequencing [27]. This approach enables the isolation of pools of B cells reactive to different antigens in parallel, with subsequent single-cell sequencing detecting oligonucleotide tags to map individual B cell reactivities to specific antigens. In studies of SARS-CoV-2 spike antigens and dengue virus, dual labeling using separately oligonucleotide- and fluor-tagged assemblies increased staining sensitivity sufficiently for exploratory detection of rare antigen-specific B cells in naturally exposed individuals [27]. This methodological advance has profound implications for understanding the breadth and specificity of humoral responses to complex viral antigens.

Flow cytometric analysis of Bovine Leukemia Virus (BLV) infection in naturally infected cows revealed marked alterations in B cell homeostasis [7]. The gp51 envelope protein was detected in both blood and milk lymphocytes, but the percentage of cells expressing gp51 in milk was much lower than in blood. Flow cytometry demonstrated a depleted number of CD4+ lymphocytes, an augmented number of CD8+ lymphocytes, a lower CD4+/CD8+ ratio, and a proliferation of CD19+ IgM+ B cells [7]. Critically, these proliferated B cells were characterized as immature, showing no tendency toward differentiation and exhibiting prolonged vitality. These findings provide mechanistic insights into the pathogenesis of enzootic bovine leukosis and the role of BLV in B cell dysregulation.

Innate Immune Recognition and Inflammatory Signaling

The role of pattern recognition receptors in viral recognition has been extensively characterized using flow cytometry. In the context of Avian Influenza Virus immunization, flow cytometric analysis revealed significant MR1 (MHC class I-related protein 1) upregulation on F4/80+ macrophages and CD11c+ dendritic cells in the peritoneal cavity, lungs, spleen, and blood at 24 hours post-immunization, sustained for at least 3 days [33]. This MR1 upregulation was notably reduced with heat-inactivated virus, highlighting a requirement for active viral replication, and could be recapitulated by poly(I:C) administration but not by Imiquimod, suggesting a role for TLR3 triggering in this setting [33]. Since MR1 presents bacterial metabolites to mucosal-associated invariant T (MAIT) cells, these findings suggest that anti-influenza vaccination may prime the immune system to combat secondary bacterial infections-a concept with significant implications for veterinary vaccine development.

Flow cytometry has also elucidated the role of inhibitory receptors in modulating antiviral immune responses. In Theiler's murine encephalomyelitis virus (TMEV) infection, CLEC12A (C-type lectin domain family 12 member A) signaling was shown to repress protective immune responses [28]. CLEC12A-deficient mice exhibited increased T cell sequestration in the brain, higher expression of pro-inflammatory cytokines (TNF-α, IL-1β) and antigen presentation genes (CD11c, CD80, MHC-I) during acute infection, leading to improved viral clearance in the hippocampus. Flow cytometric analysis revealed that CLEC12A deficiency activates splenic CD4+ and CD8+ T cells upon infection, and despite increased neuroinflammation, CLEC12A-deficient mice displayed less hippocampal damage with improved neuronal integrity [28]. These findings identify CLEC12A as a potential therapeutic target for enhancing antiviral immunity in neurotropic viral infections.

Viral Immune Evasion Strategies Revealed by Flow Cytometry

Flow cytometry has been instrumental in uncovering the molecular mechanisms by which viruses subvert host immune responses. The NS1 protein of Avian Influenza Virus has been shown to inhibit host interferon responses through multiple mechanisms, including suppression of RIG-I signaling and inhibition of TRIM25-mediated ubiquitination. Flow cytometric analysis of intracellular signaling pathways in infected cells has revealed that NS1 interacts with host proteins to block the activation of IRF3 and NF-κB, thereby impairing the induction of type I interferons and pro-inflammatory cytokines. This allows the virus to replicate to high titers before the host can mount an effective innate immune response, contributing to the high pathogenicity of certain avian influenza strains.

In the case of African Swine Fever Virus (ASFV), flow cytometry has demonstrated that the virus encodes multiple proteins that inhibit apoptosis and modulate host immune responses. ASFV infection leads to the downregulation of MHC Class I molecules on the surface of infected cells, thereby reducing recognition by cytotoxic T lymphocytes. Flow cytometric analysis of surface MHC Class I expression has been used to quantify this immune evasion mechanism and to evaluate the efficacy of vaccines that aim to restore antigen presentation. Additionally, ASFV inhibits the activation of NF-κB and the production of type I interferons, as demonstrated by flow cytometric analysis of intracellular cytokine production in infected macrophages.

The SARS-CoV-2 nucleocapsid (N) protein has been shown to drive inflammation and metabolic reprogramming through a NEAT1-dependent mechanism [22]. Flow cytometric analysis of reactive oxygen species (ROS) levels, mitochondrial membrane potential, and calcium overload in human bronchial epithelial cells stably expressing the N protein revealed that the N protein induces inflammatory responses, enhances LPS sensitivity, and triggers mitochondrial dysfunction, ER stress, and mitochondria-ER contact site (MAM) formation. The N protein promoted glycolytic reprogramming by upregulating key enzymes including GLUT1, HK2, and PKM2, with NEAT1 knockdown attenuating inflammation, glycolysis, and mitochondrial damage [22]. Mechanistically, NEAT1 silencing restored HK2-VDAC1 association and suppressed VDAC1 oligomerization, demonstrating a novel mechanism by which SARS-CoV-2 N protein exacerbates inflammation through glycolytic reprogramming and disruption of mitochondrial-ER homeostasis.

Myeloid Cell Responses and Inflammatory Pathology

Flow cytometry has provided detailed characterization of myeloid cell responses during acute viral infections. In murine cytomegalovirus (MCMV) infection, a genetic model of acute viral infection-induced tissue damage (M.H2 k/b mice) revealed increased infiltration of neutrophils to the marginal zones of the spleen, aligning with the appearance of necrosis at 2 days post-infection [31]. While TUNEL staining identified increased cell death within these clusters, apoptosis was reduced, suggesting an inflammatory death pathway in M.H2 k/b neutrophils. Multiplex ELISA identified increased IL-6 and TGF-β in the spleens of susceptible mice, and flow cytometric analysis demonstrated significant loss of marginal zone and red pulp macrophages beginning at 2 days post-infection and worsening over time [31]. The buildup of toxic lipid peroxidation byproduct 4HNE was detected early in MZMs and progressed through RPMs alongside the development of histopathology, identifying oxidative stress and macrophage loss as key drivers of tissue damage [31].

In the context of Sendai Virus infection in rats, flow cytometry has been used to study the impact of early-life stress on adult antiviral responses. Neonatal maternal separation (NMS) led to sex-specific effects on immune responses, with male NMS rats showing lower viral loads and reduced recruitment of neutrophils and inflammatory macrophages at 4 days post-infection compared to controls [30]. Both male and female NMS rats showed lower proportions of interstitial macrophages compared to their non-stressed counterparts, while proportions of CD8+ and CD4+ T lymphocytes were higher in male control rats than in female controls and reduced in male NMS rats [30]. These findings demonstrate the profound and lasting effects of developmental stress on antiviral immunity, with flow cytometry serving as a critical tool for dissecting the cellular basis of these differences.

Interferon Signaling and Antiviral States

Flow cytometric analysis of interferon signaling pathways has revealed fundamental mechanisms of antiviral immunity across diverse species. In yellowtail clownfish, the interferon-induced protein 44-like (IFI44L) homolog was identified and functionally characterized as a critical antiviral effector against Viral Hemorrhagic Septicemia Virus (VHSV) [23]. Flow cytometry revealed that AcIFI44L overexpression reduced VHSV-induced cytopathic effects and improved cell survival, while enhancing NF-κB-dependent luciferase activity and increasing NFκB1 (p50) and RELA (p65) transcript levels [14

Clinical Application and Diagnostic Performance in Veterinary Viral Diseases

The translation of flow cytometry from a research instrument to a diagnostic tool in veterinary virology has been a gradual but transformative process, driven by the need for rapid, quantitative, and multiparametric analysis of host-pathogen interactions. Unlike traditional methods such as virus isolation or endpoint PCR, flow cytometry offers the unique capacity to simultaneously assess viral antigen presence, cellular phenotype, and functional status at the single-cell level. This section critically examines the clinical application and diagnostic performance of flow cytometry across a spectrum of veterinary viral diseases, drawing on both established protocols and emerging technologies.

Direct Viral Detection and Antigen Quantification

The most direct clinical application of flow cytometry in veterinary virology is the detection of viral antigens within infected cells. This approach bypasses the need for culture amplification and provides a quantitative measure of viral burden. A landmark study in this domain demonstrated the utility of flow cytometry for the rapid detection of Bovine Viral Diarrhea Virus (BVDV) in viremic cattle. Using a pan-pestivirus monoclonal antibody specific for the p80/125 protein, the assay achieved 100% correlation with conventional virus isolation from 304 clinical blood samples [9]. The diagnostic performance was notable: the percentage of antigen-positive cells ranged from 1% to 87% in granulocytes and monocytes, and from 0% to 37% in lymphocytes, with the assay capable of detecting both low levels of antigen per cell and low percentages of antigen-positive cells [9]. Critically, the stability of the signal was robust; BVDV detection in leukocytes was not significantly influenced by blood freezing or storage at 8°C for up to six weeks, a practical advantage for sample transport from field settings [9]. This work established a foundational principle: flow cytometry can serve as a rapid (2-hour), objective, and reliable diagnostic method for identifying persistently infected (PI) animals, which are the primary reservoir for BVDV transmission.

The principle of direct antigen detection has been extended to other economically significant pathogens. For Bovine Leukemia Virus (BLV), flow cytometry has been employed to quantify expression of the gp51 envelope glycoprotein in both blood and milk lymphocytes from naturally infected cows [7]. This application is particularly valuable for understanding the pathogenesis of enzootic bovine leukosis, as it revealed a significantly lower percentage of gp51-positive cells in milk compared to blood, alongside a characteristic depletion of CD4+ lymphocytes and proliferation of immature CD19+ IgM+ B cells [7]. These findings provide a cellular correlate of the immunosuppression and lymphoproliferation that define BLV infection, offering a more nuanced diagnostic picture than serology alone.

In the context of emerging viral threats, flow cytometry has been instrumental in characterizing the cellular tropism and replication kinetics of novel pathogens. For instance, studies on Porcine Epidemic Diarrhea Virus (PEDV) have utilized flow cytometry to validate monoclonal antibodies targeting the spike protein, confirming their specificity for PEDV-infected cells and enabling the development of sensitive biosensors [4]. Similarly, the technique has been used to dissect the differential replication of Infectious Bronchitis Virus (IBV) variants in chicken macrophages, revealing that the QX strain establishes a productive infection while the M41 strain is restricted, a finding with direct implications for understanding tissue tropism and vaccine design [36].

Immunophenotyping and Functional Assessment of Antiviral Immunity

Beyond direct viral detection, the preeminent clinical strength of flow cytometry lies in its ability to profile the host immune response. This is critical for diagnosing immune-mediated pathology, monitoring vaccine efficacy, and understanding the mechanisms of protective immunity.

T Cell Responses in Livestock and Poultry

The characterization of T cell responses is central to evaluating antiviral immunity. In poultry, flow cytometry has been used to demonstrate that oral administration of a recombinant Lactobacillus plantarum strain co-expressing chicken interleukins (IL-2, IL-17B, IL-26) significantly enhances the CD4+/CD8+ lymphocyte ratio and cytokine production following IBV vaccination [15]. This provides a quantitative readout of vaccine adjuvant efficacy that is unattainable with serology alone. Furthermore, transcriptomic analysis of flow-sorted CD44+ CD8+ T cells from IBV-infected chickens revealed upregulation of genes associated with memory precursor and activated T cell status (e.g., IL6R, IL7R, NFKB1), suggesting that IBV infection drives a shift toward a memory-like phenotype [2]. This level of cellular resolution is essential for developing T cell-based protective strategies against antigenically variable viruses like IBV.

In mammalian livestock, multiparametric flow cytometry has been applied to differentiate infected from vaccinated animals (DIVA strategies) and to assess disease progression. For Bovine Tuberculosis (a bacterial disease with significant viral co-infection implications), two five-color panels were designed to identify helper, cytotoxic, activated (CD25+), and memory (CD45RO+) T cells in tuberculin-positive cattle [8]. This approach successfully detected overexpression of activation markers following antigen stimulation, demonstrating the feasibility of implementing complex immunophenotyping in a clinical veterinary setting despite the limited availability of fluorochrome-conjugated antibodies for bovine targets [8]. The same principle applies to viral infections; for example, the polyfunctionality of CD4+ T cells-simultaneously producing IFN-γ, TNF-α, and IL-17A-has been characterized in buffaloes and cattle exposed to Mycobacterium bovis, providing a high-resolution tool for discriminating infection status [12].

Natural Killer Cells and Innate Immunity

Flow cytometry is also indispensable for studying innate immune effectors. In a study of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) infection in pregnant gilts, three uterine NK (uNK) cell subsets were identified based on NKp46 expression [17]. The analysis revealed that PRRSV infection did not alter total uNK cell counts but specifically increased proliferation (Ki-67 expression) and perforin upregulation in the NKp46high subset, while simultaneously suppressing IFN-γ expression [17]. This functional dichotomy-enhanced cytotoxic potential coupled with suppressed cytokine production-provides a mechanistic explanation for the virus's ability to cause reproductive failure while evading immune clearance. Such detailed functional profiling is critical for designing interventions that restore protective NK cell functions.

Monitoring Vaccine Efficacy and Correlates of Protection

Flow cytometry has become a cornerstone for evaluating novel vaccine platforms, providing quantitative data on both humoral and cellular arms of the immune response.

mRNA and Subunit Vaccines

The development of mRNA vaccines for veterinary use has relied heavily on flow cytometry for validation. For an H3N8 Avian Influenza Virus mRNA vaccine, flow cytometry confirmed efficient expression of the hemagglutinin (HA) protein in transfected DF-1 cells, serving as a critical quality control step before proceeding to in vivo efficacy studies [39]. Similarly, a mRNA-LNP vaccine encoding the hemagglutinin of Canine Distemper Virus (CDV) was shown by flow cytometry to induce a significant increase in splenic CD4+ T cells in mice, indicating a Th1-skewed immune response [35]. In raccoon dogs, the same vaccine elicited high neutralizing antibody titers and conferred complete protection against lethal CDV challenge, with flow cytometry data providing the cellular correlate of this protection [35].

For subunit vaccines, flow cytometry is used to quantify antigen-presenting cell (APC) activation. A PEDV subunit vaccine based on the Gram-positive enhancer matrix (GEM-S) system was shown by flow cytometry to significantly increase the numbers of APCs in draining lymph nodes and enhance co-stimulatory molecule expression on dendritic cells, compared to soluble antigen alone [25]. This provided a mechanistic basis for the observed increase in systemic antibody titers and intestinal mucosal IgA, demonstrating the vaccine's ability to bridge innate and adaptive immunity.

Dendrimer Peptide and Virus-Like Particle Vaccines

The ability of flow cytometry to track rare cell populations is exemplified in studies of dendrimer peptide vaccines for Foot-and-Mouth Disease Virus (FMDV). Immunization with the B2T dendrimer peptide, which incorporates both B and T cell epitopes, led to enhanced germinal center (GC) formation and a significant increase in IgG1-positive GC B cells in draining lymph nodes, as quantified by flow cytometry [3]. Furthermore, elevated levels of IgG1-secreting B cells in the bone marrow were detected, consistent with the generation of long-lived plasma cells [3]. This level of detail is essential for understanding the durability of vaccine-induced immunity.

Virus-like particles (VLPs), such as those derived from Simian Virus 40, have been evaluated for their immunomodulatory properties using flow cytometry. Subcutaneous injection of SV40 VLPs in mice led to a significant increase in CD4+ T cells and NK cells in the spleen, without affecting immune cell populations in the lungs, liver, or kidneys [38]. This safety profile, combined with the demonstrated increase in TNF-α expression in the spleen, positions flow cytometry as a critical tool for assessing both the efficacy and biosafety of VLP-based immunotherapies.

Diagnostic Performance in Complex Disease Scenarios

Flow cytometry excels in scenarios where traditional diagnostics are confounded, such as in persistent infections, co-infections, or when assessing immune dysfunction.

Persistent Viral Infections

In a model of persistent FMDV infection, flow cytometry was used to identify a population of "bystander cells" that, while devoid of active viral replication, displayed enhanced proliferative capacity and increased susceptibility to infection [37]. This finding, which would be impossible to discern with bulk assays, revealed a critical mechanism for viral persistence: the creation of a permissive microenvironment that sustains the viral reservoir. Similarly, for Bovine Viral Diarrhea Virus, the ability of flow cytometry to detect low-level antigen expression in a small percentage of cells (as low as 1%) makes it uniquely suited for identifying PI animals that may have low-level viremia [9].

Immune Dysfunction and Immunosuppression

Flow cytometry is the gold standard for diagnosing viral-induced immunosuppression. In Bovine Leukemia Virus infection, the technique revealed a depleted number of CD4+ lymphocytes, an augmented number of CD8+ lymphocytes, a lower CD4:CD8 ratio, and a proliferation of immature CD19+ IgM+ B cells [7]. This immunophenotypic signature is diagnostic for the progressive stage of infection and correlates with the development of persistent lymphocytosis and lymphoma. The technique has also been applied to study the impact of environmental contaminants on antiviral immunity; for example, perfluorooctanoic acid (PFOA) exposure was shown by flow cytometry to significantly impair virus-specific CD8+ T cell responses and cytokine production (IFN-γ and TNF-α) following lymphocytic choriomeningitis virus infection, leading to increased viral load and liver injury [40].

Emerging Applications and Future Directions

The field is moving toward higher-dimensional analysis and integration with other modalities. The development of spectral flow cytometry panels for hamsters-including antibodies against CD44, CD62L, CD25, and CD69-is opening new avenues for studying SARS-CoV-2 pathogenesis in this critical animal model [13]. Furthermore, the combination of flow cytometry with single-cell RNA sequencing (scRNA-seq) allows for the isolation of rare antigen-specific B cells using oligonucleotide-tagged antigen assemblies, enabling the mapping of individual B cell reactivities to specific epitopes [27]. This approach has been used to profile B cell responses to SARS-CoV-2 and dengue virus, offering unprecedented resolution for vaccine development.

Finally, the concept of "flow virometry"-the analysis of individual viral particles by flow cytometry-represents a paradigm shift. This technique allows for the quantification, phenotyping, and sorting of virions based on surface antigen expression, enabling the discrimination of viral subpopulations and the correlation of surface characteristics with infectivity [14]. While still primarily a research tool, flow virometry holds immense potential for clinical diagnostics, particularly for rapidly characterizing emerging viral strains and assessing the antigenic match of vaccines.

Single-Cell Sorting and Monoclonal Antibody Discovery for Viral Pathogens

The isolation and characterization of monoclonal antibodies (mAbs) represent a cornerstone of modern veterinary virology, providing indispensable tools for viral antigen detection, serological diagnosis, therapeutic intervention, and structural biology. The integration of single-cell flow cytometric sorting with high-throughput molecular cloning has fundamentally transformed the mAb discovery pipeline, enabling the rapid generation of fully native antibody repertoires directly from antigen-engaged B lymphocytes. This technological paradigm shift bypasses the stochastic constraints of traditional hybridoma fusion and permits the interrogation of the complete humoral immune response with unprecedented precision. For veterinary pathogens-where species-specific reagents are chronically scarce and the immunological context often diverges substantially from murine or human models-this methodology carries particular transformative promise.

Methodological Framework for Single B-Cell Cloning

The foundational principle of contemporary single-cell mAb discovery rests upon the physical isolation of individual antigen-specific B cells from lymphoid tissues or peripheral blood, followed by the recovery and expression of their cognate immunoglobulin genes. The workflow typically commences with the generation of a recombinant viral antigen, often the ectodomain of a critical surface glycoprotein, expressed in a eukaryotic system to ensure appropriate post-translational modifications. For instance, Jia et al. (2025) constructed a pCAGGS eukaryotic expression plasmid encoding the C-terminal domain (CTD) of the Transmissible Gastroenteritis Virus spike protein, expressed and purified the recombinant protein from HEK-293F suspension cells, and used this immunogen to immunize BALB/c mice [1]. Upon confirmation of high serum titers (1:10⁵), splenocytes were harvested and antigen-specific memory B cells were isolated using fluorescence-activated single-cell sorting (FACS) [1]. This approach yielded 83 cognate heavy- and light-chain gene pairs from a single sorting experiment, a yield that would be logistically challenging through conventional hybridoma screening.

The isolation of antigen-specific B cells hinges upon the use of fluorescently labeled antigen probes. These probes must be meticulously validated for affinity and conformational integrity, as subtle denaturation artifacts can redirect the specificity of recovered antibodies toward irrelevant or linear epitopes. The sorting strategy typically gates on viable, single, CD19⁺ or CD138⁺ (plasma cell) lymphocytes, with antigen binding detected via a fluorophore-conjugated version of the target immunogen. The use of dual, non-competing labels (e.g., fluorophore-conjugated antigen versus a streptavidin-phycoerythrin detection system) can further reduce false-positive rates, a consideration particularly important when rare B cell specificities-such as those directed against weakly immunogenic viral proteins-are being sought.

Single-Cell RT-PCR and Variable Gene Recovery

Once individual B cells are deposited into 96- or 384-well plates containing lysis buffer and RNase inhibitors, reverse transcription and nested PCR amplification are performed using pools of primers designed to anneal to conserved framework regions within the immunoglobulin variable domains. The success of this step is critically dependent upon primer design; the IGHV and IGKV/IGLV germline repertoires of the target species must be well-characterized. For laboratory mice and rats, this poses little difficulty, but for veterinary species such as swine, cattle, or poultry, the availability of comprehensive primer sets remains limited. In the aforementioned TGEV study, the recovered paired-chain amplicons were cloned into eukaryotic expression vectors (pCAGGS) containing murine constant region domains (CL and CH), thereby generating full-length chimeric antibodies [1]. Transfection of these plasmids into HEK-293T cells resulted in the secretion of functional IgG into the supernatant within 48 hours, enabling rapid downstream screening [1].

Importantly, the single-cell methodology preserves the natural heavy-light chain pairing, which is frequently lost in phage display libraries or hybridoma fusions where chain promiscuity can yield antibodies with poor specificity or affinity. This preservation of native pairing is particularly relevant for antibodies targeting conformational epitopes on viral spike proteins, where the correct paratope orientation is essential for neutralization. Li et al. (2026) extended this paradigm to a teleost species, developing anti-CD4-1 and anti-CD8β monoclonal antibodies for rainbow trout to study immune responses to live attenuated Infectious Hematopoietic Necrosis Virus [6]. This work underscores that the single-cell sorting platform is not restricted to mammalian systems; with appropriate species-specific reagents, it can be adapted for any vertebrate with a characterized adaptive immune system.

Functional Validation and Neutralizing Activity

The successful expression of recombinant antibodies necessitates rigorous functional screening. Enzyme-linked immunosorbent assay (ELISA) against the recombinant antigen is a standard first pass, but this must be complemented by assays that confirm recognition of native viral proteins in their biological context. Indirect immunofluorescence assay (IFA) on infected cells provides critical validation, as it tests the antibody's ability to recognize viral glycoproteins in their native membrane-embedded, oligomeric state. In the TGEV study, five of eighteen ELISA-reactive clones were confirmed positive by IFA, suggesting that a substantial fraction of recovered antibodies may recognize epitopes that are occluded or structurally altered on the virion surface [1]. The final and most stringent validation is the virus neutralization assay, wherein antibodies are tested for their capacity to inhibit viral replication in permissive cells. All five IFA-positive antibodies in this study demonstrated neutralization activity against TGEV in swine testicular (ST) cells, highlighting the fidelity of the single-cell platform in recovering functionally relevant antibodies [1].

The identification of neutralizing epitopes carries immediate translational significance. For viral pathogens such as Porcine Epidemic Diarrhea Virus, which causes catastrophic neonatal diarrhea, neutralizing mAbs can inform the design of epitope-focused vaccines or provide passive immunotherapeutic reagents. A recent study by Li et al. (2026) generated a PEDV spike protein-specific mAb (isotype IgG1, κ light chain) that recognized a linear epitope spanning amino acids 696-715, localized on the surface of the spike protein's three-dimensional structure [4]. This mAb was subsequently exploited to construct a novel electrochemical immunosensor by immobilizing the antibody onto gold nanoparticle-modified screen-printed electrodes via protein A coupling [4]. The resulting sensor demonstrated high specificity for PEDV in clinical manure samples, with quantification based on changes in charge transfer resistance upon antigen binding [4].

Applications in Antigenic Profiling and Reagent Creation

Beyond diagnostic assay development, single-cell-derived mAbs are invaluable for dissecting the antigenic architecture of complex viral particles. The ability to generate a panel of mAbs targeting distinct epitopes on a single viral protein enables the construction of epitope maps, which can reveal antigenic drift over time and guide vaccine strain selection. This is particularly critical for RNA viruses with high mutation rates, such as Avian Influenza Virus and Infectious Bronchitis Virus. Wu et al. (2025) developed an mRNA-LNP vaccine encoding the hemagglutinin of H3N8 Avian Influenza Virus and confirmed protein expression in vitro using flow cytometry of transfected cells, though mAb generation against this specific subtype could further refine epitope-specific immunity [39].

The scarcity of immunological reagents for veterinary species-particularly for camelids, teleosts, and avian species-represents a major bottleneck in veterinary immunology. Single-cell mAb discovery offers a direct route to overcome this limitation. The generation of mAbs against CD4-1 and CD8β in rainbow trout [6], and the development of panels for cytokines and chemokines in hamsters [13], exemplify the expanding taxonomic reach of this technology. For example, monoclonal antibodies against hamster CD44, CD62L, CD25, and CD69 were validated by flow cytometry on activated splenocytes, enabling the tracking of T-cell activation dynamics in a species critical for modeling SARS-CoV-2 and other zoonotic infections [13]. These tools are now enabling the first comprehensive immunological studies in these non-traditional model organisms.

Advanced Multiplexing and Single-Cell Sequencing Integration

Recent innovations have married single-cell sorting of antigen-specific B cells with oligonucleotide-tagged antigen assemblies and next-generation sequencing, enabling the simultaneous profiling of B-cell reactivities against multiple viral antigens within a single experiment. Apps et al. (2025) demonstrated that B cells reactive to different SARS-CoV-2 spike variants could be isolated from human peripheral blood using fluor- and oligonucleotide-tagged antigen probes, with the oligonucleotide barcodes subsequently read out via single-cell RNA sequencing [27]. This workflow preserves the capacity for paired heavy- and light-chain recovery while simultaneously providing the full transcriptome and epitope specificity for each cell. The approach is directly translatable to veterinary virology, where it could be applied to simultaneously probe B-cell responses against, for example, the structural and non-structural proteins of Classical Swine Fever Virus or the envelope proteins of Bovine Leukemia Virus [7, 41].

The optimization of antigen:streptavidin ratios for novel viral antigens is a critical technical consideration, as imbalanced stoichiometry can lead to the formation of macromolecular complexes that trigger B-cell activation through BCR crosslinking rather than specific antigen binding, introducing artefactual sorting results [27]. For rare specificities-such as B cells recognizing conserved epitopes on the Foot-and-Mouth Disease Virus VP1 protein-the combination of dual labeling with both oligonucleotide and fluor tags increased detection sensitivity sufficiently to enable the identification of dengue-reactive cells in naturally exposed individuals, a strategy directly applicable to veterinary pathogens with low seroprevalence [3, 27].

Challenges and Future Directions

Despite its transformative potential, the single-cell mAb discovery platform faces several persistent challenges in the veterinary context. The cost of high-speed cell sorters and single-cell sequencing remains prohibitive for many diagnostic and research laboratories. The requirement for species-specific immunoglobulin PCR primers necessitates significant upfront investment for each new target species, though cross-reactive primer sets for conserved framework regions are gradually being developed. Furthermore, the antibody expression systems (typically HEK-293T or CHO cells) may not faithfully replicate the glycosylation patterns of the target species, potentially altering Fc-mediated effector functions such as antibody-dependent cellular cytotoxicity (ADCC) or complement activation. For therapeutic applications in target species-such as passive immunization of piglets against Porcine Epidemic Diarrhea Virus-the production of full-species chimeric or fully species-matched antibodies will be necessary to avoid anti-antibody responses.

Emerging technologies, including flow virometry for the direct sorting of intact viral particles based on antigenicity, may further refine the selection of mAbs with the highest affinity for native virions [14]. The ability to sort single viral particles and recover their genomic RNA for sequencing offers a complementary approach to epitope discovery that bypasses the B-cell intermediate entirely. Additionally, the integration of machine learning algorithms to predict antibody-antigen complementarity from sequence data promises to accelerate the candidate selection pipeline, reducing the need for exhaustive wet-lab screening. For veterinary virology, the continued democratization of single-cell technologies will be essential to expand the toolkit available for combating emerging and re-emerging viral pathogens across all animal taxa, from White Spot Syndrome Virus in crustaceans to Marek's Disease Virus in poultry.

Multiparametric Immunophenotyping in Vaccine Development and Evaluation

The advancement of veterinary vaccinology has been profoundly shaped by the capacity of multiparametric flow cytometry to dissect the nuanced cellular dynamics that underpin protective immunity. In the context of viral pathogens, where correlates of protection often extend beyond mere serological titers to encompass intricate networks of T-cell, B-cell, and innate lymphoid cell responses, the ability to simultaneously interrogate multiple phenotypic and functional parameters at the single-cell level is indispensable. This section provides an exhaustive examination of how high-dimensional immunophenotyping serves as a cornerstone for rational vaccine design, preclinical evaluation, and post-marketing surveillance in veterinary species.

Dissecting Vaccine-Induced T-Cell Responses: Activation, Memory, and Polyfunctionality

The evaluation of T-cell immunity is arguably one of the most critical applications of multiparametric flow cytometry in vaccine development. Classical phenotypic markers-such as CD4, CD8, CD44, CD62L, and CD45RA/RO-allow the segregation of naive, central memory (TCM), effector memory (TEM), and tissue-resident memory (TRM) subsets, each of which plays a distinct role in antiviral protection. For instance, in a landmark study evaluating a live attenuated vaccine candidate against Infectious Hematopoietic Necrosis Virus (IHNV) in rainbow trout, the development of novel monoclonal antibodies against CD4-1 and CD8β enabled the first in situ visualization of T-cell subset dynamics within melanomacrophage centers [6]. Multiparametric analysis revealed that the attenuated mutant rIHNVMut, in stark contrast to the wild-type strain, elicited a rapid, robust, and synchronized expansion of both CD4-1⁺ and CD8β⁺ T lymphocytes in the spleen and head kidney, temporally linked with upregulated Th1/CTL-signature genes (IL-2, IFN-γ, Prf1) and a sustained IgM humoral response [6]. This study exemplifies how species-specific reagent development, coupled with a multiparametric panel, can mechanistically link vaccine safety (restricted replication) with immunogenicity (coordinated cellular and humoral activation).

Furthermore, the assessment of T-cell activation status through the co-expression of markers such as CD25 (IL-2Rα), CD69, CD38, and HLA-DR provides a window into the functional quality of the vaccine response. In the context of Infectious Bronchitis Virus (IBV) in chickens, in vitro splenocyte infection with the K047-12 strain induced CD25high T cells, and subsequent transcriptomic analysis of sorted CD44⁺CD8⁺ T cells revealed upregulation of IL6R, IL7R, and NFKB1, suggesting a shift toward a memory-like transcriptional program [2]. These findings are directly translatable to vaccine evaluation; for example, when a recombinant Lactobacillus plantarum strain co-expressing chicken IL-2, IL-17B, and IL-26 was orally co-administered with an IBV vaccine, flow cytometry demonstrated significantly elevated CD4⁺/CD8⁺ T-cell ratios in peripheral blood, correlating with reduced viral loads upon challenge [15]. This underscores the power of multiparametric panels to validate novel adjuvant strategies by quantifying shifts in the T-cell compartment.

Beyond mere enumeration, polyfunctionality-the simultaneous production of multiple cytokines (e.g., IFN-γ, TNF-α, IL-2) by a single T cell-has emerged as a superior correlate of protective efficacy. In bovine tuberculosis, a multicolor flow cytometry panel was established to quantify IFN-γ, TNF-α, and IL-17A production in CD4⁺ T cells from cattle and water buffaloes following stimulation with mycobacterial antigens [12]. This approach not only discriminated infected from non-infected animals but also revealed subtle interspecies differences in the frequency of IL-17A⁺ and double-positive (IFN-γ⁺IL-17A⁺) subsets, providing critical benchmarks for evaluating candidate tuberculosis vaccines [12]. For viral vaccines, the relevance of polyfunctionality is equally profound. In a study on a dendrimer peptide vaccine against Foot-and-Mouth Disease Virus (FMDV), immunization with the B2T construct (containing both B- and T-cell epitopes) elicited robust CD4⁺ T-cell responses in the spleen, which were essential for driving germinal center (GC) reactions and the differentiation of IgG1-secreting long-lived plasma cells in the bone marrow [3]. Here, flow cytometry was instrumental in quantifying GC B cells (GL7⁺Fas⁺) and IgG1⁺ plasmablasts in draining lymph nodes and bone marrow, directly linking the T-cell epitope to the durability of the humoral response [3].

B-Cell Immunophenotyping and Germinal Center Dynamics

The humoral arm of the adaptive immune response, traditionally assessed by endpoint ELISA or virus neutralization assays, is now routinely deconstructed at the cellular level using multiparametric flow cytometry. The identification of antigen-specific B cells, plasmablasts, and plasma cells is critical for understanding the longevity and breadth of vaccine-induced antibody responses. For instance, in the development of a subunit vaccine against Porcine Epidemic Diarrhea Virus (PEDV) using the Gram-positive enhancer matrix (GEM) display system, flow cytometry was employed to quantify antigen-presenting cell (APC) numbers and co-stimulatory molecule expression (CD40, CD80, CD86) on dendritic cells in draining lymph nodes [25]. This analysis revealed that the particulate GEM-S formulation, but not soluble S protein, robustly upregulated these activation markers, correlating with a higher IgG2a/IgG1 ratio and stronger intestinal mucosal IgA production-a key correlate of protection against enteric viruses [25].

The power of modern flow cytometry is further amplified by the integration of oligonucleotide-tagged antigen assemblies, enabling the simultaneous detection of B cells specific for multiple epitopes or even different pathogens within a single sample. This technology, known as antigen tetramer or oligomer staining combined with single-cell sequencing, allows for the precise isolation of rare B-cell clonotypes and the mapping of their reactivity to specific antigens [27]. In the context of veterinary vaccine development against rapidly mutating viruses such as Avian Influenza Virus, this method could be transformative, permitting the longitudinal tracking of the B-cell repertoire in response to vaccination and boosting, and the identification of clones targeting conserved, broadly neutralizing epitopes. Moreover, the assessment of germinal center (GC) reactions using markers such as GL7, Fas (CD95), and Ki-67, in conjunction with B-cell lineage markers (B220, CD19, IgD), provides a dynamic readout of the affinity maturation process. As demonstrated in the FMDV dendrimer peptide study, the B2T vaccine significantly enhanced GC formation and increased the proportion of IgG1-positive GC B cells in draining lymph nodes, a finding that was only accessible through high-parameter flow cytometry [3]. This level of resolution is essential for differentiating vaccines that merely induce a transient antibody response from those that establish long-term humoral memory through the generation of long-lived plasma cells in the bone marrow.

Innate Lymphoid Cells: NK Cells, MAIT Cells, and the Adjuvant Effect

While much attention in vaccinology focuses on adaptive immunity, the innate immune system, particularly natural killer (NK) cells and unconventional T cells like mucosal-associated invariant T (MAIT) cells, plays a crucial role in shaping the early response to vaccination and the subsequent development of adaptive memory. Multiparametric flow cytometry has been pivotal in characterizing these populations in veterinary species. For example, in the evaluation of an mRNA-LNP vaccine encoding the hemagglutinin of Canine Distemper Virus (CDV) in raccoon dogs, the vaccine induced strong H-specific IgG responses and increased splenic CD4⁺ T cells, but the study also highlighted the potential for further investigation into NK-cell involvement [35]. In swine, the functional properties of uterine NK (uNK) cells during Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) infection have been dissected using panels including NKp46, Ki-67, perforin, CD107a, and IFN-γ [17]. This analysis revealed that PRRSV infection did not alter total uNK counts but did affect proliferation and perforin expression within the NKp46high subset, while suppressing IFN-γ expression-findings that have direct implications for the development of mucosal vaccines that must function within the unique immunological milieu of the pregnant uterus [17].

The role of MAIT cells, which recognize MR1-presented microbial metabolites, is an emerging frontier in vaccine immunology. In a murine model of influenza A virus immunization, flow cytometry demonstrated significant upregulation of MR1 on F4/80⁺ macrophages and CD11c⁺ dendritic cells in multiple tissues, and this was dependent on active viral replication and TLR3 signaling [33]. The activation of MAIT cells, measured by CD38 and HLA-DR expression upon exposure to the virus, suggests that vaccines capable of stimulating this innate-like T-cell population could provide rapid, non-MHC-restricted protection against both the target virus and secondary bacterial infections [33]. Incorporating MR1 tetramers and activation markers into veterinary vaccine evaluation panels, particularly in species such as pigs and cattle, which are natural hosts for numerous respiratory pathogens, could yield novel insights into the mechanisms of vaccine-induced heterologous immunity.

Furthermore, the antigen-independent priming of innate immunity using novel adjuvants can be rigorously monitored by flow cytometry. For example, the synthetic compound n-dodecyl-β-D-maltoside (DDM) was shown to provide broad prophylaxis against bacterial sepsis and viral infection in mice via rapid neutrophil recruitment and activation, without inducing systemic inflammation [42]. Flow cytometry was central to these findings, quantifying neutrophil infiltration, phagocytic activity, and the expression of effector genes, while neutrophil depletion experiments confirmed the central role of this cell type [42]. Such approaches underscore the expanding role of multiparametric immunophenotyping in evaluating host-directed prophylactic strategies.

Integrating Cellular and Humoral Correlates: The Example of Vaccines Against Aquaculture Pathogens

The application of multiparametric flow cytometry is particularly critical in aquaculture vaccinology, where the immune system of teleost fish diverges significantly from that of mammals, yet the principles of vaccine evaluation remain analogous. In a study of a live attenuated vaccine against IHNV, the development of anti-CD4-1 and anti-CD8β monoclonal antibodies was a monumental step forward, as it allowed for the first direct quantification of T-cell dynamics in fish lymphoid tissues [6]. This study demonstrated that the vaccine's safety (restricted replication) was inversely correlated with a robust expansion of T-cell subsets, providing a mechanistic correlate of protection that could not be obtained through viral load measurements alone [6]. Similarly, in the development of a multi-epitope vaccine against hirame novirhabdovirus (HIRRV) in Japanese flounder, flow cytometry was used to track the proliferation of CD4⁺ T and mIgM⁺ B lymphocytes in peripheral blood, spleen, and head kidney [16]. The vaccine, rGBLE, induced a delayed but sustained CD4-2⁺ T-cell response and elevated mIgM⁺ B-cell levels, which correlated with higher neutralizing antibody titers and a relative percent survival (RPS) of 85.2% following viral challenge [16]. These examples highlight how multiparametric immunophenotyping is not merely descriptive but can identify specific cellular subsets that serve as reliable correlates of protection, thereby guiding the iterative refinement of vaccine candidates.

Multiparametric Panels for Veterinary Species: Technical Considerations and Future Directions

The expansion of multiparametric flow cytometry into veterinary vaccinology has historically been constrained by the limited availability of species-specific, fluorochrome-conjugated monoclonal antibodies. However, recent efforts have begun to address this gap. For example, a study successfully designed and validated two five-color panels for the identification of T-cell subpopulations (CD4, CD8, CD25, CD45RO, and γδ TCR) in tuberculin-positive cattle, which successfully detected increased frequencies of activated (CD25⁺) and memory (CD45RO⁺) T cells [8]. The authors emphasized that similar strategies could be implemented for other species of veterinary interest, provided that cross-reactive or species-specific antibodies are available [8]. The development of new monoclonal antibodies for hamsters [13], a critical model for SARS-CoV-2, and for rainbow trout [6] exemplifies a growing trend toward reagent development for non-traditional model species. Moreover, spectral flow cytometry, which captures the full emission spectrum of each fluorochrome, offers a path to expand panel size beyond the conventional limit of 18-20 colors, enabling the simultaneous detection of 30 or more parameters. This advancement will be crucial for integrating the analysis of multiple T-cell subsets, B-cell differentiation stages, NK-cell activation states, and myeloid cell populations into a single tube, maximizing the information yield from precious clinical samples.

Finally, the coupling of immunophenotyping with functional assays, such as intracellular cytokine staining (ICS) after antigenic stimulation or the use of antigen-specific tetramers, adds a critical layer of granularity. For instance, in a study on breakthrough influenza infection in vaccinated mice, flow cytometry was used to characterize a transient lung eosinophilia characterized by a Siglec-Fhi subset, which was associated with a balanced Type 1/2 immune response and rapid viral clearance, in stark contrast to the pathological eosinophilia seen in vaccine-enhanced respiratory disease [43]. This level of phenotypic and functional resolution is essential for ensuring that novel vaccines do not induce immunopathology. As the field moves toward the development of multivalent, bionanoparticle, and mRNA-based vaccines for veterinary applications, multiparametric immunophenotyping will remain the gold standard for deconvoluting the complex cellular symphony that constitutes a protective immune response.

Data Analysis, Quality Control, and Emerging Technologies in Veterinary Flow Cytometry

The translation of flow cytometry from a specialized research instrument into a robust diagnostic and analytical tool for veterinary viral immunology demands rigorous attention to data analysis pipelines, quality control (QC) protocols, and the integration of emerging technologies. Unlike human clinical flow cytometry, which benefits from decades of standardized reagent development and automated analysis algorithms, veterinary applications must contend with species-specific immunological idiosyncrasies, a relative paucity of validated cross-reactive monoclonal antibodies, and the inherent variability of samples obtained from diverse animal populations under field conditions. This section provides an exhaustive examination of the analytical frameworks, QC imperatives, and technological innovations that underpin reliable flow cytometric investigation of antiviral immune responses in veterinary species.

Foundational Principles of Data Analysis in Veterinary Flow Cytometry

The analytical workflow for flow cytometry data in veterinary viral immunology begins with rigorous pre-processing, including compensation, transformation, and quality filtering. Compensation matrices must be meticulously established for each multicolor panel, as spectral overlap between fluorochromes-particularly when using legacy dyes like FITC and PE alongside newer polymers-can introduce substantial artifacts if not properly corrected. In studies of Bovine Leukemia Virus infection, for example, dual-color flow cytometry required careful compensation to resolve gp51 expression on CD4+ and CD8+ lymphocyte subsets, as the viral envelope protein exhibited variable fluorescence intensity across different cell populations [7]. The choice of transformation algorithm (e.g., logicle, biexponential, or hyperbolic arcsine) is equally critical, particularly when analyzing populations with extreme dynamic ranges, such as the highly granular neutrophils and monocytes that are primary targets for Bovine Viral Diarrhea Virus antigen detection [9].

Gating strategies must be species-appropriate and validated against known biological controls. For bovine immunophenotyping, the development of five-color panels targeting CD4, CD8, CD25, CD45RO, and γδ TCR required iterative optimization to resolve memory and activated T cell subsets in tuberculin-positive cattle [8]. Similarly, in rainbow trout, the recent generation of monoclonal antibodies against CD4-1 and CD8β enabled the first in situ visualization of T-cell subsets within melanomacrophage centers, but the gating strategy had to account for the unique light scatter properties of fish leukocytes, which differ substantially from mammalian lymphocytes [6]. The use of fluorescence-minus-one (FMO) controls is non-negotiable in veterinary studies, as the absence of species-specific isotype controls for many fluorochrome-conjugated antibodies necessitates alternative approaches to define positivity thresholds.

Multidimensional Data Reduction and Unsupervised Analysis

As veterinary flow cytometry panels expand beyond the traditional 4-6 colors to 12-18 parameters, manual biaxial gating becomes increasingly untenable. High-dimensional data reduction techniques, including t-distributed stochastic neighbor embedding (t-SNE), uniform manifold approximation and projection (UMAP), and principal component analysis (PCA), have been successfully applied to decipher complex immune landscapes in viral infections. In a seminal study of NK cell phenotypes in HIV-infected chimpanzees, multidimensional analysis using t-SNE and SPADE (spanning-tree progression analysis of density-normalized events) revealed that NKG2D and NKp46 were the most stable NK cell markers, while traditional markers such as CD8α, CD16, and perforin fluctuated dynamically during infection [20]. This unbiased approach corrected a long-standing misconception that chimpanzee NK cells phenotypically mirror human NK cells, demonstrating instead that CD56 expression is not a reliable lineage marker in this species.

The application of factor analysis of mixed data (FAMD) has proven particularly valuable for comparing immune responses across closely related species. In a comparative study of Bovine Tuberculosis in cattle and water buffalo, FAMD enabled the simultaneous integration of continuous variables (cytokine frequencies) and categorical variables (species, infection status) to identify that IL-17A+ and TNF-α+IL-17A+ CD4+ T cell subsets were the primary drivers of interspecies immunological differences [12]. This approach is far more powerful than univariate comparisons, as it preserves the multivariate structure of the data and can reveal subtle but biologically meaningful population shifts that would otherwise be obscured by traditional gating.

Quality Control Protocols: From Sample Acquisition to Data Integrity

The reliability of flow cytometric data in veterinary viral immunology is contingent upon rigorous QC at every stage of the workflow. Pre-analytical variables, including sample collection, anticoagulant choice, storage temperature, and transport duration, can dramatically impact cell viability and antigen expression. For detection of Bovine Viral Diarrhea Virus in viremic cattle, it was demonstrated that leukocyte-associated viral antigen remained detectable after blood freezing or storage at 8°C for up to six weeks, with no significant loss of signal intensity [9]. This remarkable stability is not universal; for Porcine Reproductive and Respiratory Syndrome Virus studies, uterine NK cell functionality, as measured by CD107a degranulation and IFN-γ expression, is exquisitely sensitive to delays in processing and requires immediate isolation and cryopreservation [17].

Instrument QC must include daily calibration with standardized beads to ensure consistent fluorescence intensity and light scatter resolution across experiments. The use of rainbow calibration particles or equivalent standards is essential for longitudinal studies, such as those tracking the evolution of T cell responses following vaccination against Infectious Bronchitis Virus [2, 15]. For flow virometry-the detection and characterization of individual viral particles-specialized QC protocols are required, including the use of size-calibrated fluorescent beads (e.g., 100 nm, 200 nm, and 500 nm) to establish the detection threshold for viral particles, which are typically an order of magnitude smaller than cells [14]. The recent development of electrochemical immunosensors for Porcine Epidemic Diarrhea Virus detection, which rely on monoclonal antibodies validated by flow cytometry, underscores the importance of QC in ensuring that antibody-based reagents perform consistently across different assay platforms [4].

Emerging Technologies: Spectral Flow Cytometry and Mass Cytometry

The advent of spectral flow cytometry represents a paradigm shift for veterinary immunology, as it circumvents many of the limitations imposed by the limited availability of species-specific fluorochrome-conjugated antibodies. Unlike conventional flow cytometry, which measures the peak emission of each fluorochrome, spectral cytometry captures the full emission spectrum across multiple detectors, allowing for the unmixing of fluorochromes with highly overlapping spectra. This technology enables the use of 30+ parameters simultaneously, which is particularly advantageous for veterinary species where antibody panels must often rely on cross-reactive reagents with suboptimal fluorochrome pairings. In studies of Avian Influenza Virus vaccination, spectral flow cytometry has been used to simultaneously track CD4+, CD8+, γδ T cells, B cells, monocytes, and dendritic cells, along with multiple intracellular cytokines and activation markers, from a single sample [33, 39].

Mass cytometry (CyTOF), which uses heavy metal isotope-tagged antibodies rather than fluorochromes, eliminates the need for compensation entirely and can theoretically measure up to 50 parameters without spectral overlap. While the high cost and limited availability of metal-conjugated antibodies for veterinary targets have restricted its adoption, pioneering studies in non-human primates have demonstrated its utility for deep immune profiling during Simian Immunodeficiency Virus infection [44]. The integration of mass cytometry with single-cell RNA sequencing (scRNA-seq) represents the cutting edge of multi-omics integration, enabling simultaneous transcriptomic and proteomic characterization of antiviral immune responses at single-cell resolution.

Single-Cell Sequencing and Multi-Omics Integration

The convergence of flow cytometry with single-cell genomics is revolutionizing our understanding of host-virus interactions in veterinary species. Fluorescence-activated cell sorting (FACS) enables the isolation of phenotypically defined cell populations for downstream scRNA-seq, providing unprecedented resolution of cellular heterogeneity. In a study of persistent Foot-and-Mouth Disease Virus infection, integrated multi-omics analysis of FACS-sorted bystander cells-those devoid of active viral replication-revealed enhanced proliferative capacity and a distinct microenvironmental signature that potentially increases viral susceptibility, thereby sustaining virus persistence within the cell population [37]. This finding would have been impossible to obtain through bulk analysis, as the bystander cell phenotype was masked by the dominant signal from productively infected cells.

The application of oligonucleotide-tagged antigen assemblies combined with single-cell sequencing has enabled multiplexed proteogenomic profiling of B cell reactivities, allowing the simultaneous mapping of antigen specificity, B cell receptor sequence, and transcriptomic state from thousands of individual cells [27]. This technology has direct applications for veterinary vaccine development, as it can identify rare B cell clones with neutralizing activity against viruses such as Porcine Transmissible Gastroenteritis Virus [1] and Avian Influenza Virus [39]. The recent generation of monoclonal antibodies against hamster cytokines and T cell activation markers [13] and against rainbow trout CD4-1 and CD8β [6] demonstrates that the reagent gap for veterinary species is gradually closing, enabling more sophisticated single-cell analyses.

Artificial Intelligence and Machine Learning in Data Interpretation

The exponential increase in data dimensionality from modern flow cytometry platforms demands computational approaches that can identify patterns beyond human cognitive capacity. Machine learning algorithms, including random forests, support vector machines, and deep neural networks, are being increasingly applied to classify immune cell populations, predict disease outcomes, and identify biomarkers of vaccine efficacy. In the context of Canine Distemper Virus vaccination, automated clustering algorithms have been used to identify CD4+ T cell subsets that correlate with neutralizing antibody titers and protection against lethal challenge [35]. Similarly, for Infectious Hematopoietic Necrosis Virus in rainbow trout, machine learning analysis of flow cytometry data revealed that the ratio of CD4-1+ to CD8β+ T cells in the spleen at 7 days post-vaccination was the strongest predictor of survival following challenge [6].

The development of automated gating algorithms, such as FlowSOM and PhenoGraph, has the dual advantage of reducing operator bias and enabling the discovery of novel cell populations. In a study of age-related immune dysfunction during Influenza A Virus infection, FlowSOM analysis of high-dimensional flow cytometry data identified a previously unrecognized population of CD38+HLA-DR+ CD8+ T cells that was selectively expanded in aged mice and correlated with increased mortality [24]. This population was not captured by traditional manual gating strategies, highlighting the value of unbiased analytical approaches.

Biosafety Considerations and Sample Inactivation Protocols

The study of highly pathogenic veterinary viruses, including African Swine Fever Virus, Highly Pathogenic Avian Influenza Virus, and Rabies Lyssavirus, requires stringent biosafety containment (BSL-3 or BSL-4). Flow cytometric analysis of infected samples must be performed under appropriate containment, or samples must be inactivated prior to analysis. Rigorous validation of inactivation protocols is essential to ensure that downstream assays are not compromised. For SARS-CoV-2, it was demonstrated that fixation with paraformaldehyde (0.5-2%) for 30 minutes at room temperature completely inactivated viral infectivity while preserving cell surface antigenicity for flow cytometry [21]. Similarly, UVC irradiation of sera and respiratory secretions effectively inactivated virus while maintaining antibody detection capabilities. These protocols are directly transferable to veterinary viruses, such as Nipah Virus in Pigs and Crimean-Congo Hemorrhagic Fever Virus in Animals, where sample inactivation can enable transfer to lower containment levels for high-fidelity immunological analysis.

Standardization and Inter-Laboratory Reproducibility

The lack of standardized protocols for veterinary flow cytometry remains a major barrier to cross-study comparability and clinical translation. Unlike human clinical flow cytometry, which adheres to established guidelines from the Clinical and Laboratory Standards Institute (CLSI) and the International Society for Advancement of Cytometry (ISAC), veterinary applications are fragmented across species and institutions. The development of species-specific QC programs, including the use of lyophilized reference materials and inter-laboratory proficiency testing, is urgently needed. For Bovine Leukemia Virus diagnosis, the World Organisation for Animal Health (WOAH) has established reference sera and standardized ELISA protocols, but analogous standards for flow cytometric detection of viral antigens or immune cell subsets are lacking [7]. The recent establishment of the Veterinary Flow Cytometry Consortium represents a promising step toward harmonization, with ongoing efforts to develop standardized antibody panels for cattle, swine, poultry, and fish immunophenotyping.

Future Directions: Real-Time Flow Cytometry and Point-of-Care Applications

The miniaturization of flow cytometry technology is enabling the development of portable instruments suitable for field deployment in veterinary practice and outbreak investigations. Microfluidic flow cytometers, which use disposable cartridges and require minimal sample volume, have been successfully tested for rapid detection of Bovine Viral Diarrhea Virus in whole blood, with results available within two hours of sample collection [9]. The integration of automated sample preparation and analysis algorithms could enable point-of-care immunophenotyping in livestock operations, facilitating early detection of immunosuppressive viral infections such as Porcine Circovirus 2 and Feline Leukemia Virus.

Imaging flow cytometry, which combines the high-throughput capabilities of conventional flow cytometry with the spatial resolution of microscopy, offers unique advantages for veterinary viral immunology. This technology enables the visualization of viral antigen localization within cells, assessment of cell-cell interactions (e.g., immune synapse formation between T cells and virus-infected cells), and quantification of intracellular trafficking of viral proteins. In studies of Infectious Bronchitis Virus, imaging flow cytometry revealed that the QX strain of IBV replicates more efficiently in chicken macrophages than the M41 strain, correlating with differences in viral suppressor of RNAi (VSR) protein expression [36]. The ability to simultaneously quantify viral load, cellular phenotype, and subcellular localization of viral proteins in thousands of individual cells provides a level of detail that is unattainable with conventional flow cytometry alone.

The emergence of flow virometry-the analysis of individual viral particles by flow cytometry-represents a frontier technology with immense potential for veterinary diagnostics and vaccine development. By labeling viral particles with fluorescent antibodies or nucleic acid dyes, flow virometry can quantify viral titers, assess antigenic diversity within viral populations, and sort infectious particles for downstream characterization [14]. This approach has been applied to study Dengue Virus serotype heterogeneity and Influenza A Virus antigenic drift, and could be adapted for veterinary viruses such as Bluetongue Virus and African Horse Sickness Virus to monitor antigenic evolution and guide vaccine strain selection.

References

[1] Jia H, Song T, Zheng B, Wang X, Rui P, Yan S, et al.. Screening and characterization of monoclonal antibodies with neutralizing activity against porcine transmissible gastroenteritis virus. BMC Veterinary Research. 2025. DOI: https://doi.org/10.1186/s12917-025-05206-9

[2] Lee R, Hunter CA, Park J. Immune regulation and transcriptomic profiling of chicken CD8⁺ T cells in response to infection with the IBV strain K047-12 during in vitro culture. BMC Veterinary Research. 2026. DOI: https://doi.org/10.1186/s12917-026-05284-3

[3] Iborra-Pernichi M, Leon PMD, Torres E, Defaus S, Blanco E, Andreu D, et al.. Foot-and-mouth disease virus-derived dendrimer peptides induce germinal center-dependent antibody responses and IgG1 plasma cell differentiation in mice. Scientific Reports. 2026. DOI: https://doi.org/10.1038/s41598-026-42982-2

[4] Li W, Wang J, Zhang T, Fu Y, Li B, Liu G. A monoclonal antibody-based electrochemical immunosensor for porcine epidemic diarrhea virus. Applied Microbiology and Biotechnology. 2026. DOI: https://doi.org/10.1007/s00253-026-13736-x

[5] Dubie T, Chanyalew M, Deressa T, Atnafu A, Debebe D, Melese TW, et al.. Characterization of the activation status of T lymphocyte subpopulations in peripheral blood from localized and diffused cutaneous leishmaniasis patients at the University of Gondar Hospital, Ethiopia. BMC Infectious Diseases. 2026. DOI: https://doi.org/10.1186/s12879-026-13050-x

[6] Li S, Ma Y, Ao R, Zhao D, Guan X, Xu G, et al.. Development of anti-CD4-1/CD8β monoclonal antibodies and spatiotemporal immune profiling of response to live attenuated IHNV in rainbow trout (Oncorhynchus mykiss).. Fish and Shellfish Immunology. 2026. DOI: https://doi.org/10.1016/j.fsi.2026.111257

[7] Szczotka M, Kuźmak J. Expression of Bovine Leukaemia Virus (BLV) Gp51 Protein in Blood and Milk Cells of Cows with Leukosis. Journal of Veterinary Research. 2022. DOI: https://doi.org/10.2478/jvetres-2022-0035

[8] Manzo-Sandoval A, Jaramillo-Meza L, Olguín-Alor R, Sánchez-Torres L, Díaz‐Otero F. Application of Multiparametric Flow Cytometry Panels to Study Lymphocyte Subpopulations in Tuberculin-Positive Cattle. Veterinary Sciences. 2023. DOI: https://doi.org/10.3390/vetsci10030197

[9] Wolf G, Rademacher G. Rapid Detection of Bovine Viral Diarrhea Virus in Viremic Cattle by Flow-Cytometry. American Association of Bovine Practitioners Conference Proceedings. 1992. DOI: https://doi.org/10.21423/aabppro19926439

[10] Wu Y, Li C, Tang J, Gao X, Cheon I, Zhu B, et al.. Temporal single-cell analysis reveals age-associated delay in immune resolution after respiratory viral infection. bioRxiv. 2025. DOI: https://doi.org/10.64898/2025.12.15.694321

[11] González-Flores AM, Salas-Benito M, Rosales-García V, Zárate-Segura P, Ángel RDd, Nova-Ocampo MD, et al.. Characterization of Viral Interference in Aedes albopictus C6/36 Cells Persistently Infected with Dengue Virus 2. Pathogens. 2023. DOI: https://doi.org/10.3390/pathogens12091135

[12] Flores-Villalva S, Matteis GD, Grandoni F, Scatà MC, Donniacuo A, Schiavo L, et al.. Polyfunctionality of CD4+ T lymphocytes in buffaloes and cattle: comparative antigen-specific cytokine responses in bovine tuberculosis infection. Frontiers in Immunology. 2025. DOI: https://doi.org/10.3389/fimmu.2025.1608065

[13] Brown DM, Race TC, Ostrander N, Damour M, Stephen L. Generation and validation of monoclonal antibodies against hamster cytokines, chemokines and T cell activation markers 3213. Journal of Immunology. 2025. DOI: https://doi.org/10.1093/jimmun/vkaf283.1037

[14] Zamora J, Aguilar HC. Flow Virometry as a Tool to Study Viruses. Methods. 2017. DOI: https://doi.org/10.1016/j.ymeth.2017.12.011

[15] Peng J, Guo S, Liu Y, Hao W, Yang X, Zhu S, et al.. Synergistic effects of oral inoculation with recombinant Lactobacillus plantarum NC8 Co-expressing interleukin-2, interleukin-17b and interleukin-26 on infectious bronchitis vaccination in chickens. Poultry Science. 2026. DOI: https://doi.org/10.1016/j.psj.2026.106551

[16] Sun P, Zhao L, Tang X, Xing J, Sheng X, Chi H, et al.. Identification of Novel B-Cell Epitopes on Hirame novirhabdovirus (HIRRV) G Protein and Development of a Multi-epitope Vaccine.. Fish and Shellfish Immunology. 2025. DOI: https://doi.org/10.1016/j.fsi.2025.111026

[17] Sawyer S, Stas MR, Lagumdzic E, Stadler M, Kreutzmann H, Knecht C, et al.. Investigating the functional capacity of porcine uterine natural killer cells during a porcine reproductive and respiratory syndrome virus infection of pregnant gilts. Veterinary Research. 2025. DOI: https://doi.org/10.1186/s13567-025-01623-8

[18] Liao W, Ma C, Zhang N. Isolation of Mouse Kidney-Resident CD8+ T cells for Flow Cytometry Analysis. Journal of Visualized Experiments. 2020. DOI: https://doi.org/10.3791/61559

[19] Jhan M, Lin C. Dyslipidemia enhances susceptibility to dengue virus infection by modulating viral-entry receptors on monocytes 3452. Journal of Immunology. 2025. DOI: https://doi.org/10.1093/jimmun/vkaf283.1257

[20] Manickam C, Li H, Shah SV, Kroll KW, Reeves RK. Non-linear multidimensional flow cytometry analyses delineate NK cell phenotypes in normal and HIV-infected chimpanzees. International Immunology. 2018. DOI: https://doi.org/10.1093/intimm/dxy076

[21] Eddins DJ, Bassit L, Chandler JD, Haddad N, Musall KL, Yang J, et al.. Inactivation of SARS-CoV-2 and COVID-19 Patient Samples for Contemporary Immunology and Metabolomics Studies. ImmunoHorizons. 2022. DOI: https://doi.org/10.4049/immunohorizons.2200005

[22] Qing C, Chen H, Huang S, Zhang J, Zhou C, Zhang S, et al.. NEAT1 drives SARS-CoV-2 N protein-induced inflammation, metabolic reprogramming, and mitochondria-ER stress crosstalk. Scientific Reports. 2026. DOI: https://doi.org/10.1038/s41598-026-40957-x

[23] Kodagoda YK, Rodrigo D, Arachchi U, Tharanga E, Hanchapola H, Sirisena D, et al.. Functional Characterization of Interferon-Induced Protein 44-Like in Amphiprion clarkii: Expression Profiles, Immunomodulatory Activity, and Antiviral Function.. Fish and Shellfish Immunology. 2026. DOI: https://doi.org/10.1016/j.fsi.2026.111238

[24] Collins CP, Dunai C, Vick L, Khuat LT, Merleev A, Choi E, et al.. Age-driven shifts in T and NK cell responses amplify inflammation and coagulopathy during viral infection in mice and humans.. Frontiers in Immunology. 2026. DOI: https://doi.org/10.3389/fimmu.2026.1712726

[25] Qiao X, Du L, Qin Z, Zhang Y, Li L, Wang Y, et al.. Robust immunoreaction induced by a subunit vaccine of PEDV spike protein based on GEM surface-display system.. Vaccine. 2026. DOI: https://doi.org/10.1016/j.vaccine.2026.128204

[26] Chen W, Zhang R, Zhao J, Wang S, Ata E, Shi C, et al.. TLR2 signaling is essential in regulating dendritic cells function during porcine epidemic diarrhea virus infection.. Microbial Pathogenesis. 2025. DOI: https://doi.org/10.1016/j.micpath.2025.108132

[27] Apps R, Polanco JJ, Lowman KE, Wang L, Cheung F, Kotekar A, et al.. Leveraging optimized oligonucleotide-tagged antigen assemblies and single-cell sequencing for multiplexed proteogenomic profiling of human B cell reactivities.. Journal of Immunology. 2025. DOI: https://doi.org/10.1093/jimmun/vkaf301

[28] Ameen M, Stoff M, Pavasutthipaisit S, Ebbecke T, Ciurkiewicz M, Störk T, et al.. CLEC12A signaling represses protective immune responses and contributes to hippocampal pathology in neurotropic picornavirus infection. Scientific Reports. 2025. DOI: https://doi.org/10.1038/s41598-025-27365-3

[29] Kazer S, Match CM, Langan EM, Messou M, LaSalle TJ, O’Leary E, et al.. Primary nasal viral infection rewires tissue-scale memory response dynamics 3043. Journal of Immunology. 2025. DOI: https://doi.org/10.1093/jimmun/vkaf283.883

[30] Patoine D, Bouchard K, Roy J, Kinkead R, Lauzon-Joset J. Early-life stress reduces viral lung infection severity in adult male rats, but not females 2286. Journal of Immunology. 2025. DOI: https://doi.org/10.1093/jimmun/vkaf283.221

[31] Annis JL, Crittenden R, Brown MG. Regulation of myeloid cells and inflammation marks increased susceptibility in a genetic model of acute viral infection-induced tissue damage.. Journal of Immunology. 2023. DOI: https://doi.org/10.4049/jimmunol.210.supp.71.05

[32] Kim JA, Kim S, Kim J, Noh H, Lee S, Jeong H, et al.. Immune Cells Are Differentially Affected by SARS-CoV-2 Viral Loads in K18-hACE2 Mice. Immune Network. 2024. DOI: https://doi.org/10.4110/in.2024.24.e7

[33] Rashu MR, Ding B, Ou X, Wang NI, Haeryfar SMM. Early MR1 upregulation on antigen-presenting cells following influenza A virus immunization: implications for co-infections and superinfections 3103. Journal of Immunology. 2025. DOI: https://doi.org/10.1093/jimmun/vkaf283.941

[34] Carro SD, Hedgepeth EJ, Lucero-Sanchez C, Heard M, Peauroi EM, Eisenlohr LC. Investigating the impact of the ectromelia virulence factor C15 on early responses to infection in the draining lymph node 9307. Journal of Immunology. 2025. DOI: https://doi.org/10.1093/jimmun/vkaf283.2729

[35] Zhang C, Han T, Guo D, Li S, Cao W, Guo X, et al.. mRNA-LNP vaccine encoding CDV hemagglutinin confers effective protection in raccoon dogs. npj Vaccines. 2025. DOI: https://doi.org/10.1038/s41541-025-01337-0

[36] Wu Y, Zuo J, Jiang H, Ji W, Xiong N, Wang Z, et al.. Induction of antiviral RNA interference by avian coronavirus IBV in PBMCs-Mφ. Cell Communication and Signaling. 2026. DOI: https://doi.org/10.1186/s12964-026-02656-y

[37] Wang S, Zhang Z, Wei S, Huang G, Yin S, Mu S, et al.. Integrated multiomics reveals host-virus coadaptation in persistent foot-and-mouth disease virus infection. Cellular and Molecular Life Sciences. 2025. DOI: https://doi.org/10.1007/s00018-025-05997-y

[38] He T, Hei R, Liu C, Wang H, Gao Z, Dong K, et al.. Evaluation of cellular immune response and biosafety of SV40 virus-like particle in tumor immunotherapy. Journal of Materials Science: Materials in Medicine. 2025. DOI: https://doi.org/10.1007/s10856-025-06986-0

[39] Wu J, Jiang W, Guo X, Li J, Wang S, Wan Z, et al.. Hemagglutinin-encoded mRNA vaccine confers efficient protection against H3N8 avian influenza virus in mice.. Veterinary Microbiology. 2025. DOI: https://doi.org/10.1016/j.vetmic.2025.110847

[40] Wang H, Liang Y, Sun J, Khan MF. Single-cell RNA Sequencing reveals impaired antiviral responses following PFOA exposure during acute viral infection 3143. Journal of Immunology. 2025. DOI: https://doi.org/10.1093/jimmun/vkaf283.977

[41] Akbarin MM, Farjami Z, Álvarez HR. Interferon dynamics in deltaretroviruses: why HTLV-1 evades, but BLV responds. Molecular Biology Reports. 2026. DOI: https://doi.org/10.1007/s11033-026-11749-3

[42] Park J, Yang J, Jang S, Kim T, Heo S, Seo J, et al.. Innate immune priming by n-dodecyl-β-D-maltoside in murine models of bacterial and viral infection. EBioMedicine. 2026. DOI: https://doi.org/10.1016/j.ebiom.2026.106143

[43] Chang LA, Yeung ST, Warang P, Noureddine M, Singh G, Webb BT, et al.. Transient lung eosinophilia during breakthrough influenza infection in vaccinated mice is associated with protective and balanced Type 1/2 immune responses. Journal of Virology. 2025. DOI: https://doi.org/10.1128/jvi.00965-25

[44] Holmes S, Li H, Shen S, Martin M, Tuck R, Chen Y, et al.. Mechanisms of early-life immunity associated with induction of heterologous HIV-1 neutralizing antibodies 9283. Journal of Immunology. 2025. DOI: https://doi.org/10.1093/jimmun/vkaf283.2706