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Neutrophils are the first line of defense against pathogen invasion in the human body, excluding the skin, and are an important component of the innate immune defense. They are abundant in the circulatory system and contain abundant antimicrobial granules in their cytoplasm. They respond rapidly to pathogen invasion and infiltrate into inflammatory sites, playing a crucial role in pathogen clearance []. For a long time, it has been widely believed that neutrophils primarily combat pathogen invasion through phagocytosis, oxidative bursts, and degranulation []. However, this understanding expanded significantly with the introduction of a landmark study. In 2004, NETs were first identified and defined as a killing mechanism employed by neutrophils in response to specific stimuli, which are effective against multiple pathogenic bacteria. Over the subsequent two decades, this discovery has captured widespread attention among researchers, with research expanding to encompass other pathogens, fungi, protozoa, and, notably, viruses—with the first direct evidence of NETs targeting viruses emerging in 2012 []. Concurrently, research models have been extended to animals like fish and zebrafish, further confirming the cross-species conservation of this function. NETs are now established as a conserved immune mechanism spanning infection defense, immune regulation, and pathological injury, offering a novel perspective for understanding the immune functions of neutrophils [,,,].

The process of NET formation is referred to as NETosis, a form of programmed cell death distinct from apoptosis and necrosis. Within minutes of neutrophil stimulation and NETosis initiation, actin filaments begin to disassemble, halting cell movement and helping neutrophils anchor themselves at the site of inflammation. Subsequently, this is followed by the remodeling of vimentin filaments, endoplasmic reticulum vesicle formation, and chromatin decondensation. Once the nuclear membrane disintegrates, chromatin and cytoplasmic granules mix together to form intracellular NETs. Finally, the plasma membrane ruptures, releasing intracellular NETs into the extracellular space where they exert their immune-activating functions [,]. The essence of NETs is a fibrous network structure composed of a decondensed DNA scaffold and neutral granule-derived proteins attached to this scaffold, with a diameter of 15–17 nm. These derived proteins are highly diverse, including histones, citrullinated histones, serine proteases, myeloperoxidase, calmodulin, antibiotics, defensins, and cytoskeletal proteins, among others. However, the composition of NETs may vary depending on the stimulus neutrophils receive []. Once released into the extracellular space, NETs combat pathogen invasion by enveloping, capturing, and sustaining a pressure-driven microenvironment with high concentrations of antimicrobial proteins [,,].

The classic pathway for NET release is known as suicidal NETosis, which relies on reactive oxygen species (ROS) generated by NADPH oxidase to activate the key enzyme—peptidyl arginine deiminase 4 (PAD4). PAD4 exhibits high ROS and calcium ion dependence, and its role in mediating the citrullination of histone arginine residues leads to a reduction in the positive charge of histones, thereby decreasing the electrostatic attraction between histones and negatively charged DNA and causing histone–DNA separation, nucleosome disassembly, and ultimately chromatin decondensation []. Patients with chronic granulomatous disease (CGD) have impaired NADPH oxidase complex 2, impairing or eliminating the ability of neutrophils to form NETs through ROS. These patients are extremely susceptible to fungal infections [,]. In some cases, the ROS produced by mitochondria are also sufficient to trigger NETosis [,]. In addition, there are non-canonical pathways for NET release that do not require neutrophil death, known as vital NETosis, which often activates PAD4 by inducing an increase in intracellular calcium concentration through the activation of calcium ion channels on the cell surface [,]. In this case, the extracellular release of NETs no longer depends on the rupture of the plasma membrane but rather on the transport of NETs to the extracellular space via nuclear budding and vesicles. At this point, neutrophils with intact cell membranes still retain a certain degree of phagocytic activity [,].

In previous studies, researchers generally deemed that NETs primarily play an immune role in bacterial and fungal infections. However, growing evidence suggests that they also play an important role in antiviral immunity. The antiviral mechanisms of NETs are more complex compared to their roles against other microorganisms. In addition to directly killing viral particles through antimicrobial proteins and restricting viral spread by enveloping them with sticky DNA structures, NETs can also exert antiviral immune effects indirectly by activating other immune cells, such as activating plasmacytoid dendritic cells (pDCs) [] and lowering the activation threshold of T lymphocytes to enhance the body’s antiviral adaptive immune response []. However, it is worth noting that as toxic substances are released into the internal milieu, NETs can also impose a burden on the body and cause severe tissue damage while performing their antiviral functions [,]. Therefore, elucidating the relationship between NETs and viral infection is crucial for understanding and utilizing their immune functions. This article will review the role of NETs in antiviral immunity and related advances.

We conducted literature searches following the PRISMA statement guidelines. The databases searched included PubMed, Web of Science, Embase, and other relevant databases, spanning the period from March 2004 (when NETs were first identified) to July 2025. Search terms included “neutrophil extracellular traps,” “NETs,” and “viral infection,” among others.

The inclusion criteria were as follows: (1) Basic science or clinical studies on the interaction between NETs and viruses. (2) Original studies (e.g., in vitro experiments, animal models, clinical studies) or high-quality reviews.

The exclusion criteria were as follows: (1) Duplicate publications. (2) Abstracts, meeting summaries, and similar publications that do not provide complete data. (3) Any literature with conflicts of interest.

The titles, abstracts, and full texts of the retrieved literature were independently screened by two researchers, who cross-validated the results and evaluated the quality of the included studies. Finally, 110 articles that met the criteria were included.

Recent studies have shown that the mechanisms by which viruses induce neutrophils to produce NETs exhibit high diversity, with different viral types and infection sites significantly influencing the response pathways of NETosis (). In most cases, neutrophils recognize viral nucleic acids or structural proteins through multiple receptors, thereby triggering NET formation. In certain cases, neutrophils are recruited to the microenvironment by various inflammatory mediators in the context of viral infection, indirectly inducing NET formation. Notably, platelets, as blood cells that become activated early in certain viral infections, can also bridge neutrophils and activate NET formation through multiple mechanisms.

Overall, these pathways do not exist in isolation or operate independently; rather, they coordinate NET formation through temporal coordination, signal crosstalk, or microenvironmental adaptation. Certain pathways may be rapidly activated in the early stages of infection to limit viral spread, while others remain persistently activated in the middle to late phases to counteract ongoing viral replication. Furthermore, the dominant activation patterns of these pathways adaptively adjust across different tissue microenvironments based on local signaling conditions, such as inflammatory mediators and hormone levels. This synergistic nature represents a key strategy for neutrophils in combating viral infections, offering multidimensional intervention targets for antiviral therapies targeting NETs. Specific mechanisms are elaborated in the subsequent sections.

The literature indicates that neutrophils can recognize human immunodeficiency virus (HIV) ssRNA via TLR7 and TLR8, subsequently activating the NADPH oxidase complex through the MyD88 pathway, inducing ROS production, and triggering the formation of NETs []. The nucleocapsid protein (N protein) and spike protein (S protein) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can induce NET generation through multiple receptor-mediated pathways. Neutrophil surface receptors such as Dectin-1 and CLEC5A recognize linear glycosylated epitopes on the N protein and trimeric conformation-dependent glycosylated epitope on the S protein, recruiting the FcγRγ chain to activate the Syk kinase, which subsequently initiates downstream activation signals and induces neutrophils to produce non-ROS-dependent NETs []. Additionally, studies have shown that anti-S IgG1 antibodies produced by the host against the S protein can bind to Fcγ receptors on the surface of neutrophils with high affinity, activating the Syk-p38 MAPK signaling pathway through receptor cross-linking, thereby activating neutrophil oxidative burst and inducing the release of NETs []. Chikungunya virus (CHIKV) and respiratory syncytial virus (RSV) both induce NET formation via the PRRs-ROS axis, but their specific mechanisms differ significantly. When infecting mice and humans, CHIKV activates neutrophil NET generation by activating TLR7 []. RSV viral particles and their F protein can also induce NET release via a concentration gradient, but the core mechanism relies on the activation of the TLR4, ERK, and p38 MAPK signaling pathways []. Unlike CHIKV and RSV, Zika virus (ZIKV) non-structural protein 1 (NS1) mediates ROS-independent NET formation via TLR4 [].

Different from the above mechanism that depends on PRR activation signaling pathways, multiple viruses can also trigger neutrophil NETosis through functional receptors on the cell surface.

Neutrophils can be activated by the HIV-1 envelope glycoprotein (Env) through the CCR5 and CXCR4 co-receptor pathway, leading to the activation of the phospholipase C-mediated calcium signaling pathway, resulting in a rapid increase in intracellular calcium concentration, which then activates PAD4, thereby inducing ROS-independent NETosis. In peripheral blood neutrophils, calcium ion signaling activated by the CCR5 and CXCR4 co-receptors dominates the early response, while ROS signaling activated by the TLR7/8 pathway dominates the late response [,]. It should be noted that aging can attenuate neutrophils’ responsiveness to calcium signaling, resulting in delayed NET release []. Nonetheless, neutrophils in the female reproductive tract have impaired responses to calcium signals, and the formation of HIV-1-induced NETs in this region mainly depends on the activation of late TLR8 signaling. This could explain why women are susceptible to HIV infection after high-risk sexual behavior, and this pathway is further impaired by aging []. Additionally, studies have shown that neutrophils in the cervix and endometrium release NETs more efficiently than peripheral blood neutrophils, which further highlights the important role of NETs in preventing HIV infection in the female reproductive tract []. However, this appears to be partially inconsistent with the notion of calcium signaling defects in neutrophils in the female reproductive tract, which requires further investigation.

Additionally, hantavirus (HTNV)-induced NETs depend on the functional cell surface receptor β2 integrin (CD18), whose heterodimeric subunits CR3 (CD11b/CD18) and CR4 (CD11c/CD18) jointly mediate the viral infection process in neutrophils. Studies have shown that neutrophils in β2 integrin gene knockout mice completely lose their ability to respond to HTNV stimulation, and thus they are unable to induce NET formation by activating NADPH oxidase to produce ROS [].

In addition to the direct activation of neutrophils by viruses, neutrophils can also produce NETs in response to various indirect stimuli during viral infection.

Hepatitis B virus (HBV) infection leads to the downregulation of MHC-I molecule expression levels in hepatocytes, rendering the inhibitory receptors on NK cell surfaces unable to recognize their ligands and thus becoming disabled. This process will activate NK cells, which release perforin and granzyme B. Perforin forms small pores in the hepatocyte membrane, facilitating the entry of granzyme B into the cell. Once granzyme B enters hepatocytes, it will induce the activation of caspase-8 and the cleavage of GSDMD, leading to hepatocyte pyroptosis. The integrity of the plasma membrane is disrupted during pyroptosis, resulting in the release of High Mobility Group Box 1 (HMGB1) into the extracellular environment. HMGB1 will activate TLR4 signaling, which in turn triggers the release of NETs by neutrophils []. At the same time, HBV infection can upregulate the expression of S100A9 in hepatocellular carcinoma cells. Upon secretion, this protein can activate NADPH oxidase through TLR4/RAGE receptors, thereby promoting the formation of NETs []. In addition, in patients with HBV-induced fulminant viral hepatitis (FVH), the expression of Neutrophil-specific fibrinogen-like protein 2 (FGL2) and an ion channel protein called mucolipoprotein 3 (MCOLN3) is upregulated in neutrophils. FGL2 can directly interact with MCOLN3, regulating calcium ion influx and initiating autophagy, thereby leading to the formation of NETs []. It is worth noting that the multiple mechanisms by which HBV induces NET formation may result in heterogeneous NET composition, which depends on the course of viral hepatitis. Hepatitis patients with elevated levels of NET markers, such as citrullinated histone (Cit H3) and HMGB1, may indicate HBV infection, while concurrent increases in autophagy-related proteins may be associated with the progression of fulminant hepatitis. This suggests that NETs have potential as biomarkers for liver disease stratification [,,]. Infection with parvovirus B19 (B19V) induces the production of B19V-VP1u IgG antibodies in the host. These antibodies can induce ROS-dependent NETosis by activating the cAMP/PKA signaling pathway in neutrophils, but this process does not depend on PAD4, suggesting that other non-classical NETosis pathways may exist in the context of B19V infection [].

Also, the inflammatory environment formed after viral infection is often enriched with high concentrations of inflammatory factors, such as IL-1β, IL-6, IL-8, and TNF-α, among others [,]. On the one hand, neutrophils are recruited to the site of infection by these inflammatory mediators in the host body. On the other hand, these inflammatory mediators enhance their oxidative burst capacity, indirectly inducing the ability to generate NETs. Taking varicella-zoster virus (VZV) infection as an example, VZV activates a strong type I interferon (IFN-α/β) response in the host after infection, leading to the release of large amounts of inflammatory factors and chemokines, which induce the explosive release of NETs []. Similarly, rhinovirus (RV) infection specifically stimulates epithelial cells to release IL-33, which is a unique alarm molecule produced by epithelial cells. On the one hand, IL-33 promotes neutrophil chemotaxis; on the other hand, it binds to ST2 on the surface of neutrophils, activating the PLCγ2–calcium signaling pathway and inducing the release of NETs. In patients with asthma complicated by RV infection, this activation pattern is even more pronounced, ultimately leading to the exacerbation of asthma symptoms []. In studies of influenza A virus (IAV), alveolar macrophages recognize viral RNA through Z-DNA-binding protein 1 (ZBP1), triggering RIP3-dependent necroptosis and the release of IL-1α/β, which promotes neutrophil chemotaxis, activates their IL-1R receptors, and subsequently promotes NET formation [].

In the case of viral infection, platelets are usually in a highly activated state [,]. Research indicates that activated platelets play an important role in the generation of NETs [].

H1N1 is a subtype of IAV. In mice infected with the H1N1 influenza virus, large numbers of platelets and neutrophils were observed to accumulate in the pulmonary blood vessels []. Research shows that H1N1 infection induces the expression of tissue factor (TF) in lung cells, activating the plasminogen activator. The plasminogen activator activates PAR4 by cleaving proteases on the surface of platelets, promoting platelet aggregation and activation. Activated platelets release chemokines and microparticles, promoting the recruitment of neutrophils. Additionally, through the binding of surface P-selectin to neutrophil surface P-selectin glycoprotein ligand 1 (PSGL-1), they trigger the activation of neutrophil surface integrin CD18, enhancing their binding and activating the NF-κB and MAPK signaling pathways within neutrophils, thereby inducing the release of NETs []. Other studies have shown that in dengue virus (DV) infection, the NS1 of DV can directly activate platelets, promoting platelet degranulation through the TLR4 signaling pathway and releasing P-selectin and von Willebrand factor (VWF), enhancing platelet–neutrophil interactions and inducing neutrophils to release ROS-independent NETs. Inactivated NS1 co-incubated with platelets failed to induce this effect []. Additionally, after DV activates platelets, they release exosomes (EXOs) and microvesicles (MVs), which induce NET release through the CLEC5A and TLR2 signaling pathways, respectively [].

The above mechanisms indicate that platelets not only play a key role in coagulation disorders associated with viral infections, but they also serve as an important link between viral infections and NET-related immune responses. The excessive formation of NETs not only exacerbates inflammatory responses, but it also may accelerate multi-organ damage and coagulation disorders in patients with dengue hemorrhagic fever and COVID-19 [,]. Therefore, in the study of virus-related diseases, it is crucial to explore the balance between platelet activation and NET release.

NETs represent a crucial mechanism by which neutrophils exert their antiviral functions, employing multiple synergistic pathways to counter viral invasion (). Central to this process is the entrapment of viruses within a concentrated microenvironment enriched in cytotoxic proteins, which suppress viral replication and dissemination or directly compromise viral structural integrity. Additionally, NETs can activate other immune cells, triggering a cascade amplification of antiviral immunity. These mechanisms not only highlight the critical role of neutrophils in combating viral invasion but also reveal the dynamic regulatory function of NETs as immune signaling hubs, offering a novel perspective on virus–host interactions.

NETs can mechanically bind and capture viruses, preventing their spread []. The DNA scaffold of NETs is highly viscous, and its decondensation process significantly increases the exposed surface area in the extracellular environment []. Concurrently, the highly expressed fibronectin in the extracellular matrix contains DNA-binding sites, which facilitate the anchoring of NETs and help them resist the effects of blood flow shear stress, thereby enhancing the virus-capturing capacity of NETs [,,]. Additionally, histones embedded in the NET structure are rich in positively charged amino acids, which can adsorb negatively charged viral particles and envelopes through electrostatic interactions [,].

Multiple neutrophil-derived proteins that are attached to NETs can directly kill viruses, inhibit viral replication, or block viral transmission. According to research reports, histones H1 and H2A in NETs, along with myeloperoxidase (MPO) and α-defensin, can inhibit HIV-1 transcription and disrupt the integrity of viral envelopes []. In RSV infection, MPO inhibits the in vitro replication of RSV isolates, and this effect can be reversed by MPO inhibitors []. Additionally, serine proteases and bactericidal/permeability-increasing protein (BPI) in NETs can recognize, bind, and cleave critical sites of the RSV F protein, thereby blocking RSV fusion with host cell membranes and intercellular spread [,].

Neutrophil-derived proteins can also exert antiviral effects indirectly by stimulating and activating the antiviral responses of other immune cells. The literature indicates that histones and HMGB1, as DAMPs, can activate other immune cells to release pro-inflammatory cytokines to trigger immune responses [,] or activate pDCs through TLRs to initiate the type I IFN antiviral response, thereby activating a cascade amplification of antiviral immunity []. Additionally, NETs can enhance the speed and extent of the body’s antiviral immune response by lowering the activation threshold of T lymphocytes [].

NETs are important immune defense weapons of neutrophils, but viruses have also evolved to have powerful mechanisms to evade host immune responses. They have developed various strategies to evade the cytotoxic effects of NETs, thereby creating favorable conditions for their survival, replication, and spread within the host (). The main escape strategies include inhibiting neutrophil activation, blocking the release pathways of NETs, interfering with neutrophil metabolism, and disrupting the structural framework of formed NETs. Notably, different viral types exhibit significant differences in their escape strategies. This dynamic interplay between viruses and hosts not only reveals the critical role of NETs in antiviral immunity but also, by elucidating these viral escape mechanisms, provides some new insights into the development of antiviral therapies targeting NETosis regulation.