Antiviral Phytoremediation for Sustainable Wastewater Treatment

Zure, Diaiti; Drizo, Aleksandra; Sung, Meng-Hau; Mehari, Amanuel; Maiguo, Eko; Kuo, David H-W

doi:10.3390/su18010523

Abstract

Enteric viruses in wastewater remain a persistent public health threat. Conventional treatments often achieve only modest viral log₁₀ reductions and can generate toxic disinfection byproducts, but high-energy advanced processes are often unaffordable. Antiviral phytoremediation, which involves virus removal mediated by plants and their rhizosphere microbiota, offers a low-cost, low-energy alternative; however, it has scarcely been studied. A bibliometric analysis of ~23,000 wastewater treatment studies (1976–2025) identified only 30 virus-targeted records within plant-based treatment branches, representing ~0.13% of the total corpus. This critical review structures antiviral phytoremediation into a four-barrier framework: (i) sorption/filtration, (ii) rhizosphere-mediated inactivation, (iii) plant internalization, and (iv) intracellular degradation. Pilot and full-scale studies provide strong support for the first two barriers, whereas evidence for internalization and intracellular degradation is limited, mainly laboratory-based, and often inferred from molecular rather than infectivity assays. Standalone constructed wetlands typically achieve ~1–3 log₁₀ virus reductions, but hybrid configurations that combine wetlands with complementary processes achieve ~3–7 log₁₀ reductions, with performance varying between enveloped and non-enveloped viruses and across climates. This review distills design principles for cost-effective hybrid systems and identifies methodological and governance priorities, positioning rigorously designed phytoremediation as a scalable part of climate- and pandemic-resilient wastewater infrastructure.

Keywords:

phytoremediation; viral removal; wastewater treatment; hybrid systems; sustainable remediation

1. Introduction

Human pathogenic viruses, including enteroviruses, noroviruses, and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), persist in wastewater collection systems and are disseminated through multiple environmental pathways (Figure 1), substantially threatening public health [1,2]. Millions of viral particles from infected individuals enter municipal sewer systems daily, where they can remain infectious for periods ranging from hours to months depending on the viral strain, temperature, and organic content [1,2,3]. Without adequate treatment, these viruses contaminate receiving waters, agricultural soils, and aquaculture and seafood production systems [3,4].

Figure 1. Transmission pathway of pathogenic enteric viruses. Viruses transmit from infected to susceptible individuals through environmental routes. Transmission occurs when bodily fluids (sweat, droplets, tears, blood, feces, saliva) spread via different pathways (air, water, solids, vectors). Susceptible individuals may contract infections through multiple entry points (eyes, nose, mouth, blood, skin). Arrows show the direction of viral transmission from infected to susceptible individuals. Route-label colors (Water, Air, Solid, Vector) are used only for visual distinction. Red icons indicate infected individuals or groups, and blue icons indicate uninfected (susceptible) individuals or groups.

Gastrointestinal viruses (i.e., noroviruses, adenoviruses, hepatitis A viruses) are highly resistant to traditional disinfectants (e.g., chlorination), ultraviolet (UV) irradiation, and several advanced treatment processes [5,6]. Advanced technologies, such as membrane bioreactors (MBRs) and advanced oxidation processes (AOPs), typically achieve higher viral inactivation than conventional secondary treatments; however, their performance remains virus-specific. Reported MBR log₁₀ removals range from ~1–7 across different viral pathogens, with ~4–6 log₁₀ for adenoviruses and ~5–6 log₁₀ for norovirus genogroup II [7,8], corresponding to ~97–100% removal, but still allowing infectious virions to pass into downstream environments. The widespread deployment of such systems in resource-constrained- regions is hindered by high capital expenditure, substantial energy demands (~0.4–5.0 kWh·m⁻³ of treated wastewater), and operational complexity, which require skilled staff and robust tracking infrastructure [9,10]. These constraints are acute in areas where wastewater infrastructure expansion lags population growth and climate variability exacerbates water scarcity.

Documented disease outbreaks linked to wastewater reuse disproportionately affect farmworkers, vulnerable consumers, and communities near disposal sites, with children and immunocompromised populations experiencing elevated morbidity and mortality [11]. The detection of SARS-CoV-2 ribonucleic acid (RNA) in wastewater from diverse regions has shown the value of wastewater-based epidemiology for disease surveillance [12,13,14]. Simultaneously, persistent viral signals in treated effluents underscore that existing treatment trains do not reliably eliminate viruses and that additional low-energy antiviral barriers are needed, particularly where wastewater is recycled for irrigation or groundwater recharge [15].

Nature-based treatment systems, such as constructed wetlands (CWs), have been deployed globally and are often described as highly effective in removing conventional pollutants (nutrients and bacterial pathogens) and various emerging contaminants (pharmaceuticals and microplastics) across a wide range of climates [16,17]. In contrast to chemical disinfection, which can generate potentially carcinogenic by-products, such as trihalomethanes (THMs) and haloacetic acids (HAAs), that persist in treated effluents and receiving waters [18], CWs and related phytoremediation systems rely on plant–microbe–substrate interactions and have low energy and chemical inputs. However, the blanket perception of high effectiveness for CWs masks substantial uncertainty and variability in viral removal performance, reflecting both a limited antiviral evidence base and significant methodological heterogeneity.

Plants are the functional units of CWs and support multiple complementary contaminant removal pathways described under the umbrella of phytoremediation [19,20]. These include rhizofiltration (immobilization of contaminants via adsorption and biofilms on root surfaces), phytoextraction (selective uptake of metals and their translocation into harvestable biomass) [21], phytodegradation (enzymatic breakdown of contaminants within plant tissues), and rhizodegradation (microbially mediated degradation in the rhizosphere stimulated by plant root exudates) [22,23,24]. Model macrophytes, such as Lemna minor (Common Duckweed) and Phragmites australis (Common Reed), typically achieve removal efficiencies of ~51–100% for heavy metals (concentration dependent), ~70–99% for excess nitrogen (N) and phosphorus (P), and ~70–100% for culturable bacterial pathogens across various CWs configurations [25,26,27]. These elevated removal efficiencies are often attributed to bioactive secondary metabolites (i.e., polyphenols, terpenoids, organic acids) that (a) have direct antimicrobial properties or (b) stimulate beneficial rhizospheric microbial consortia that antagonize pathogenic microorganisms [28,29].

Plants also have innate antiviral defense mechanisms, including non-host- resistance pathways and salicylic acid-dependent inducible defenses that inhibit viral replication and movement within plant tissues [30,31,32]. Preliminary studies suggest that certain macrophytes may show enhanced antiviral activity. For example, Pennisetum purpureum (Elephant grass) has been used effectively for nutrient removal in CWs [33], while Perilla frutescens (Shiso, Beefsteak Plant, Perilla Mint, Chinese Basil, Korean Kkaennip/Deulkkae) produces caffeic-acid derivatives and polyphenols that interact with viral capsid proteins, modulate host immune signaling, and exhibit in vitro efficacy against SARS-CoV-2 [34,35]. However, most of these insights are derived from plant–virus systems or pharmacological studies rather than from rigorous evaluations of human virus removal in engineered water treatment.

To delineate the extent of underdevelopment of antiviral phytoremediation, we conducted a bibliometric analysis of ~23,000 peer-reviewed publications on wastewater-treatment methods/techniques (1976–2025) using the Dimensions platform (Dimensions, Digital Science & Research Solutions Ltd., London, UK; available at https://app.dimensions.ai accessed on 31 March 2025). Dimensions was selected because it is a linked research database that connects publications with other research outputs (e.g., grants, patents, and policy documents) and supports large-scale research analytics [36,37,38]. Importantly, the map indicates that virus-targeted phytoremediation studies are extremely rare (Figure 2): only 30 records in total were virus-specific within plant-based treatment branches (CW phytoremediation: 24; Hybrid CW [HCW] phytoremediation: 6), representing ~0.13% of the full wastewater-treatment corpus (30/23,235). Within phytoremediation, viral endpoints accounted for ~1.5% of CW phytoremediation papers (24/1575) and ~7.4% of HCW phytoremediation papers (6/81), underscoring the marginal attention to antiviral phytoremediation relative to other targets. By contrast, phytoremediation research is dominated by heavy metals and bacterial pathogens, while antiviral phytoremediation remains underrepresented despite mounting evidence of viral contamination in wastewater worldwide. This asymmetrical representation is not a benign gap; it exposes substantial voids in mechanistic understanding of plant–virus interactions (enveloped and non-enveloped viruses) and highlights the paucity of field-scale evaluations of antiviral phytoremediation and hybrid nature-based treatment trains.

Figure 2. Bibliometric map connecting conventional (CT) and advanced wastewater-treatment (AT) domains to nature-based options (constructed wetlands [CW]; hybrid CW [HCW]; phytoremediation [Phyto]) and target contaminants (heavy metals [HM], pathogen [Patho], other pollutants [OP]). Phyto is shown as bridging both domains because it is reported both as a CT low-tech approach in some applications and as an AT polishing/hybrid component in integrated treatment trains. “CT” and “AT” here denote bibliometric research domains rather than strict engineering classifications of individual unit processes. Patho types: bacteria (Bac), viruses (Vir), parasitic protozoa (Parapro), and helminths (Helm). Grey shaded bands represent bibliometric domains within the wastewater-treatment literature (CT, CW, HCW, AT, and their overlap), while green-highlighted connections denote phytoremediation links to general pathogen and virus research clusters, which support the antiviral phytoremediation theme. Data source: Dimensions Research Database (https://app.dimensions.ai), accessed on 31 March 2025.

A critical reading of this sparse literature raises several cross-cutting research questions that structure this review:

(1): How do plant exudates and rhizospheric microbiota interact co-metabolically with viruses, and which physicochemical mechanisms (e.g., specific binding to capsid proteins, envelope disruption, oxidative damage) dominate under different conditions?
(2): How do these mechanisms differ between enveloped and non-enveloped viruses and across representative taxa (e.g., adenoviruses, noroviruses, enteroviruses, rotaviruses, SARS-CoV-2 and surrogates)?
(3): How do plant species and community compositions (e.g., L. minor, P. australis, P. frutescens, P. purpureum) compare in antiviral performance under standardized hydraulic and environmental conditions?
(4): How do methodological choices, particularly the reliance on molecular detection (PCR, qPCR, RT-PCR) rather than infectivity assays and the use of inconsistent sampling and normalization protocols, bias the reported viral removal efficiencies and mechanistic interpretations?
(5): To what extent can hybrid systems that integrate CWs with complementary technologies (e.g., filtration, disinfection, advanced oxidation) close the performance gap between nature-based and high-energy engineered solutions for viruses at field scale?
(6): Which traits could be targeted through genetic engineering or synthetic biology (in plants and associated microbiota) to enhance antiviral performance, and what are the ecological and regulatory constraints on deploying such changes?
(7): How do antiviral phytoremediation and its hybrid configurations perform when evaluated through techno-economic analysis (TEAs) and life-cycle assessment, particularly in low-income and climate-vulnerable settings?

To address these questions, this review focuses on human enteric and respiratory viruses relevant to wastewater and its reuse. Specifically, we focused on non-enveloped enteric viruses (i.e., adenoviruses, noroviruses, enteroviruses, and rotaviruses) and a limited set of enveloped respiratory viruses such as SARS-CoV-2 and commonly used surrogates, because these groups (a) account for a substantial fraction of wastewater-borne infection risks, (b) are frequently tracked in treatment and reuse studies, and (c) span the contrasting virion architectures (enveloped vs. non-enveloped) that influence sorption, persistence, and inactivation [1,2,3,4,5,6,7,8,12,13,14,15]. Plant viruses and other agricultural plant pathogens are considered only insofar as they illuminate general plant antiviral defenses that may be harnessed in water treatment contexts.

Building on the bibliometric diagnosis of an underdeveloped evidence base, this critical review pursues four objectives. First, we synthesize the mechanistic pathways underlying antiviral phytoremediation from the molecular to the ecosystem scale, organizing the disparate literature into a four-barrier framework: (i) physical sorption and filtration; (ii) rhizosphere-mediated inactivation; (iii) plant internalization; and (iv) intracellular degradation. Second, we examine how these mechanisms translate into system-level performance across different CW configurations and hybrid treatment trains, highlighting where robust field-scale evidence exists and where findings rest on preliminary or methodologically constrained studies. Third, we distill design principles and practical applications of antiviral phytoremediation, identifying field-tested configurations and operational strategies that have achieved significant viral reductions. Fourth, we interrogate the remaining barriers, including methodological limitations, regulatory blind spots, and economic and governance challenges, and outline evidence-based research directions needed to move antiviral phytoremediation from a niche polishing option toward a globally relevant part of climate-resilient, pandemic-prepared wastewater infrastructure.

By integrating perspectives from plant biology, environmental microbiology, wastewater engineering, and systems ecology, this review positions antiviral phytoremediation not as a cure-all but as a technically feasible, potentially scalable, and environmentally resilient element of nature-based infrastructure. A more mechanistically grounded and critically evaluated understanding of plant-mediated virus removal is essential for such systems to contribute credibly to fair public health protection, climate adaptation, and environmental justice across diverse socioeconomic contexts.

2. Mechanistic Basis of Antiviral Phytoremediation

2.1. Phytoremediation Mechanisms

Phytoremediation in planted treatment systems operates through several concurrent pathways within the plant–substrate–microbe continuum: rhizofiltration (physical interception and interfacial partitioning), phytostabilization (immobilization within the rhizosphere), rhizodegradation and phytodegradation (microbially mediated and plant enzymatic transformation), and phytoextraction and phytovolatilization (uptake, sequestration, and for a limited set of compounds, volatilization). The rhizosphere, a narrow zone of water and substrate directly influenced by root exudates and associated microbiota, is the central reaction space where these processes converge [39,40,41]. Although these pathways are well known from nutrient and metal studies, they also define the interfaces at which viruses encounter plant systems and underpin antiviral phytoremediation (Figure 3B).

Figure 3. Conceptual antiviral phytoremediation of viral pathogens in wastewater. Letters denote: (A) Influent containing waterborne contaminants and viruses passes through the rhizosphere (plant–substrate–microbe continuum) to produce treated effluent. (B) Key phytoremediation processes include rhizofiltration (physical interception and sorption to roots/media), phytostabilization (immobilization and reduced mobility), rhizodegradation (microbially mediated transformation supported by root exudates and biofilms), and phytodegradation (plant enzymatic transformation and sequestration in tissues); phytoextraction and limited phytovolatilization are also indicated (for viruses, attenuation is via internalization and intracellular degradation rather than true volatilization). (C) Proposed virus-specific mechanisms include sorption–filtration, rhizosphere-mediated chemical and enzymatic inactivation (e.g., reactive oxygen species and enzymes), viral internalization via apoplastic or endocytosis-like pathways, and intracellular antiviral degradation pathways. Color coding: red icons indicate infectious virions; broken red icons indicate inactivated virions; purple layers show biofilms/microbes; blue elements show water/flow; brown elements show substrate/media; green structures show plant roots/tissues and root exudates.

2.1.1. Rhizofiltration

Root mats, periphytic biofilms, and porous media assemble into tortuous filters that remove suspended and colloidal particles through straining, attachment, and sedimentation. Dissolved and nano-colloidal solutes partition to organic and mineral surfaces via electrostatic attraction, hydrophobic interactions, and cation bridging [39,40]. Plant traits, such as high fine-root surface area and vigorous biofilm growth, increase the surface area, but root exudates modulate near-root pH and charge, enhancing sorption and co-precipitation [23,42]. For viruses, these interfaces act as the primary contact barrier, determining whether virions are physically kept long enough for subsequent chemical and biological inactivation.

2.1.2. Phytostabilization

Phytostabilisation reduces contaminant mobility and bioavailability through sorption to roots and media, precipitation reactions (e.g., phosphate- or carbonate-mediated), and organic ligand complexation within the rhizosphere [39,42,43]. Plants with deep or fibrous root systems create extensive sorptive matrices and microzones with distinct redox potentials and pH conditions that favor the immobilization of metals and some polar organics. Root cell walls and vacuoles serve as sinks for ions and for polar compounds. Although viruses are not stabilized in the classical geochemical sense, the persistent attachment of virions to root and biofilm matrices functionally parallels phytostabilisation by reducing their mobility and exposure pathways.

2.1.3. Rhizodegradation and Phytodegradation

In the rhizosphere, plant exudates (organic acids, amino acids, sugars, and phenolics) serve as substrates for heterotrophic microorganisms and modulate local redox conditions, fostering consortia capable of transforming dissolved organics and co-metabolizing contaminants (rhizodegradation) [23,44,45]. Biofilms on roots and media secrete oxidases, peroxidases, and hydrolases that accelerate depolymerization and humification, but oxygen release via aerenchyma creates redox mosaics that support coupled nitrification–denitrification and sulfur/iron cycling [39,40]. Within plant tissues, endogenous enzymes (peroxidases, dehalogenases, nitrilases) transform absorbed compounds (phytodegradation), and some products are sequestered in the cell walls or vacuoles [20,42]. For viruses, these chemically active microenvironments and enzyme fields provide opportunities for capsid, envelope, and genome damage once virions are captured near the roots.

2.1.4. Phytoextraction and Phytovolatilization

Phytoextraction involves the removal of dissolved ions and some polar organics via root uptake, followed by intracellular binding to organic acids or thiol-rich peptides and compartmentalization within vacuoles or cell walls [20,41,46]. Periodic harvesting exports sequestered contaminants and promotes below-ground turnover. Phytovolatilization contributes to a limited set of volatile elements and compounds (e.g., certain species of mercury and selenium), where biochemical reduction or transformation generates volatile forms released via transpiration [47]. For viruses, true phytoextraction and volatilization have not yet been shown; instead, the internalization of virions into root or shoot tissues and their subsequent intracellular degradation represent a similar but mechanistically distinct pathway, as discussed in Section 2.2.

2.2. Antiviral-Specific Mechanisms

Antiviral phytoremediation can be conceptualized as a four-barrier system in which plants and their associated microbiomes attenuate viral contaminants through (1) sorption–filtration, (2) rhizosphere-mediated inactivation, (3) viral internalization, and (4) intracellular degradation (Figure 3). This framework extends classical phytoremediation ideas to viruses by focusing on how plant-driven mass transfer, surface interactions, and immune-like responses reduce the concentration of infectious virions in complex wastewater matrices [23,48,49].

Historically, virus removal in wetlands and planted systems emerged as an adjunct to studies on bacteria, helminths, and parasitic protozoa in waste stabilization ponds and CWs, with early work relying on enteric virus indicators and bacteriophage surrogates [48,49,50,51]. Only recently, especially during and after the SARS-CoV-2 pandemic, have researchers begun to ask targeted questions about plant-mediated antiviral processes, including the differences between enveloped and non-enveloped viruses. Despite this shift, the number of dedicated antiviral phytoremediation studies remains small, and many rely on molecular detection (PCR, qPCR, RT-PCR) without corresponding infectivity assays, complicating mechanistic interpretation.

2.2.1. Sorption–Filtration at Plant, Biofilm, and Substrate Interfaces

Sorption–filtration is the initial barrier that intercepts virions on high-surface-area matrices such as roots, biofilms, and porous substrates [50,51]. At the molecular level, three interaction modes dominate:

(1): Electrostatic interactions: Capsid proteins present charged amino acid residues (e.g., lysine, arginine, aspartate, glutamate) whose net surface charge depends on the pH relative to the virus isoelectric point. Root cell walls, extracellular polymeric substances (EPS), and mineral grains carry carboxyl, hydroxyl, and phenolic groups typically negatively charged at circumneutral pH values. Therefore, the attraction or repulsion between virions and these surfaces is governed by pH, ionic strength, and multivalent cations [39,50,51,52,53].
(2): Hydrophobic interactions: Exposed non-polar patches on capsid proteins and, for enveloped viruses, hydrophobic lipid domains in the viral envelope can interact with hydrophobic regions of root- and biofilm-associated polymers, promoting adsorption and aggregation.
(3): Cation bridging: Divalent cations (e.g., Ca²⁺, Mg²⁺) can bridge negatively charged viral surfaces and carboxyl-rich EPS or root exudates, enhancing attachment.

In CWs, plant-supported biofilms and mineral surfaces provide dominant specific surface areas, similar to the matrices that immobilize dissolved metals via sorption and complexation [52,53]. Field and pilot studies generally report ~1–3 log₁₀ reductions in viral markers attributable primarily to physical capture and adsorption, with performance modulated by hydraulic retention time (HRT), root/biofilm density, virion surface chemistry, ionic strength, and organic matter content [54,55]. However, many of these studies measure genome copies rather than infective units, so the reported log₁₀ reductions may overestimate true inactivation, stressing the need for infectivity-based validation.

2.2.2. Rhizosphere-Mediated Inactivation

Beyond capture, the rhizosphere acts as a chemically reactive, antiviral zone. Root exudates (organic acids, phenolics, flavonoids) and microbial metabolites shape redox conditions and generate reactive species and enzymes that can damage viral parts [23,28,56,57]. Mechanistically:

Reactive oxygen species (ROS), such as H₂O₂, ·OH, and O₂·⁻, can oxidize amino acid side chains (e.g., methionine, cysteine, and tryptophan) in capsid proteins, induce lipid peroxidation in viral envelopes, and promote strand breaks in viral genomes.
Phenolics and flavonoids (i.e., caffeic-acid derivatives, chlorogenic acid, rutin, and other polyphenols) can bind to capsid proteins and viral enzymes through hydrogen bonding and π–π interactions, potentially blocking receptor-binding sites or fusion domains and destabilizing protein conformations [28,34,35,56].
Enzymes produced by biofilms and root-associated- microbes, such as peroxidases, laccases, proteases, and nucleases, can catalyze the oxidative polymerization of phenolics, generate additional ROS, and directly cleave proteins and nucleic acids [23,45,58].

Non-enveloped enteric viruses with rigid icosahedral capsids (e.g., noroviruses, adenoviruses, enteroviruses) generally require higher ROS doses and prolonged contact times to achieve comparable attenuation to enveloped viruses, whose lipid envelopes are more susceptible to oxidative and enzymatic attacks [8,59,60]. Experimental evidence for rhizosphere-mediated inactivation includes in vitro virucidal assays with plant extracts and exudates and reductions in infective phages in planted versus unplanted beds [23,56,57,58]. However, the direct attribution of log₁₀ reductions to specific exudates or enzymes in full-scale systems is rare, reflecting a major mechanistic knowledge gap.

2.2.3. Viral Internalization (Cell Entry and Early Destabilization)

Following surface capture, some virions may be internalized into the root tissues. The proposed entry routes include:

Movement with water through apoplastic pathways and intercellular spaces,
Endocytosis-like uptake at the root epidermis or cortex, and
Entry occurs via emerging lateral roots, lenticels, or damaged tissues [61,62].

Enveloped viruses appear more prone to early destabilization during or after entry due to the fragility of their lipid envelopes, but non-enveloped viruses, reinforced by robust protein capsids, usually persist longer and resist early chemical degradation [8,59,63]. Plant chitinases and β-1,3/1,6-glucanases (glycoside hydrolase family 19 [GH19]), along with lectins that recognize N-acetylglucosamine motifs, can target glycoprotein domains on viral envelopes or associated glycoproteins, contributing to particle disassembly [23,61]. Heterologous expression of antiviral membrane proteins such as IFITM3 (interferon-induced transmembrane protein 3) in model plants has been proposed as a way to modulate endosomal lipid dynamics and enhance uptake-linked destabilization [64,65].

Most evidence for internalization in phytoremediation contexts comes from the detection of viral RNA/DNA in root tissues (PCR, qPCR, or fluorescence-labeled particles). These methods cannot always distinguish between virions inside living cells, virions adsorbed in cell walls or apoplastic spaces, and degraded fragments. There is little evidence that human enteric or respiratory viruses replicate in macrophytes. Thus, while internalization is a plausible intermediate barrier, its frequency, depth, and contribution to net infectivity loss in field systems remain poorly understood.

2.2.4. Intracellular Inactivation (Genome Silencing and Proteolysis)

If virions or their genomes enter plant cells, a suite of intracellular antiviral responses can be activated. Key processes include:

RNA interference (RNAi): Small interfering RNAs guide sequence-specific cleavage of viral RNA, leading to post-transcriptional gene silencing [66,67,68].
Ubiquitin–proteasome pathways: ubiquitination marks viral proteins for degradation by the 26S proteasome, reducing the pool of functional structural and non-structural proteins.
Autophagy: autophagy-related (atg-dependent) pathways can engulf viral particles or replication complexes into autophagosomes that fuse with lytic compartments [66,67,68].

Plant secondary metabolites also participate in this process. For example, the bioflavonoid rutin from Ocimum basilicum (Sweet Basil) binds to structural proteins across diverse viruses with favorable binding energies (−7 to −10 kcal·mol⁻¹), potentially inhibiting attachment, fusion, and genome release (phytoinactivation) [69]. Peroxidases and proteases further contribute to capsid degradation under oxidative conditions [23,45]. Chemically, these processes involve peptide-bond hydrolysis in capsid proteins, oxidative modification of amino acids, and phosphodiester bond cleavage in nucleic acids, ultimately rendering virions non-infective.

However, virtually all detailed mechanistic insight into RNAi, ubiquitin–proteasome interactions, and autophagy comes from plant–plant-virus systems, not from human enteric or respiratory viruses in CWs. Reported ~1–3 log₁₀ additional viral reductions attributed to intracellular degradation therefore rest on indirect or surrogate evidence, often inferred from decreases in genome copies in plant tissues rather than from direct infectivity measurements [66,67,68,69,70,71]. From a systems perspective, intracellular pathways likely set the upper limit for true inactivation; however, upstream barriers (capture and rhizosphere chemistry) regulate the encounter frequency and loading of this cellular machinery [39,72]. Rigorous infectivity-based studies are needed before intracellular degradation can be treated as a reliably measured design feature.

2.3. Comparative Analysis of Viral Particles and Heavy Metals

2.3.1. Removal Mechanisms

Plants and plant–microbe systems remove heavy metals and viruses through superficially similar stages: interception at interfaces, transformation in the rhizosphere, and organism-level processing. However, the underlying physicochemical and biochemical mechanisms diverge, with important design implications (Table 1). Heavy metals, typically present as hydrated ions or complexes, are removed mainly via sorption and complexation to roots, biofilms, and mineral media, followed by uptake, chelation (e.g., by organic acids and phytochelatins), and vacuolar sequestration, culminating in phytostabilization and phytoextraction [73,74,75]. Metals cannot be destroyed; therefore, management focuses on speciation and immobilization [76].

Viruses, nanoscale colloids composed of proteinaceous capsids with or without lipid envelopes, are first immobilized through electrostatic and hydrophobic interactions, then inactivated chemically and enzymatically in the rhizosphere, and for a subset, further processed through internalization and intracellular degradation [77,78,79]. Unlike metals, viral particles are supramolecular assemblies whose infectivity can be neutralized by capsid or envelope damage and genome cleavage, even if their physical presence persists [78,80,81]. Non-enveloped viruses with tightly packed icosahedral capsids show high resistance to desiccation, pH fluctuations, and moderate oxidants, but enveloped viruses are more vulnerable to lipid-targeting agents and ROS [72,82]. At interfaces, adsorption parameters also differ: for model bacteriophages and coronaviruses, Freundlich adsorption coefficients span ~2 × 10³–2.7 × 10⁵ mL·g⁻¹, influenced by ionic strength, DOC, and temperature [83], but divalent metal cations typically follow more predictable surface complexation trends dominated by pH and competing ligands [84]. The design for viral control emphasizes charge neutralization, aggregation aids, and chemically active rhizospheres, whereas metal removal emphasizes pH/redox control and ligand management.

2.3.2. Factors Influencing Removal Efficiency

Pilot- and field-scale macrophyte systems commonly report ~1–2 log₁₀ reductions in viral markers from physical capture alone, with higher total reductions when rhizosphere inactivation and intracellular pathways are engaged (Table 1) [85]. Plant traits that increase the active surface area (fine root- density and healthy periphyton) and hydraulics that prevent short-circuiting enhance encounter rates and viral attenuation [86]. Temperature fluctuations affect virion adsorption equilibria and reaction kinetics, contributing to greater seasonality in virus removal than that typically observed for metals [87]. In contrast, heavy metal removal is often higher and more consistent under comparable retention times, often reaching ~70–100% in well-maintained wetlands or rhizofiltration units when speciation favors sorption and plant uptake capacities are not saturated [88].

Virus removal is sensitive to virion architecture (enveloped vs. non-enveloped), DOC, and competing colloids. Elevated DOC levels can shield virions from adsorption sites and scavenge ROS, thus reducing the effectiveness of both physical and chemical barriers. Chemical amendments can partially narrow this performance gap. Polyphenols, such as rutin, bind to viral surface proteins with favorable free energies (−7 to −11 kcal·mol⁻¹), promoting virion aggregation and enhancing capture, while upstream charge-stabilization processes (e.g., electrocoagulation and optimized flocculation) increase early-stage retention within root-associated matrices [79,89,90]. Maintaining young, metabolically active plant stands with a balanced nutrient status and moderate redox heterogeneity supports the sustained production of antiviral metabolites and robust metal-binding capacity [91,92,93].

2.3.3. Design Considerations for Combined Virus and Metal Removal

When wastewater has both viruses and dissolved metals, the system design must integrate complementary barriers while managing potential interactions (Table 1). Organic acids and flavonoids that contribute to viral inactivation also chelate metals and change speciation; this generally favors metal immobilization but can increase competition with natural organic matter for adsorption sites, requiring careful control of influent chemistry and retention time [94,95]. Dense biofilms that capture virions also provide ligands for metal binding, but pose operational challenges related to clogging versus surface area preservation [75,96,97].

Tracking requirements also differ. Viral control must be assessed by infectivity assays or confirmed surrogates to confirm true inactivation, but metal control is evaluated via dissolved/particulate speciation and plant tissue burdens to assess stability against remobilization [70,98]. For example, macrophyte beds based on Pistia stratiotes (Water Lettuce) have achieved ~0.5–1.0 log₁₀ reductions in bacteriophage surrogates per pass while simultaneously lowering dissolved metal concentrations [79,99,100,101]. Coupling such systems with polishing steps (UV, ozone, ferrate) can increase viral removal to ~3–7 log₁₀, while maintaining or enhancing metal removal [102,103,104,105]. Cost and reliability considerations often favor modular hybrid systems, where downstream activated carbon or nanofiltration units buffer against peaks in organic load or viral spikes and control trace organics that interfere with adsorption and enzymatic activities [106,107]. Adaptive tracking and rule-based control, flow equalization, intermittent aeration, and targeted coagulant dosing help sustain performance under seasonal and load variabilities [108,109,110,111,112].

2.3.4. Emerging Innovations for Enhanced Removal Performance

Three innovation streams target the differential challenges posed by viruses and metals. First, engineered plant–microbe consortia can partition antiviral and metal-binding functions; for example, Streptomyces and Bacillus strains producing lytic enzymes and exopolysaccharide-rich periphyton enhance both virion inactivation and metal capture [113,114]. Second, host trait enhancement, via choice of genotypes with large fine-root areas and elevated antioxidant and proteostasis capacities, or prospectively via genetic engineering, could improve intracellular viral degradation while maintaining metal tolerance [115,116]. Third, upstream physicochemical aids tailored to viral colloids, such as mild electrocoagulation for charge neutralization, reduce viral loading in biological stages and stabilize performance under cold or high DOC conditions while remaining compatible with metal removal pathways [117,118,119].

Artificial intelligence (AI) and machine learning (ML) tools offer additional optimization. AI applications in CWs and related nature-based systems have focused on predicting and controlling bulk water quality parameters (e.g., BOD, nutrients, and turbidity) and operational set-points [120,121]. No published studies have shown AI-guided optimization explicitly targeting viral endpoints in phytoremediation systems. Therefore, we consider AI as a promising but under-explored approach, with the potential to coordinate flow, aeration, and dosing regimens in response to virological surrogates once appropriate tracking frameworks are in place. Positioning antiviral phytoremediation within broader circular bioeconomy agendas will require integrating virus control with resource recovery functions, as highlighted in recent work on wastewater-derived energy, nutrients, and value-added products [122,123,124]. Collectively, these emerging approaches illustrate how the mechanistic differences between metals and viruses can be translated into complementary barriers. However, they also expose the fragility of the current antiviral evidence base and the need for rigorous infectivity-centered field validation.

Table 1. Virus and heavy metal removal mechanisms in plant-microbe rhizosphere system.

Mechanism/ Parameter	Virus (Antiviral Mechanism)	Heavy Metal (Co-Removal)	Performance Metrics	Critical Variables	Example Systems	Ref.
Primary removal	electrostatic/hydrophobic adsorption; aggregation; enzymatic inactivation	ion exchange; surface complexation; chelation; precipitation	virus: ~1–2 log₁₀ capture, up to 7 log₁₀ with polishing; metal: ~70–100% removal	charge density, DOC, pH, root potential, ionic strength	Pistia stratiotes, Typha latifolia, Phragmites australis beds	[75,77,85]
Rhizosphere biochemistry	exudate oxidation, proteolysis; polyphenol virion destabilization (ΔG ≈ −10 kcal/mol)	organic acid complexation; phytochelatin synthesis; redox cycling	inactivation rate: k = ~0.02–0.07 h⁻¹ (25 °C); infectivity loss: 65 ± 12% (48 h)	root activity, flavonoid flux (~0.8–1.5 mg g⁻¹ DW), microbial profile, T-sensitivity	natural/changed wetlands	[79,91,92]
Particle stability	capsid/envelope disruption, genome cleavage; enveloped viruses removed ~2–5× better	speciation-dependent stability; vacuolar sequestration post-uptake	enveloped removal: >90%; RNA decay: ~65–84% (~48–72 h)	temperature, pH, oxidative potential, virion charge	macrophyte–biofilm systems	[79,82]
Adsorption/ partitioning	Freundlich Kₙ = ~2 × 10³–2.7 × 10⁵ mL g⁻¹; mean capture: 58 ± 20%	surface complexation log K = ~4–8 (pH-driven)	capture efficiency: ~58 ± 20% (n = 16); K = ~10³–10⁴ mL g⁻¹ for bacteriophages	ionic strength, DOC competition, surface pKa, hydrophobicity	rhizofiltration, periphyton-root systems	[83,84]
HRT requirement (d)	~3–6 d for viral attenuation; ideal ~5–10 d	~2–4 d for metal sorption equilibrium	virus: ~1–2 log₁₀ per stage; metal: ~70–100% removal	flow uniformity, aeration regime, recirculation, temperature effects	hybrid wetland + UV, VSSF units	[88,103]
Chemical aids	polyphenols (ΔG); electrocoagulation (EC) ~2–3× capture boost	biochar, zeolite, Fe(OH)₃, molecular imprinted polymers composites	virus capture: +1–1.5 log₁₀ gain with EC; metal removal: +15–30% with media	coagulant dose (FeCl₃ ~5–20 mg/L), pH, oxidation reduction potential (ORP)	modular wetland-filter hybrids	[90,106,125]
Microbial contribution	lytic enzymes, quorum-regulated proteases (Bacillus); ROS generation	extracellular polymeric substance matrix, siderophore secretion, biosorption	+0.5–1.0 log₁₀ increment; peroxidase activity ~25–60%	microbial diversity, nutrient ratio (C:N:P ≈ 100:10:1), rhizosphere age	engineered consortia	[41,113,114]
Seasonal sensitivity	strong T-dependence (−0.3 log₁₀ per 10 °C drop); dissolved organic carbon (DOC) competition	moderate; resilient under redox/pH shift	winter: keeps ~70–85% of summer rate with thermal buffering	temperature, DOC level, biofilm maturity, flow fluctuation	aerated/intermittent-flow constructed wetland systems	[86,87,126]
AI control	adaptive flow/dosing for dual targeting; real-time viral prediction (~12–18 h lead time)	dynamic ligand control via real-time speciation	±10% variance reduction under fluctuating loads	pH/ORP sensors, metabolite biosensors, AI feedback	smart AI-integrated wetlands	[111,120]

3. Recent Advancements in Antiviral Phytoremediation

3.1. Plant Selection and Optimization

3.1.1. Strategic Choice of Antiviral Plants via Mechanisms to Traits

Selecting plants for antiviral phytoremediation should be grounded in the four-barrier framework outlined in Section 2 (sorption/filtration, rhizosphere-mediated inactivation, internalization, intracellular degradation) and mapped to specific plant traits. Key traits include: (i) high fine-root specific surface area and dense periphyton for efficient sorption–filtration; (ii) metabolically active rhizospheres that release phenolics and flavonoids and support enzyme-producing microbiota for chemical and enzymatic inactivation; (iii) tissues exhibiting robust endocytosis-like activity and proteostasis to support internalization and intracellular degradation; and (iv) shoot–root architectures compatible with the target hydraulics and redox zoning [51,79,127].

Systems that maintain young, rapidly growing stands with vigorous root turnover and periphyton renewal provide more stable viral attenuation across seasons than over-aged canopies with diminished below-ground activity [40,128]. Seasonal variability and influent chemistry further refine plant selection: species that sustain exudation and peroxidase/oxidase activity at lower temperatures and tolerate DOC fluctuations without impairing biofilm health mitigate performance declines during winter and high DOC shocks [129,130]. Hydraulic compatibility is equally important: VSSF wetlands favor species that maintain porosity and resist clogging under intermittent loading, but free water- surface (FWS) beds focus on emergent canopies that enhance light and oxygen delivery for oxidative chemistry [131,132,133,134].

3.1.2. High-Performance Antiviral Plant Species

Evidence from bench, pilot, and field studies has identified several benchmark macrophytes that combine extensive, cleanable surface areas with chemically active rhizospheres.

P. australis acts primarily as a capture- and hydraulics-dominant structural species, providing large fine-root areas (>300 cm²·g⁻¹), strong oxygen transfer, and high bed porosity (~25–35%) in VSSF and baffled FWS systems. This consistently supports diverse, functionally rich rhizobacterial communities linked to pathogen degradation while preserving hydraulic conductivity [40,135,136].

Typha spp. (Cattail, Bulrush/Reedmace, Cumbungi, Raupō) and Cyperus spp. (Chufa/Tiger Nut, Fox Sedge, Papyrus) offer similar structural functions and periphyton support, with robust tolerance to nutrient and DOC variability, which is typical of municipal influents [135,136,137]. In polycultures with Phragmites, they add functional redundancy and seasonal buffering.

O. basilicum contributes to antiviral rhizosphere chemistry through high phenolic and flavonoid exudate production (~0.8–1.5 mg·g⁻¹ DW) and elevated oxidase/peroxidase activity, supporting chemical inactivation, particularly of enveloped viruses. Rutin from O. basilicum exhibits favorable binding free energies (−9.7 to −10.9 kcal·mol⁻¹) against multiple viral surface proteins, promoting virion aggregation and helping with capture within root and biofilm matrices [69,79,138,139,140].

Strobilanthes cusia (Assam Indigo, Chinese Rain Bell, Pink Strobilanthes/Vein Leaf) produces indole alkaloids such as tryptanthrin (IC₅₀ ~10–50 µM against coronavirus NL63 protease), alongside elevated RNase and protease activities, suggesting a role in intracellular enzymatic defense when deployed in warm, shallow beds or floating systems [100,139,140].

P. stratiotes provides rapid rhizofiltration capacity via extensive adventitious roots and fast biomass production, typically achieving ~0.5–1.0 log₁₀ reductions per pass in rhizofiltration units and contributing to ~3–5 log₁₀ removal when coupled with UV polishing [79,141,142]. However, its invasive potential and sensitivity to low DOC/high shear stress require careful containment and hydraulic control.

Collectively, these findings indicate that no single species optimizes all four of the antiviral barriers. State-of-the-art designs therefore favor polycultures. For example, tricultures of Phragmites–Typha–Ocimum exploit trait complementarity: large surface area and hydraulic robustness from structural reeds, combined with exudate-driven antiviral chemistry from aromatic species, yielding ~2.8 ± 0.5 log₁₀ reductions and ~85 ± 10% infectivity loss over multi-year operation [135,143,144]. A comparative summary of the macrophytes is presented in Table 2.

Table 2. Plant traits and viral removal in constructed wetland systems.

Scientific Name (Common Name)	Functional Traits (Key Mechanisms)	Optimal Configurations	Viral Removal Performance (log₁₀ Reduction)	Co-Removal Benefits	Critical Constraints	Ref.
Monoculture systems
Phragmites australis (Common reed)	fine root area (>300 cm² g⁻¹); dense periphyton; strong O₂ transfer; high porosity (~25–35%)	VSSF (intermittent loading); baffled FWS	1.2 ± 0.3 log₁₀ (capture-dominant); field stability ~60–75%	high N/P removal (~70–99%); stable heavy metal uptake (~70–90% Zn/Cu)	seasonal dormancy (winter); requires periodic harvest (~2–4 × yr⁻¹); establishment time 4–6 wks	[40,135,136]
Ocimum basilicum (Sweet basil)	high phenolic/flavonoid exudates (0.8–1.5 mg g⁻¹ DW); elevated oxidase/peroxidase activity	horizontal/free-water flow with aeration; mixed beds (2:1 ratio)	2.3 ± 0.4 log₁₀ (chemical inactivation); +40% for enveloped viruses	volatile oil antimicrobial effects; phenolic anti-biofilm agents; biomass valorization potential	high T-sensitivity (~20–30 °C ideal); short lifespan (requires replacement ~2–3 × yr⁻¹)	[79,139,140]
Strobilanthes cusia (Assam indigo)	indole alkaloid production (Tryptanthrin 10–50 µM IC₅₀); elevated RNase/protease activity	floating macrophytes; warm shallow beds (~20–28 °C)	+0.7 ± 0.2 log₁₀ gain over baseline (intracellular enzymatic defense)	medicinal/commercial value co-product potential; strong nucleic acid hydrolysis capability	tropical requirement (dies < 10 °C); limited geographic deployment; alkaloid bioaccumulation risk	[100,139,140]
Pistia stratiotes (Water lettuce)	extensive adventitious root system; rapid biomass production; high transpiration	floating-bed systems; rhizofiltration units	~0.5–1.0 log₁₀ per pass; ~3–5 log₁₀ in CWs–UV hybrid (high sorption capacity, K = ~10³–10⁴ mL g⁻¹)	high heavy metal uptake (~70–85%); scalable for rapid deployment	Invasive potential (requires containment); sensitive to low DOC/high shear; capture-dominant mechanism	[79,141,142]
Optimized polyculture systems
Phragmites + Typha + Ocimum (triculture)	trait complementarity: max surface area + diverse exudate chemistry + functional redundancy	coupled VSSF–free-surface system	2.8 ± 0.5 log₁₀ reduction; 85 ± 10% infectivity loss (capture–inactivation synergy)	superior stability; buffering seasonal/load variations; showed performance over 3+ yrs	higher complexity in operation and maintenance (O&M); longer initial establishment (~8 wks); requires strict nutrient control	[135,143,144]

3.1.3. Selection Criteria and Screening Workflow

A robust plant selection workflow integrates (1) desk-based pre-screening, (2) bench assays, and (3) pilot-scale verification:

(1): Pre-screening filters candidate species based on local availability, invasiveness risk, hydraulic compatibility (root porosity, aerenchyma), and evidence of antiviral metabolites or vigorous rhizosphere metabolism [143,145].
(2): Bench assays measure metrics aligned with the four-barrier framework: short-term sorption coefficients (mL·g⁻¹) on roots and periphyton; exudate-driven loss of infectivity for enveloped versus non-enveloped viral surrogates; early internalization markers such as viral RNA decay within root tissues over ~24–72 h; and indicators of intracellular degradation, including peroxidase, protease, and RNase activities and stress-response gene expression under realistic ionic strength and DOC levels [61,146,147].
(3): Pilot verification in the intended hydraulic configuration confirmed (a) stability of log₁₀ reductions across seasons and loading rates, (b) maintenance requirements (harvesting frequency, clogging control), and (c) ecological safeguards (no pathogen amplification or spread of non-native taxa). Comparative tests among Phragmites–Typha–Cyperus mixtures consistently reveal complementary hydraulic and rhizosphere chemistry. Incorporating a minority fraction (~10–30% stem density) of antiviral-rich aromatic species (e.g., O. basilicum, S. cusia) can enhance inactivation without compromising hydraulic conveyance [143].

3.1.4. Optimization Strategies and Pretreatment Integration

Three recurrent optimization strategies enhance plant-mediated antiviral performance:

(1): Biomass age management: Maintaining young, metabolically active stands through staged planting and periodic harvesting preserves a high root-specific surface area and fresh periphyton [148].
(2): Rhizosphere stabilization: Ensuring balanced nutrient supply and moderate redox heterogeneity (e.g., intermittent aeration in VSSF wetlands) sustains the production of enzymes and ROS important for capsid and envelope damage [149,150,151].
(3): Gentle pretreatment: Pairing plant stages with mild pretreatment that enhances early viral capture without damaging roots or biofilms, such as low-dose electrocoagulation or optimized coagulation to neutralize charge and aggregate virions before root zone contact, improves first-barrier performance, particularly under high DOC or low temperatures [152,153,154].

Interplanting antiviral-rich species with structural macrophytes and incorporating upstream destabilization steps (iron-based coagulants or low-dose electrocoagulation) can increase first-barrier retention and reduce the intracellular processing burden [152,153,154]. Maintaining operational flexibility for seasonal rebalancing (e.g., transient aeration changes and selective harvesting) keeps the plant–microbe assembly near its ideal performance [129,155].

3.2. System Optimization

3.2.1. Hydraulics and Contact Optimization

Virus attenuation in planted treatment systems is limited by contact efficiency; thus, optimizing flow architecture to maximize uniform interaction between influent and active interfaces (roots, biofilms, media) is critical (Figure 4). In VSSF beds, the influent should be evenly distributed via multiple ports or perforated headers, with hydraulic loading rates (HLR) matched to bed conductivity and maintained porosity to reduce short-circuiting. In FWS cells, baffles and staged islands straighten flow paths and reduce wind-driven recirculation that bypasses root zones [54,156]. Design heuristics for municipal-strength wastewater target an HRT sufficient to achieve ~1–2 log₁₀ removal via primary sorption/filtration, followed by an additional ~0.5–1.0 bed volume for rhizosphere chemical and intracellular attenuation [127,157,158]. Under elevated organic loads or low temperatures, incorporating a ~20–40% HRT safety margin or a compact pretreatment stage (e.g., coagulation, roughing filtration) stabilizes upstream capture and mitigates desorption risk [126,159]. Because viral removal scales with the interfacial area, plant species and media that maintain a high specific surface area (fine roots, cleanable media) and resist compaction are essential [160]. Operational strategies, such as alternating day dosing in VSSF systems (to re-aerate pores and support biofilm recovery), rotating inlet zones (to reduce localized clogging), and periodic tracer tests (to reassess effective HRT), help sustain contact efficiency. If effective HRT falls below ~70% of the design or residence time variance increases, remedial actions (hydro-flushing, media scarification, partial media replacement) should be implemented before irreversible performance decline [161].

Figure 4. Hydraulics and contact optimization for virus attenuation in vertical subsurface flow (VSSF) beds and free-water surface (FWS) cells. (A) VSSF plan and cross-section showing perforated influent distribution to promote a uniform wetting front, increase contact with filter media/biofilms/roots, and reduce short-circuiting. (B) FWS plan view showing baffles and staged vegetated islands that straighten flow paths, suppress wind-driven recirculation, and direct flow through root-zone “hotspots” for enhanced sorption/filtration and rhizosphere attenuation. Tracer testing and traffic-light diagnostics indicate declining effective HRT and maintenance triggers; stress conditions may require added HRT safety margin or compact pretreatment (e.g., coagulation/roughing filtration. Colors: solid blue arrows denote main water flow paths, green zones indicate active interfaces (roots, biofilm, media), and red dashed arrows indicate bypass or short-circuit flow paths.

3.2.2. Rhizosphere Chemistry and Redox Management

Sustaining a metabolically active and chemically reactive rhizosphere is the second major optimization lever, as it underpins capsid and envelope damage and accelerates intracellular antiviral responses (Table 3). Intermittent aeration in VSSF systems, with ON/OFF cycles of ~1–2 h and dissolved oxygen (DO) maintained above ~2 mg·L⁻¹ near the inlets, supports oxidative enzymes and ROS generation while preserving anoxic niches deeper in the bed for denitrification and redox balance [44,162,163]. In FWS cells, shallow shelves with emergent canopies enhance light and oxygen penetration, promoting peroxidase activity, exudation and periphyton development [164].

Nutrient balance critically influences antiviral metabolite flux and the dynamics of microbes. Maintaining C:N:P ratios of ~100:10:1 avoids carbon starvation, which suppresses enzyme production and prevents eutrophic blooms that smother roots [165,166]. Temperature-responsive regulation in beneficial Pseudomonas spp. and quorum sensing in biofilms modulate in situ metabolite release [165,166,167,168]. Plant immune pathways, such as salicylic acid/NPR1 signaling and redox-sensitive cues, link systemic acquired resistance (SAR) to local oxidative bursts and enzymatic defenses [169,170,171,172]. During cold seasons, increasing water levels to improve thermal buffering and reducing HLR to preserve contact time can partially compensate for slower kinetics [126,173].

Based on the limited virus-specific data and broader experience from nutrient and bacterial removal, the working physicochemical windows for antiviral phytoremediation are ~DO > 2 mg·L⁻¹ near inlets, rhizosphere ORP in the range +100 to +250 mV, pH ~6.5–8, and water temperatures preferably above ~10 °C [162,163,164,165,166,167,168]. These ranges are heuristic rather than optimized for viruses; few studies systematically vary these parameters while tracking viral infectivity, so these values should be treated as starting points to be refined by site-specific tracking and future research.

Therefore, routine tracking should focus on functional indicators. Standard physicochemical profiling (DO, ORP, pH, conductivity) should be complemented by periodic assays of peroxidase and protease activities or proxies for exudate production. Sustained declines in enzymatic activity justify stand rejuvenation via selective harvesting or adjusted aeration before downstream performance losses are established [174].

3.2.3. Biomass and Media Maintenance

Optimized phytoremediation systems maintain a renewing surface characterized by abundant fine roots, refreshed periphyton, and unclogged pores (Table 3). Staggered harvesting, which involves removing ~20–40% of above-ground biomass per event two to four times annually, depending on growth rate, promotes root turnover and exudation while preventing thatch build-up [175,176]. The rapid removal of senescent litter is essential because its decomposition elevates DOC and fosters anoxic mats that impair viral capture and enzymatic activity [177,178].

Media stewardship is also essential. Well-graded aggregates resistant to compaction and with limited fines (<1–2 mm, except in thin reactive layers) provide tortuous flow paths and maintain permeability [179]. When the head loss increases, back-flushing with clean water or air-pulse scouring during offline periods may restore the conductivity. Persistent clogging near inlets may require dosing line rotation, raising or flipping near-surface media, or replacing the top ~10–15 cm of media to restore hydraulic conductivity and root penetration [180,181]. Excessive biofilm accumulation can slough during load shocks, releasing colloids and associated viruses; moderate shear via pulsed dosing and avoidance of abrupt HLR changes can help mitigate this. Where recurrent sloughing persists, a small upstream roughing filter can capture the sloughed material before it enters the planted beds [180,181].

3.2.4. Pretreatment, Polishing, and Seasonal Adaptation

Gentle pretreatment stabilizes the initial viral capture barrier without damaging the plants or biofilms (Table 3). Practical options include low-dose coagulant addition (e.g., FeCl₃ at ~5–20 mg·L⁻¹, tuned via zeta potential) to neutralize charge and promote virion aggregation, and mild electrocoagulation in compact tanks at ~1–2 mA·cm⁻² [125,182,183,184]. Both approaches could yield ~1–1.5 log₁₀ gains in early capture and increase DOC tolerance, particularly under cold or high DOC conditions.

For stringent compliance, particularly when non-enveloped viral surrogates dominate or during peak viral loads, macrophyte stages are most effective when paired with compact polishing units such as UV reactors (with confirmed fluence), ferrate or ozone micro-dosing, or short granular activated carbon (GAC) contactors. Hybrid treatment trains commonly show additive viral inactivation, boosting overall removal from ~1–3 log₁₀ in plant-only systems to ~3–7 log₁₀ when UV and/or AOP polishing are included [51,185,186,187,188]. Oxidant placement is critical: to avoid phytotoxicity, oxidants should be placed downstream of planted units, or if upstream stabilization is necessary, applied at low doses with short contact times and rapid quenching [187,188].

As of 2025, most reported CW–UV or CW–AOP hybrids operate at decentralized or pilot scales, from <10 m³·d⁻¹ (household/community systems) to O(10³) m³·d⁻¹ [51,185,186,187,188]. Scaling to larger flows is constrained more by wetland area requirements than by UV/AOP reactor capacity. UV doses in the range of ~10–48 mJ·cm⁻² achieve complete inactivation of many enveloped viral surrogates and substantial reductions of more resistant non-enveloped viruses; AOP doses (e.g., ozone ~0.2–0.5 mg·L⁻¹, ferrate ~0.5–1 mg·L⁻¹) are generally selected to balance virucidal efficacy with minimal phytotoxicity and manageable by-product formation [44,189,190,191,192,193,194,195]. However, virus-specific performance data at full scale remain sparse, and most studies rely on genome copies or surrogates, highlighting the need for more rigorous infectivity-based evaluations.

Explicit seasonal operating envelopes further enhance the resilience of the system. In summer, the HLR can be increased within capacity, biomass removal intervals shortened, and brief daytime aeration used to suppress anoxic mats [144,196]. In cold temperate climates, coupling wetlands with low-enthalpy geothermal systems can stabilize winter temperatures, sustain ~70–85% of summer performance, and reduce the need for greenhouse enclosures [126,173,197,198].

Table 3. Design and operational drivers of viral attenuation in constructed wetlands.

Factor/Strategy	Target Parameters	Key Action/Specification	Performance Metric	Mechanistic Rationale	Ref.
Hydraulic loading rate (HLR)	HLR & distribution uniformity	VSSF: 0.05–0.15 m³ m⁻² d⁻¹; perforated manifold dosing	~1–2 log₁₀ removal; HRT = ~4–15 d	maximizes root–water contact and filtration efficiency; prevents short-circuiting	[54,156,190]
Flow configuration	flow pattern & dead-zone control	baffled FWS/staged islands; dispersion index > 0.7	HRT efficiency ~70–95%; channeling causes up to −30% loss	promotes plug flow (extended home time); increases uniform virion–biofilm interaction	[158,191]
Hydraulic retention time (HRT)	retention stability & redundancy	design: ~4–10 d (+20–40% safety margin for low T)	stable up to 3 log₁₀ removal	sustains contact time for adsorption/inactivation kinetics; reduces desorption risk	[126,159]
Rhizosphere aeration	intermittent air cycles & DO	ON/OFF ~1–2 h cycles; DO > 2 mg L⁻¹ at inlet	enzyme gain ~20–45%; redox maintained (+50 to +200 mV)	boosts oxidative/enzymatic antiviral activity (peroxidases, ROS); prevents anoxic clogging	[44,162]
Redox/nutrient balance	C:N:P Ratio & ORP	C:N:P ≈ 100:10:1; ORP target: +100–+250 mV	infectivity loss 2.5 ± 0.4 log₁₀	optimizes synthesis of antiviral exudates and enzymatic function; stabilizes microbial consortia	[165,166]
Temperature buffering	seasonal heat retention	raise water depth ~10–20% (winter); optional geothermal loop (<10 °C differential)	keeps ~70–85% of summer rate; viral loss: −0.3 log₁₀ per 10 °C drop	counteracts T-dependent reduction in enzymatic/adsorption kinetics; ensures year-round stability	[126,173]
Biomass management	harvest fraction & frequency	remove ~20–40% biomass ~2–4 × yr⁻¹	+0.5–0.8 log₁₀ improvement post-harvest	renews roots and exudation capacity (young plants are more active); prevents DOC release from senescence	[175,176]
Pretreatment (chemical)	charge neutralization & aggregation	FeCl₃ 5–20 mg L⁻¹ or EC ~1–2 mA cm⁻²	+1–1.5 log₁₀ viral gain (primary capture); +20% DOC tolerance	strengthens primary capture by neutralizing negative virion charge; flocculation enhances settling/adsorption	[125,182]
Polishing/disinfection	secondary oxidation	UV ~30–60 mJ cm⁻²; ferrate ~0.5–1 mg L⁻¹; ozone ~0.2–0.5 mg L⁻¹	~5–7 log₁₀ total removal; low phytotoxicity	eliminates residual, recalcitrant infectivity (non-enveloped viruses); ensures safety for reuse standards	[185,188]

3.3. Hybrid Antiviral Plant Systems and Technology Integration

3.3.1. Rationale and Design Principles

Hybrid systems integrate plant-mediated antiviral mechanisms (sorption/filtration, rhizosphere inactivation, internalization, and intracellular degradation) with engineered pretreatment and polishing barriers to enhance log₁₀ removal, stabilize performance under cold or high DOC conditions, and reduce land footprints for urban applications (Figure 5). Typically, vertical or subsurface CWs are preceded by charge-stabilization units (electrocoagulation or optimized coagulant dosing) and followed by UV reactors or GAC contactors. In applications requiring high reuse standards, MBRs may serve as alternative or additional polishing stages [7,70,189,199].

Figure 5. Hybrid antiviral plant system for virus attenuation. System components include bed architecture, active interfaces (roots, porous media, biofilms), and staged removal via primary sorption/filtration (HRT target ~1–2 log₁₀) followed by rhizosphere attenuation (additional bed-volume HRT). Performance indicators include effective HRT, RTD (residence time distribution) variance, clogging/ponding, and channelized flow; operational controls and remedial actions (alternating dosing, inlet rotation, tracer testing, hydro-flushing, media scarification, partial media replacement) limit bypass and maintain predictable log removal. Colors: Solid blue arrows show the main water flow path from influent through pretreatment, the planted bed, and polishing stage to effluent, whereas the colored compartments represent charge-stabilization pretreatment, rhizosphere-based attenuation in the planted bed, and disinfection/filtration processes in the polishing stage.

This design logic exploits mechanistic complementarity: pretreatment enhances early virion capture and reduces colloidal load; planted beds provide sustained biochemical inactivation; and polishing units secure the loss of residual infectivity, especially for robust non-enveloped viruses [70,189]. Across pilot studies, hybrid plant-based systems have consistently outperformed single-stage planted systems under variable influent conditions. For example, macrophyte–UV sequences have converted stable ~2–4 log₁₀ reductions in planted stages into ~3–7 log₁₀ overall removal, while also reducing land requirements relative to plant-only configurations [51,185,186,187,188,196]. These gains are maximized when upstream mixing and contact are well-engineered and when downstream polishing units are sized to residual pathogen loads, not influent peaks [200,201].

3.3.2. UV- and AOP-Enhanced Macrophyte Systems

UV provides a compact, energy-efficient- polishing step that inactivates RNA and DNA viruses primarily via nucleic acid photodamage. Performance depends on the delivered fluence, water transmittance, and shielding by particulates and colored DOC [44,189,190,191,192,193,194]. Typical UVC doses of ~10–48 mJ·cm⁻² can achieve complete inactivation of many enveloped viral surrogates and significant reductions in non-enveloped viruses; for example, SARS-CoV-2 exhibits ~50% reduction within ~1.4 min at laboratory irradiance levels [189,190,191]. When combined with planted beds, UV treatment addresses residual virions that persist after rhizosphere-mediated inactivation, providing a final safety barrier for reuse.

AOPs (ozone, ferrate, UV/H₂O₂) applied pre- or post-UV further improve performance by degrading UV-absorbing chromophores and reducing DOC, thus improving UV transmittance and reducing competition for sorption sites on roots and periphyton. Constructed wetland–ultraviolet (UV)–hydroxyl radical (HO·) trains have shown the concurrent removal of pesticides and total organic carbon (TOC), illustrating how modest pre-oxidation can enhance downstream enzymatic efficacy [44,191,195]. However, humic substances and melanoidins can absorb UV light and quench reactive species, requiring modest coagulation or roughing filtration upstream of UV reactors in such matrices.

From a practical perspective, UV/AOP–CW hybrids are state-of-the-art for decentralized antiviral treatment, but their broader deployment is limited by operational and maintenance (O&M) complexity (lamp cleaning and replacement, oxidant management), sensitivity to water quality, and cost in low-resource settings. Crucially, most published performance data relate to surrogates or molecular markers, not infectivity, so claims of complete inactivation must be interpreted cautiously.

3.3.3. Genome Editing and Synthetic Biology for Plant–Microbe Defense

Targeted genome editing and microbiome engineering offer conceptually attractive routes to strengthen biological antiviral barriers, especially rhizosphere inactivation and intracellular degradation, without sacrificing hydraulic performance. CRISPR-Cas-mediated editing in aquatic and wetland plants has been used to modify antioxidant capacities, ion transport, and exudation profiles, which are associated with improved contaminant removal and stress tolerance [202,203]. Synthetic biology approaches have enabled the design of engineered rhizobacterial consortia that partition functions, such as lytic enzyme secretion, ROS generation, and extracellular polymeric substance production, potentially augmenting virion capture and capsid damage at the root interface [204,205].

However, these advances remain largely at the proof-of-concept stage. Genome-edited macrophytes and engineered consortia have been evaluated mainly in microcosms or greenhouse experiments, often with non-viral endpoints. No full-scale antiviral phytoremediation system deploys genetically modified plants or microorganisms, and their translation to the field is constrained by biosafety concerns, GMO regulations, and ecological risks [206,207,208]. Emerging risk mitigation- strategies, such as confined pilot trials and microbial kill-switch circuits, may eventually enable cautiously controlled deployments; however, genome editing and synthetic biology should be viewed as promising research directions rather than ready-to-implement solutions.

3.3.4. Digital/AI Tracking and Scaling Feasibility

Digital tracking technologies (optical sensors, DOC probes) coupled with rule-based control have improved hydraulic and redox stability in some planted systems [121]. Early AI-assisted frameworks have been used to predict and optimize flows, HRT, aeration duty cycles, and coagulant dosing based on bulk water quality data [120,121]. However, no published studies have reported AI control explicitly optimized against viral infectivity or surrogates in CWs. AI should be regarded as an emerging tool with the potential to coordinate decentralized hybrid systems once reliable virological surrogates and robust sensor networks are available.

Scaling analyses highlight major non-technical barriers, such as land scarcity, fragmented governance, financing constraints, and limited O&M capacity [209,210]. Modular decentralized wastewater treatment (DEWATS) hybrids can alleviate footprint constraints and enable phased implementation; however, durable operation depends on community engagement, capacity building, and long-term support for maintaining critical parts (e.g., UV lamps, sensors, and control software) [209,210]. In many low-resource settings, minimum-viable hybridization configurations such as coagulation + planted bed + compact UV reactor, may offer the best reliability-to-cost ratio, with AOPs or membranes reserved for applications demanding stringent reuse standards. Here again, the limiting factor is not conceptual design but institutional and financial capacity, suggesting that TEAs and governance analyses are as critical as mechanistic optimization.

4. Practical Applications and Implementation

4.1. Constructed Wetland Systems (CWs)

4.1.1. Wetland Configurations and Hydraulic Design

CWs are engineered ecosystems that integrate capture, rhizosphere inactivation, viral internalization, and intracellular degradation under controlled flow regimes (Figure 6). Different CW configurations emphasize different subsets of the four antiviral barriers.

Figure 6. Schematic overview of major constructed wetland (CW) configurations and a hybrid treatment train. (A) Free water surface (FWS) wetland showing attenuation via sedimentation, sorption/filtration on biofilms and root surfaces, modest rhizosphere chemistry effects, and sunlight-driven photolysis/UV exposure. (B) Horizontal subsurface flow (HSSF) wetland highlighting steady filtration through a gravel/media bed, adsorption, and suboxic–anoxic redox conditions along the flow path. (C) Vertical subsurface flow (VSSF) wetland with intermittent dosing, unsaturated flow and aerobic microzones that enhance filtration and rhizosphere oxidation, supporting downstream internalization and degradation processes. (D) Example sequential hybrid CW train combining VSSF and FWS stages with polishing (e.g., compact UV) to meet reuse-oriented effluent standards, with seasonal operation adjustments indicated (e.g., winter HRT increase).

Free water surface (FWS) cells combine light exposure, oxygenation, and photochemical pathways with plant–biofilm interfaces. They primarily reinforce barrier 1 (sorption/filtration) via root and biofilm surfaces and provide a modest barrier 2 through rhizosphere chemistry and photolysis in the open water column [201,211].

Horizontal subsurface flow (HSSF) wetlands provide steady filtration through saturated porous beds, strengthening barrier 1, but often operate under suboxic or anoxic conditions that limit oxidative rhizosphere processes (barrier 2) [77,201,212].

Vertical subsurface flow (VSSF) wetlands, intermittently dosed via perforated manifolds, maintain aerobic microzones, high specific surface areas, and short diffusion distances. These conditions support both robust barrier 1 and enhanced barrier 2, and create the most favorable preconditions for barriers 3–4 (internalization and intracellular degradation) by sustaining active root and microbial metabolism [213].

Unit-process wetlands can arrange these configurations sequentially so that phototransformation and microbial degradation are staged to address co-located pathogens and trace organics in municipal effluents [214,215]. However, densely vegetated FWS systems may suffer from canopy shading and recirculation, which suppress photolysis and reduce first-barrier- contact, often resulting in <1 log₁₀ viral removal without hydraulic correction [201]. HSSF wetlands are prone to anoxic conditions; column and sand-bed studies have reported diminished adsorption and slower inactivation under anaerobic conditions, consistent with weaker electrostatic fixation and reduced oxidative chemistry [77,201,212]. VSSF designs can deliver ~2–3 log₁₀ reductions at HRTs of ~5–10 days when well aerated and dosed [54,213].

Field practice combines well-distributed dosing, baffling, and staged cells to maintain effective HRTs near design values and stabilize contact during fluctuations in organic matter or temperature [110,201]. Design heuristics recommend sizing the initial planted stage to achieve ~1–2 log₁₀ viral reduction via capture/filtration (barrier 1), followed by an additional ~0.5–1.0 bed volume to support rhizosphere and intracellular processes (barriers 2–4) beyond the efficiency plateaus set by pore saturation and biofilm maturation [62,201,216]. Cold season operation benefits from modest HRT increases and flow equalization to counteract reduced reaction rates [126,217]. Available data suggest that, for standalone CWs, winter viral log₁₀ removals are typically ~0.5–1.0 lower than summer at constant HRT, implying a ~30–60% reduction in performance; however, most of these estimates are extrapolated from broader pathogen datasets and are not yet underpinned by long-term infectivity measurements [110,126,173,217].

4.1.2. Substrate, Vegetation, and Rhizosphere Dynamics

Substrate selection governs both hydraulics and microbiology (Table 4). High porosity and biofilm-supportive media increase the interfacial area and microbial colonization, reinforcing barrier 1, while appropriate hydraulic loading rates limit short-circuiting [218]. Reactive media, such as steel slag derivatives, particularly basic oxygen furnace slag, can contribute to pathogen reduction via transient high pH microzones (pH ≈ 10.6–11.4) and enhanced phosphorus capture [219,220]. These pH excursions may also enhance barrier 2 by damaging capsid proteins and viral envelopes; however, robust virus-specific evidence is limited, and pH management and safety assessments are essential.

Alternate day loading in VSSF wetlands promotes re-oxygenation and nitrification–denitrification cycling; during rest periods, redox rebound and substrate re-oxygenation maintain enzyme activity that supports antiviral chemistry (barrier 2) [213]. Plant selection must balance hydraulic integrity and biochemical potency. P. australis and Typha spp. provide aerenchyma-driven oxygen transfer, strong root mats, and stable periphyton, reinforcing barrier 1 and enabling barrier 2, but aromatic and medicinal taxa (e.g., O. basilicum, S. cusia) enrich the rhizosphere with polyphenols and other antiviral metabolites that strengthen barrier 2 and potentially barriers 3–4 [135,221]. Root-associated biofilms structured by exudates (organic acids, amino acids, and fatty acids) enhance nutrient cycling and contaminant attenuation, and plant growth-promoting rhizobacteria (PGPR) coordinate division of labor metabolism, including lytic enzymes relevant to viral inactivation [41,218].

Polyculture wetlands leverage complementarity and functional redundancy by mixing structural macrophytes with antiviral-rich species, widening seasonal operating windows, and buffering influent variability (see previous Table 2) [145,222]. Cold climate adaptations include greenhouse enclosures, selection of cold-tolerant species, and experimental use of CRISPR-edited macrophytes with enhanced stress tolerance. However, genome-edited plants remain at the proof-of-concept stage and require rigorous biosafety reviews before open-system deployment [217,223].

Table 4. System configurations and innovations for antiviral hybrid wetlands.

Components	Configuration Description	Primary Antiviral Mechanisms	Demonstrated Performance	Innovation Value/Application	Ref.
FWS wetland	shallow vegetated channels (~0.3–0.6 m); open photic zone	photolysis, oxidation, biofilm sorption (low shear)	~0.5–1.0 log₁₀ baseline; up to 2.0 with baffling	simple, low-cost system; sensitive to temperature and climate variability	[201,211]
HSSF wetland	saturated porous bed; laminar flow	filtration and anoxic biofilm degradation (stable pH)	1.0 ± 0.3 log₁₀ (n = 15); high stability across pH changes	filtration-dominant removal; good hydraulic control; limited oxidative capacity	[77,212]
VSSF wetland	intermittent dosing (alt-day); aerated percolation	adsorptive capture and oxidative decay on roots/media (high O₂)	~2–3 log₁₀ at HRT ~5–10 d	high efficiency (~2–3× HSSF); reduces land area; requires mechanical dosing/aeration	[54,213]
Multistage hybrid CWs	sequential VSSF–FWS or VSSF–UV trains (multi-barrier approach)	combined filtration, oxidation, photolysis, enzymatic action	~3–7 log₁₀ total removal (highest efficacy)	meets stringent reuse standards; functional redundancy buffers system failures	[110,158]
Substrate innovation	gravel, slag, zeolite, biochar, ferric media	enhanced adsorption; pH ~10–11 microzones; ROS generation	+10–30% extra removal from reactive layers	increases specific surface area; biochar adds catalytic/adsorptive properties; controls metal mobility	[218,219,220]
Vegetation selection	Phragmites, Typha, Ocimum, Strobilanthes (targeted functional traits).	O₂ release, enzyme induction, antiviral metabolite exudation	~2–4 log₁₀ (field mean); 85–95% infectivity loss	shifts CWs from simple filtration to biochemically active reactors; cost-effective performance boost	[135,223]
Digital tracking/AI	IoT sensors (DO, ORP, metabolites, microbial activity)	predictive control and early alerting (machine learning integration)	~12–18 h lead time before viral breakthrough prediction	improves reliability/uptime; enables adaptive dosing/flow control; important for fluctuating loads.	[224,225]
Synthetic biology integration	engineered microbial consortia & biosensors (PGPR, lytic strains)	self-regulated enzymatic capture loops; enhanced proteolysis	+1–2 log₁₀ added potential (proof-of-concept)	high potential for targeted virus/pathogen removal; highly specific mechanism; requires regulatory acceptance	[136,226,227]

4.1.3. Operational Management, Seasonal Performance, and Digital Tracking

Operational management must maintain a renewing surface state characterized by staged harvesting (~20–40% of above-ground biomass per event), removal of senescent litter, and periodic media maintenance to prevent pore blockage and anoxic mats that undermine barriers 1 and 2 [62,228]. Routine functional tracking (DO, ORP, pH, conductivity, head loss) should be complemented by periodic enzyme activity assays (peroxidase, protease) or exudate markers to detect declines in rhizosphere reactivity (barrier 2) before performance degradation becomes difficult to reverse [213,218].

ML models trained on multi-season plant, hydraulic, and chemical data have shown high predictive accuracy (R² ≈ 0.85–0.95) for nitrogen and COD and can be augmented with virtual samples under sparse field data [224]. Root exudate biomarkers integrated with artificial intelligence (AI) have been proposed as early warning indicators for viral breakthrough (lead times ~12–18 h), and field-deployable CRISPR–Cas12a assays can detect viral RNA within ~30 min to support event-driven operational controls [224,225]. These digital approaches have been shown mainly for bulk water quality; no published systems have yet optimized operations explicitly against viral infectivity endpoints, so their antiviral utility remains prospective rather than confirmed.

Seasonal operational playbooks can enhance the reliability. Winter mode typically reduces hydraulic loading rates by ~10–30%, increases water depth for thermal buffering, and lengthens rest periods to maintain HRT, but summer mode tightens harvesting intervals and may incorporate brief daytime aeration to suppress anoxic mats [110]. Even with such measures, current data indicate that winter virus removals are often ~0.5–1.0 log₁₀ lower than summer for stand-alone CWs [110,126,173,217], highlighting the importance of downstream polishing, where stringent reuse standards are applied.

4.1.4. Implementation, Lifespan and Governance

Pilot projects in resource-limited- contexts have shown the feasibility and co-benefits of CWs, including livelihood opportunities and local stewardship. For example, participatory projects in the Philippines illustrate how co-design- and community O&M can improve system longevity and compliance [229]. With routine maintenance (periodic media rehabilitation and replanting), CWs are typically designed for a service life of ~15–25 years [217,228]. However, most antiviral performance datasets span only ~3–5 years or less, so extrapolating long-term virus removal from short-term- data remains speculative.

Persistent adoption gaps arise from regulatory exclusion, limited long-term pathogen tracking, and fragmented governance. Updated guidance emphasizing adaptive management and performance verification, including viral infectivity metrics, would accelerate mainstreaming [201,230]. Synthetic biology and microbiome engineering offer potential tools for augmenting CW robustness: engineered microbial consortia could partition lytic enzyme production, ROS generation, and extracellular polymer synthesis to enhance capture and inactivation (barriers 1–2), while biosensors and gene-circuit sentinels could provide online diagnostics [226,227]. However, such approaches remain limited to laboratory and microcosm studies and face substantial biosafety and regulatory hurdles.

A pragmatic implementation pathway emphasizes minimum-viable hybridization: low-dose coagulation upstream of planted beds combined with compact UV downstream, while simultaneously developing data and governance frameworks that certify viral infectivity loss alongside conventional water quality metrics [135,136,201]. Collectively, these strategies translate mechanistic insights into deployable, resilient antiviral CWs, hydraulically disciplined, biochemically active, digitally supervised, and socially embedded, capable of delivering ~3–7 log₁₀ virus reduction when appropriately paired with polishing [110,158,201].

4.2. Modular and Scalable Designs

4.2.1. Modular Treatment Architectures

Modular treatment architectures assemble plant-based units and compact physicochemical steps into standardized modules that can be scaled, duplicated, or reconfigured as load demands and reuse targets evolve (Figure 7) [231,232]. This approach aligns with decentralized wastewater (DEWATS) strategies for dense urban and dispersed peri-urban contexts, enabling phased implementation and plug-in upgrades that reduce capital risk and accelerate service provision [104]. Governance analyses highlight the role of modularity in lowering institutional barriers by enabling performance-verified subunits, for example, a planted bed combined with UV as a minimum viable hybrid, that communities can adopt and expand as financing and operational capacity mature [104].

Figure 7. Conceptual modular hybrid antiviral planted treatment systems enabling scalable deployment and reuse-oriented performance. (A) Standardized “minimum viable hybridization” treatment train coupling compact charge-stabilization/roughing (coagulation or electrocoagulation) with a compact planted unit (intensified VSSF bed or baffled FWS cell; barriers 1–2: sorption–filtration and rhizosphere inactivation; target ~2–4 log₁₀ reduction) and downstream polishing (barrier 4: UV or GAC; optimized for residual load) to achieve an overall target of ~3–7 log₁₀ reduction. (B) Compact urban integration concepts (rooftop/podium wetland; linear green-corridor cells) emphasizing staged contact zones, pulsed/alternating dosing, and minimal land-use conflict. (C) Mobile and adaptive containerized units with interchangeable cartridges, rule-based/event-driven control, and rapid pathogen analytics to manage variable influent conditions. (D) Future enhancements using advanced materials and biotechnology (e.g., 3D-printed scaffolds, redox-active fillers/micronutrients, engineered plant–microbe consortia, and biosensing) to increase reactive surface area and real-time performance feedback. Color: blue arrows show the main wastewater flow path across standardized treatment modules; green elements highlight planted or green-infrastructure units; orange and purple modules indicate compact urban, mobile/containerized, and advanced materials/biotechnology options.

The design follows the antiviral barrier framework: an initial compact charge stabilization or roughing step (coagulation, electrocoagulation) enhances barrier 1 (early virion capture); planted units provide barriers 1–2 (sorption–filtration and rhizosphere inactivation); and a downstream UV or GAC module ensures loss of infectivity for recalcitrant, non-enveloped viruses (barrier 4) [233,234,235]. Standardized hydraulic and electrical interfaces between modules (header geometry, bypass loops, and 24 V control systems) help with field assembly and maintenance by local operators [236,237]. Module sizing targets stable partial reductions of ~2–4 log₁₀ in the planted stage, with enough headroom for seasonal performance. Polishing units are sized to treat residual pathogen loads rather than influent peaks, thus optimizing energy use and footprint [204,238].

4.2.2. Compact Wetland Designs for Urban Areas

Compact CWs, including intensified VSSF beds, baffled FWS cells, and unit-process wetlands, maximize the specific interfacial area and narrow residence time distributions, enhancing viral contact efficiency within limited land areas [94,204]. Urban pilots have shown that high-porosity media, multi-port influent dosing, and short flow paths can maintain effective first-barrier capture and rhizosphere chemistry under varying organic loads, particularly when combined with downstream UV polishing [236,237,239]. Rooftop and podium-deck wetlands can exploit gravity-fed pulsed dosing and solar exposure to support periphyton, but green corridors along drainage channels convert linear hydraulics into staged contact zones with minimal land-use conflict [240,241].

To mitigate performance declines during cold seasons or high-DOC events, modular pretreatment stages (coagulation or electrocoagulation cartridges) stabilize virion capture upstream of compact beds, and UV or GAC skids secure compliance during peak viral loads [242]. Operations remain straightforward: alternating day dosing preserves media porosity and oxygen supply, and quarterly tracer tests verify that effective HRT remains near design values despite biofilm growth [243,244].

4.2.3. Mobile and Adaptive Treatment Units

Trailer-mounted or containerized modular units offer rapid deployment for outbreak response, festivals, or disaster relief, with flexibility for redeployment as demand shifts [236,245]. To operationalize mobile modules as decision-ready assets, rapid pathogen analytics (e.g., RT-RPA–CRISPR–Cas12a assays) can detect viral RNA within ~30 min, connecting with low-power- field readers for event-driven operational control [246,247,248]. These data streams can support rule-based changes in flow equalization, aeration cycles, and coagulant dosing, helping to maintain antiviral efficacy despite episodic influent variability [210,249]. Given the variable influent chemistry in mobile deployments, interchangeable upstream destabilization cartridges (coagulant or electrocoagulation) and downstream UV modules preserve reliability when non-enveloped viral surrogates predominate or colored dissolved organics attenuate UV transmittance [165,250].

4.2.4. Advanced Materials and Bio-Digital Rhizosphere Enhancements

Additive manufacturing (3D printing) enables the production of root zone- scaffolds and advanced media with high, cleanable surface areas and engineered pore networks that resist compaction and clogging under pulsed loading, improving initial viral capture and periphyton stability within a compact footprint [251]. Incorporating redox-active fillers or slow-release micronutrients into these scaffolds may sustain enzymatic activity and ROS generation, which are critical for capsid and envelope damage, although virus-specific performance data are still limited [105,252].

Synthetic biology and biosensing layers further enhance the antiviral capacity and support closed-loop control. Engineered plant–microbe consortia may partition lytic enzyme secretion, EPS production, and ROS generation, while gene circuit-based biosensors provide real-time reporting of stress or viral surrogate signals [253,254]. In decentralized contexts, such biosignals could trigger simple control systems that autonomously adjust dosing or aeration, complementing modular governance frameworks [255,256,257]. These approaches are at an early experimental stage, field-scale antiviral demonstrations are lacking, and regulatory acceptance remains a significant barrier.

Therefore, a pragmatic near-term pathway focuses on minimum-viable hybridization constructed from standardized modules: upstream destabilization via cartridge coagulation or mild electrocoagulation, a compact planted bed (intensified VSSF or baffled FWS), and a small UV skid. This configuration is equity-oriented, scalable, and capable of achieving ~3–7 log₁₀ overall viral reductions when tuned to residual pathogen loads rather than influent peaks, as in several pilot-scale demonstrations [70,194,258].

4.3. Hybrid Treatment Systems and Economic Considerations

4.3.1. Cost–Performance and Governance Alignment

Hybrid treatment integrates plant-based barriers with compact physicochemical processes to convert the partial reductions achieved in planted units (capture and rhizosphere inactivation) into compliance-grade removals while reducing land use and operational complexity [105,258]. Treatment targets should be specified based on reuse and effluent standards, clarifying the critical role of hybrid systems in providing final polishing for agricultural reuse or urban non-potable applications [259,260].

In resource-constrained environments, single-step advanced treatment systems are often hampered by high capital costs, energy demands, and institutional fragmentation. Modular hybridization distributes performance requirements across smaller, verifiable units and can reduce oversizing for winter operations, as downstream UV/AOP can buffer seasonal declines in CW performance [259,260]. For small to medium flows (<~5000 m³·d⁻¹), decentralized CW–UV hybrids typically show lower specific operational expenditure (OPEX) and energy use than MBRs or MBR–AOP systems at comparable viral log₁₀ reduction targets, particularly where land costs are moderate and labor is available [194,261,262,263,264]. However, most published cost data are not virus-targeted; they are based on conventional water quality metrics, so their extrapolation to antiviral performance must be made cautiously.

Governance mechanisms, such as clear reuse standards, performance-based procurement, and community-based O&M contracts, further enhance feasibility by aligning incentives between regulatory agencies and operators [262,265]. Without such frameworks, hybrid systems risk underperforming, regardless of their intrinsic technical potential.

4.3.2. Plant-Based and Physicochemical Hybrid Systems

Field and pilot-scale- studies indicate that macrophyte treatment stages with Typha and Phragmites can achieve ~2.0–3.5 log₁₀ reductions in viral surrogates under optimized hydraulics. Subsequent UV disinfection typically provides an additional ~1–3 log₁₀ reduction, depending on the fluence and water transmittance [70,266]. Floating wetland configurations using Eichhornia crassipes (water hyacinth) combined with compact UV units have achieved ~1.5–2.1 log₁₀ extra reduction for norovirus surrogates while maintaining relatively low capital expenditure (CAPEX) and offering options for biomass valorization [267,268,269]. Where turbidity and colored DOC reduce UV effectiveness, upstream light touch charge destabilization (coagulants or mild electrocoagulation) enhances early viral capture and restores downstream UV efficiency [270,271].

For applications demanding higher assurance, electrochemically enhanced membrane bioreactors (e-MBRs) have achieved ~2.8–5.0 log₁₀ reductions in SARS-CoV-2 through combined filtration and in situ oxidation. When deployed as polishing units after the planted stages, e-MBRs can be downsized to treat residual viral loads, improving energy efficiency relative to full-flow application [7,199]. The overall design sequence leverages mechanistic complementarity: upstream destabilization for capture (barrier 1), planted beds for biochemical inactivation (barriers 1–2, with contributions to 3–4), and UV, AOPs, or membranes for final infectivity loss (barrier 4) [154].

4.3.3. Financing, O&M Economics, and Scale-Up

Hybridization supports stepwise financing: starting with a minimum viable hybrid (e.g., coagulation cartridge + planted bed + small UV unit) and incrementally adding modules as reuse standards tighten or flows increase [263]. Blended financing, municipal budgets, green bonds, microfinance for community O&M, and Public–Private Partnership (PPPs), can bridge CAPEX gaps and secure maintenance commitments [264]. Lifecycle cost assessments (LCAs) should account for avoided land purchase, reduced need for winter oversizing, and revenue or cost offsets from biomass valorization (e.g., compost, biochar), which can improve the net present value of plant-centric systems [7,199].

System reliability depends on lean but consistent O&M: periodic harvesting, inlet rotation, cartridge replacement for pretreatment, UV sleeve cleaning, lamp replacement on monthly to annual cycles, and simple sensor-based feedback (DO, ORP, head loss) to trigger rule-based operational changes [110,272,273,274]. Digital tracking can reduce operational risks by forecasting influent perturbations and optimizing set-points, provided that the tools remain accessible to local operators and do not introduce new failure modes [110,275].

Regarding rentability, current evidence suggests that well-designed hybrid CWs can be cost-competitive with or cheaper than advanced mechanical alternatives on a per log₁₀ removal basis for small–medium flows, particularly when energy prices are high and land is available. However, the absence of virus-specific LCAs and TEAs is a significant limitation, and robust economic comparisons remain an open research need rather than a settled conclusion.

4.3.4. Equity, Biosafety, and Major Limitations Compared with Alternatives

The fair deployment of hybrid systems requires modular governance and context-sensitive technology choices, including appropriately sized UV units, affordable consumables, and avoidance of hard-to-source reagents [276,277,278]. Emerging synthetic biology innovations, engineered plant–microbe consortia partitioning lytic enzymes, ROS generation, and EPS production, could enhance planted stage virus removal and reduce polishing requirements, but they demand rigorous biosafety assessment, continuous tracking, and, where appropriate, genetic kill switches [276,279,280]. These approaches remain largely experimental and are not yet ready for routine implementation.

The major limitations of antiviral phytoremediation, even in hybrid form, include:

(1): Large land requirements relative to compact advanced processes are required.
(2): Seasonal and climatic sensitivity, with winter performance typically ~0.5–1.0 log₁₀ lower than summer, unless mitigated.
(3): Incomplete and variable log₁₀ removals for robust non-enveloped viruses in standalone CWs (~1–3 log₁₀) make polishing essential for high-risk uses.
(4): High dependence on local O&M capacity (harvesting, clogging control, component maintenance).
(5): There is limited insight into the degradation products and long-term stability of antiviral function, given the short duration of most studies.

Compared to chlorination, hybrid CW systems avoid carcinogenic disinfection by-products (THMs, HAAs) and reduce chemical dependence; however, chlorination remains cheaper per m³ and can achieve high log₁₀ removals when carefully managed [5,6,18]. Compared to standalone UV, CW hybrids reduce lamp loading by handling much of the particulate and organic burden biologically, thus improving UV robustness. Compared to MBRs and other advanced processes, CW hybrids offer lower energy demand and fewer by-products, but at the cost of land, slower response times, and greater variability [7,8,9,10,44,189,190,191,199,259,260,261,262]. Antiviral phytoremediation is best viewed as a central component of low-energy, multi-barrier treatment trains rather than a universal replacement for engineered disinfection.

5. Challenges and Prospects

5.1. Current Challenges and Persistent Gaps

5.1.1. Variability of Rhizosphere Antiviral Activity

Antiviral phytoremediation is constrained by the intrinsic stability of many non-enveloped viruses and the variable nature of plant–microbe antiviral interactions. Enveloped virions are generally susceptible to oxidative chemistry and enzymatic degradation in the rhizosphere, but non-enveloped- particles with robust icosahedral capsids often persist, yielding only ~1–3 log₁₀ reductions in well ran CWs unless supplementary treatment steps are included [281,282,283]. This structural asymmetry implies that systems relying predominantly on barrier 1 (physical capture via roots, biofilms, and porous media) are vulnerable to desorption or downstream breakthrough under fluctuating hydraulic conditions [284,285,286].

Rhizosphere antiviral activity is not a fixed property; it fluctuates with temperature, nutrient supply, and redox conditions, which modulate the synthesis of antiviral metabolites and lytic enzymes in the rhizosphere. These environmental drivers generate seasonal plateaus in viral inactivation, even when contact times are apparently adequate [287,288]. In colder seasons and in influent with high DOC, reduced exudation rates and slower enzyme turnover weaken barrier 2 (rhizosphere-mediated inactivation) and downstream barriers 3–4 (internalization and intracellular degradation), so that physical capture dominates [289,290,291]. Winter viral removals are commonly ~0.5–1.0 log₁₀ lower than those in summer, even at similar HRTs [110,126,173,217].

The diversity of viruses further complicates predictive modeling and design. Persistence and inactivation kinetics differ markedly between strains (e.g., rotaviruses vs. enteroviruses), so plant–microbe assemblages designed for one surrogate may perform poorly against others unless systems are explicitly designed to create multilayered, mechanistically distinct barriers [220,292]. Treating virus removal as a single scalar performance metric conceals these differences and risks overconfidence in systems confirmed on limited surrogate sets.

Collectively, these biological constraints indicate that hybrid systems and seasonally adaptive operations are essential, particularly in cold or DOC-rich environments. The single-barrier reliance on capture or rhizosphere chemistry is unlikely to deliver robust antiviral performance across virus classes and seasons [154,293,294].

5.1.2. Engineering and Operational Constraints

From an engineering perspective, antiviral phytoremediation systems are limited by how much viruses encounter and live at reactive interfaces. Even modest dead zones or hydraulic short-circuiting can reduce effective HRT, halving expected contact times and reducing first-barrier- viral removal to <~1–2 log₁₀, regardless of plant traits [154,295,296]. Clogging, which occurs through inlet obstruction, pore occlusion by fines, and biofilm aging, reduces media porosity and interfacial area, thus lowering virus capture and suppressing rhizosphere activity. Restoring hydraulic performance often requires disruptive interventions (media replacement, scarification), which are rarely budgeted or planned for in long-term O&M [40,230].

Material selection imposes additional trade-offs. Alkaline industrial by-products and reactive substrates may transiently enhance pathogen reduction via high pH microzones and adsorption; however, they require tight pH control to avoid phytotoxicity and downstream regulatory non-compliance [297,298]. Biologically favorable, high-surface-area media support biofilms and enzymatic processes but can accelerate clogging if dosing and harvesting protocols do not maintain a young active surface [110,299]. Operational discipline, alternating day dosing in VSSF beds, seasonal HLR adjustment, selective harvesting, and tracer-based verification of HRT is seldom standardized beyond research pilots, creating a persistent gap between laboratory-grade performance and field reliability [110,300]. Without codified operational playbooks and robust training, antiviral phytoremediation may be perceived as unreliable for reuse.

5.1.3. Tracking, Standards, and Methodological Gaps

The most serious constraint of antiviral phytoremediation is methodological. Many demonstration studies report substantial reductions in viral RNA/DNA but do not assess infectivity, which is the definitive metric of public health- protection [301,302]. In particular:

Viral internalization and intracellular degradation in full-scale systems are almost exclusively inferred from molecular data (PCR/qPCR detection of genomes in plant tissues) rather than from direct infectivity assays or microscopy [61,62,63,301].
PCR/qPCR signals cannot distinguish intact virions from adsorbed or degraded fragments; thus, viral genomes in roots do not demonstrate functional internalization or inactivation.
Several studies have reported larger log₁₀ reductions in genome copies than in plaque-forming units, suggesting that molecular assays can overestimate true inactivation [302,303].

Barriers 3–4 (internalization and intracellular inactivation) remain conceptually plausible but empirically under confirmed in real wastewater CWs. Treating them as fully measured and designable features is premature. Many published high removal claims rely on short-term, small-scale tests with surrogate viruses under controlled conditions, with limited replication and no long-term performance tracking.

Standardized, infectivity-centered protocols integrating surrogate selection, dose response relationships, and side-by-side qPCR and infectivity assays remain scarce in field deployments, hampering meta-analysis and conservative design [303]. There is also a lack of agreed verification frameworks: minimum HRT testing, enzyme activity proxy panels, and explicit specification of event-driven UV or AOP backstops are rarely built into regulatory approvals [304,305].

Economic and governance data show similar gaps in the literature. Few studies have provided virus-focused life cycle cost analyses that include seasonal operations, routine media maintenance, and analytical tracking, making it difficult to assess the long-term affordability of decentralized systems, where modular hybrids are most relevant [306,307]. Pathways for translating promising enhancements such as biochar amendments that stabilize redox and adsorption, or synthetic biology approaches that reinforce rhizosphere and intracellular barriers into permitting and biosafety frameworks for open systems are largely undefined [308,309].

Addressing these gaps will require the coordinated development of infectivity-based tracking protocols, operational playbooks, and regulatory guidance for novel materials and engineered consortia, alongside long-term field trials that explicitly track antiviral performance over multi-year horizons.

5.1.4. Fate of Degradation Products and Mineralization Potential

From a chemistry perspective, antiviral phytoremediation generates two broad classes of transformation products:

(1)

Virus-derived degradation products include

(a): Peptides and amino acids from capsid and envelope proteins,
(b): Nucleotides and nucleobases from viral genomes, and lipids and fatty acids from the envelopes.

These are biogenic molecules readily assimilated or further mineralized by the rhizosphere and planktonic microbial communities. There is no evidence that viral degradation yields persistent or uniquely hazardous organic by-products; the main risk is incomplete inactivation, not toxic transformation products [20,78,80,81].

(2)

Transformation products of plant-derived antiviral compounds and co-contaminants, such as:

(a): Oxidized and polymerised polyphenols (e.g., quinone-type intermediates, humic-like macromolecules),
(b): Partial oxidation products of co-occurring micropollutants (e.g., pharmaceuticals). Existing studies suggest that polyphenols and flavonoids usually undergo humification and partial mineralization, contributing to soil and sediment-like organic matter [20,42,310,311]. Intermediate quinone-type species can be redox-active; however, systematic ecotoxicity assessments in CW effluents are rare, and the specific contribution of antiviral exudates to overall effluent toxicity is essentially unquantified.

In principle, the organic matter embodied in viral particles is amenable to near-complete mineralization (CO₂, H₂O, biomass) across multi-stage plant–microbe systems. Almost no studies have closed the carbon and nitrogen mass balance for viral inputs, and the fate of co-contaminant transformation products is more concerning than that of viruses themselves. This highlights the need for integrated fate and effects studies that combine targeted and untargeted- chemical analyses with infectivity assays and ecotoxicological tests.

5.2. Prospects and Emerging Innovations

5.2.1. Genetic Engineering to Enhanced Antiviral Activities

Genetic engineering offers targeted strategies to enhance the two primary biological antiviral barriers, rhizosphere inactivation (barrier 2) and intracellular degradation (barrier 4), without compromising hydraulic performance. CRISPR–Cas genome editing could, in principle, modulate the antioxidant capacity, exudation profiles, and proteostasis pathways that are critical for capsid damage and genome cleavage [312]. Plants engineered for increased late endosomal restriction, ROS generation, and protease/RNase activity may reduce the contact time required for enveloped viruses and, to a lesser extent, for non-enveloped viruses.

Complementary approaches include the expression of antiviral decoy receptors or lectin-like proteins that bind viral surface motifs extracellularly, promoting aggregation and capture within the root or periphyton matrix before internalization [313,314]. Localizing such proteins in root exudates or apoplastic spaces could intercept virions at key interfaces while reducing growth penalties. However, the practical status of these strategies is nascent:

The genetic modification of wetland plants and rhizobacteria has been shown mainly in laboratory or greenhouse experiments, often with stress tolerance or metal uptake endpoints rather than antiviral performance [202,203].
No full-scale antiviral phytoremediation system operates with genome-edited macrophytes or engineered microbial consortia.
For many reuse applications, well-designed polyculture CW–UV hybrids have achieved ~3–7 log₁₀ viral reductions without genetic engineering, suggesting that editing is not a universal prerequisite.

Genetic engineering may become necessary or attractive in demanding niches, such as cold or saline climates with stringent reuse standards, exceptionally high viral loads, or severe land constraints, where incremental gains in barriers 2–4 could significantly reduce the size or energy burden of the polishing units. So translational research should focus on bench-to-pilot pipelines that link genetic modifications to barrier-specific metrics (enzyme activities, ROS fluxes) and infectivity loss for both enveloped and non-enveloped surrogates under realistic ionic strength, DOC, and temperature regimes [315]. Until such pipelines are shown and regulatory approvals are obtained, gene editing should be treated as a research frontier, not as an immediate design tool.

5.2.2. Biosafety and Biocontainment Strategies

Any genetic enhancement program must integrate robust biocontainment strategies. These include conditional containment systems, sterile triploid or polyploid cultivars, and chloroplast-targeted transgenes that reduce gene flow while preserving plant vigor and remediation efficacy [316,317]. Spatial confinement via biofilm-centric cultivation and physical barriers can further restrict dispersal while supporting dense periphyton communities that enhance antiviral activity [318].

Risk assessment frameworks should incorporate off-target evaluations, ecological interaction studies, staged field releases, environmental DNA tracking, and rollback plans, treating engineered consortia as time-limited interventions subject to periodic re-licensing [319]. Ethical oversight and community consent models developed for other nature-based solutions provide useful templates for transparent decision-making and shared stewardship during pilot deployments [320,321]. Governance toolkits that combine technical safeguards (sterility, kill switches), operational controls (harvest scheduling, inlet rotation), and social contracts (community O&M agreements) will be critical for any responsible scaling from greenhouse experiments to neighborhood-scale implementations [322,323].

5.2.3. Advanced Materials and Autonomous Systems

Next-generation media and root zone scaffolds fabricated via additive manufacturing can provide tunable porosity gradients and high, cleanable surface areas, enhancing early viral capture while resisting compaction and clogging under variable hydraulic loads [324]. Incorporating redox-active fillers or micronutrient dopants may sustain enzyme production and ROS generation, boosting rhizosphere antiviral activity without inducing phytotoxicity [325,326]. Biomimetic self-cleaning surfaces inspired by lotus leaf morphology show promise for fouling resistance in compact pretreatment and polishing cartridges upstream or downstream of planted units [327,328].

AI-enabled tracking is emerging as a potential control layer for modular and decentralized systems. Edge computing sensor networks that monitor DO, ORP, head loss, and proxies for exudate or enzyme activity can feed predictive models to anticipate clogging and viral breakthrough, enabling condition-based maintenance that reduces downtime by ~30–60% [328,329]. TEAs forecasting and adaptive operational dispatch, adjusting UV or coagulant dosing based on residual rather than influent peaks, could lower OPEX while maintaining compliance during seasonal performance lows [154,330]. However, it is important to emphasize that no published CW systems use AI to optimize explicitly against viral infectivity endpoints, so AI is a promising but unproven tool for antiviral control.

In land-constrained settings, intensified vertical subsurface flow (VSSF) units, baffled FWS cells, and minimum viable hybrid systems (combining destabilization, planted beds, and small UV units) provide scalable building blocks with simplified failure modes, suitable for peri-urban corridors and informal settlements [94].

5.2.4. Policy, Climate Resilience and Circular Value

Policy alignment is essential for mainstreaming antiviral phytoremediation practices. Harmonized verification protocols, including minimum HRT testing, infectivity-based endpoints, and enzyme activity proxy panels, integrated into national manuals and international guidelines, would help close the gap between laboratory validation and field approval [1,331]. Without such frameworks, regulators have little basis for comparing CW-based hybrids with established disinfection technologies.

Climate resilience requires both biological and infrastructural adaptation. Drought-tolerant species such as Vetiveria zizanoides (Vetiver/Vetiver Grass, Khus-khus/Khas, Miracle Grass, Xiang-Gen-Cao/Yan-Lan-Cao) and P. purpureum can maintain fine-root areas and exudation under water scarcity, while salt-tolerant taxa and hybrid desalination–phytoremediation systems address coastal intrusion and salinization pressures [332]. Circular economy strategies, including biochar production from macrophyte biomass (surface area ~200–400 m²·g⁻¹), nutrient recovery, and the development of local soil amendment- markets, can enhance the lifecycle value and stabilize redox and adsorption properties when biochars are judiciously applied as media amendments [310,311]. However, robust assessments of potential contaminants in valorized products (e.g., trace organics and metals) are still rare.

Integrated roadmaps that combine technology, finance, and policy, starting with minimum viable hybrid systems and scaling via modular blocks under community O&M and transparent performance verification, offer the most credible pathway toward fair, climate-resilient antiviral phytoremediation deployment [333]. Achieving this will require technological innovation and confronting the deep evidentiary gaps and methodological inconsistencies that limit confidence in plant-based virus removal.

6. Conclusions

Antiviral phytoremediation has the potential to convert hydraulic contact into loss of viral infectivity through a coordinated set of plant-mediated barriers. By organizing the scattered literature into a four-barrier framework comprising sorption/filtration, rhizosphere-mediated inactivation, internalization, and intracellular degradation, this review clarifies where the mechanisms are already robust and where they remain speculative.

Pilot- and field-scale evidence strongly supports barriers 1 (sorption/filtration) and 2 (rhizosphere-mediated inactivation). Well-designed constructed wetlands (CWs) consistently enable the physical capture of roots, biofilms, and porous media; under favorable conditions, they enable the function of chemically active rhizospheres that generate ROS, phenolics, and lytic enzymes capable of damaging viral capsids and envelopes.

Barriers 3 and 4 (internalization and intracellular degradation) are conceptually plausible but empirically fragile in real-world wastewater systems. Most supporting data are obtained from plant–virus model systems or PCR/qPCR detection of viral genomes in plant tissues, which cannot distinguish intact virions from adsorbed or degraded fragments and rarely include infectivity assays. Treating these barriers as fully measured design levers is premature.

Across reported systems, standalone CWs typically achieve ~1–3 log₁₀ reductions in viral surrogates or genome copies, with enveloped viruses being generally more susceptible than non-enveloped enteric viruses. Hybrid treatment trains that combine planted systems with compact polishing units (e.g., UV, ferrate/ozone, or membranes) can reach ~3–7 log₁₀ total reductions, especially when CWs pre-remove particulates and dissolved organic carbon, so that polishing stages treat residual viral loads rather than influent peaks. Relative to chlorination, UV, MBRs, or AOPs, CW-based hybrids operate at lower energy demand and contain fewer disinfection by-products, but require larger land areas and are more sensitive to seasonal and biological variability.

Antiviral phytoremediation remains a data-poor and methodologically heterogeneous field. Heavy reliance on molecular endpoints, short-term trials, inconsistent surrogate selection, and sparse long-term and economic data constrain robust design and policy uptake. These priorities, anchored in the four-barrier framework, outline a coordinated research and governance agenda:

(a)

Mechanistic and methodological consolidation

Infectivity-centered studies that explicitly link plant traits, exudate chemistry, and microbial consortia to changes in viral infectivity across barriers 1–4 should be focused on, with clear differentiation between enveloped and non-enveloped viruses.
Standardized protocols for surrogate selection, combined qPCR/infectivity assays, and minimum testing for HRT and contact efficiency should be developed to enable rigorous cross-study comparisons and conservative design criteria.

(b)

Long-term and seasonal field evaluations

Implement multi-year monitoring of CWs and CW-based hybrids across climatic regimes, with explicit characterization of winter performance, high dissolved organic carbon conditions, and clogging dynamics to define realistic seasonal performance envelopes.
These data can be used to derive design safety margins and operational strategies that maintain viral protection under stress conditions, rather than only under ideal summer operations.

(c)

Techno-economic and life cycle assessment

Conduct virus-specific techno-economic and life cycle assessments comparing CW-based hybrids with chlorination, UV-only, and MBR or AOP trains, accounting for land, energy, disinfection by-products, operation and maintenance, and co-benefits such as nutrient recovery and biomass valorization.
These analyses were applied to identify settings where antiviral phytoremediation is genuinely cost-effective and where higher-energy options are justified by risk profiles or land constraints.

(d)

Governance, standards, and modular deployment

Integrate infectivity-based endpoints, relevant enzyme activity proxies, and minimum viable hybrid configurations (e.g., coagulation plus CW plus compact UV) into national and international guidelines for wastewater treatment and reuse.
Promote modular, decentralized architectures with standardized interfaces and clear operation and maintenance playbooks to support community-level operations, staged financing, and adaptive upgrades as performance requirements evolve.

(e)

Emerging tools and cautious innovation

Explore genetic engineering, engineered microbial consortia, advanced media, and data-driven control as tools to strengthen specific antiviral barriers, progressing through transparent bench-to-pilot pipelines under stringent biosafety oversight.
These emerging tools should be considered as complements to, not substitutes for, robust hydraulics, appropriate plant selection, and sound operational discipline, and large-scale deployment should be delayed until antiviral benefits are shown under realistic wastewater conditions.

If these research and policy priorities are pursued systematically, antiviral phytoremediation can evolve from a conceptually attractive but empirically under-anchored niche into a credible and scalable component of climate-resilient and pandemic-prepared wastewater infrastructure. Realizing this potential will require technological innovation and deliberate efforts to close the evidentiary gaps highlighted in this review and align regulatory, economic, and community frameworks with the realities of plant-based virus control.

Author Contributions

D.Z. contributed to conceptualization, investigation, analysis, visualization, writing—original draft, and writing—review & editing. M.-H.S., A.M. & E.M. contributed to writing—review & editing. A.D. contributed to supervision and writing—review & editing. D.H.-W.K. contributed to supervision, visualization, writing—review & editing, resources, project administration, and funding acquisition. All authors have read and agreed to the published version of the manuscript.

Funding

This study was funded by the Ministry of Science and Technology, Taiwan (MOST 105-2221-E-029-002 and 106-2221-E-029-003).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data are contained within the article.

Acknowledgments

This study was conducted at the Department of Environmental Science and Engineering, Tunghai University, with gratitude to the School of Forestry, PNG University of Technology, for providing space and resources for data analysis and writing. During the preparation of this manuscript, the authors used BioRender (web-based platform; https://biorender.com, accessed 1 November 2025; BioRender Inc., Toronto, ON, Canada), NanoBanana Pro (Gemini 3.0 Pro image generation model, accessed November 2025; Google DeepMind, London, UK), and OriginPro 2023 (Origin 2023/2023b; OriginLab Corporation, Northampton, MA, USA) for generating, editing, and analyzing illustrative images and data visualizations; the bibliometric data for this review were retrieved from the Dimensions database (web-based research analytics platform; https://app.dimensions.ai, accessed on 31 March 2025; Dimensions, Digital Science & Research Solutions Ltd., London, UK).

Conflicts of Interest

The authors declare no conflicts of interest.

Abbreviations

AOPs	Advanced Oxidation Processes
AI	Artificial Intelligence
CAPEX	Capital Expenditure
C:N:P	Carbon:Nitrogen:Phosphorus Ratio
COD	Chemical Oxygen Demand
CRISPR	Clustered Regularly Interspaced Short Palindromic Repeats
CRISPR-Cas	Clustered Regularly Interspaced Short Palindromic Repeats [CRISPR]-associated [Cas] nuclease
CWs/CW	Constructed Wetland Systems
DEWATS	Decentralisation of Wastewater Treatment
DO	Dissolved Oxygen
DOC	Dissolved Organic Carbon
EC	Electrocoagulation
e-MBRs	Electrochemically Enhanced Membrane Bioreactors
EPS	Extracellular Polymeric Substance
FWS	Free-Water Surface (or Free-Water Surface cells/systems)
GAC	Granular Activated Carbon
GH19	Glycoside Hydrolase Family 19
GMO	Genetically Modified Organism
HAA	Haloacetic Acids
HLR	Hydraulic Loading Rate
HO·	Hydroxyl Radical
HRT	Hydraulic Retention Time
HSSF	Horizontal Subsurface Flow
IC₅₀	Inhibitory Concentration 50%
IFITM3	Interferon-Induced Transmembrane Protein 3
MBRs	Membrane Bioreactors
ML	Machine Learning
O&M	Operation and Maintenance
ORP	Oxidation-Reduction Potential
PGPR	Plant Growth-Promoting Rhizobacteria
PPP	Public–Private Partnerships
RNA	Ribonucleic Acid
RNAi	RNA Interference
RNase	Ribonuclease
ROS	Reactive Oxygen Species
RT-RPA	Reverse Transcription Recombinase Polymerase Amplification
SA	Salicylic Acid
SARS-CoV-2	Severe Acute Respiratory Syndrome Coronavirus 2
SSF	Subsurface-Flow
THM	Trihalomethanes
TOC	Total Organic Carbon
UV	Ultraviolet
UVC	Ultraviolet C
VSSF	Vertical Subsurface-Flow

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Figure 1. Transmission pathway of pathogenic enteric viruses. Viruses transmit from infected to susceptible individuals through environmental routes. Transmission occurs when bodily fluids (sweat, droplets, tears, blood, feces, saliva) spread via different pathways (air, water, solids, vectors). Susceptible individuals may contract infections through multiple entry points (eyes, nose, mouth, blood, skin). Arrows show the direction of viral transmission from infected to susceptible individuals. Route-label colors (Water, Air, Solid, Vector) are used only for visual distinction. Red icons indicate infected individuals or groups, and blue icons indicate uninfected (susceptible) individuals or groups.

Figure 2. Bibliometric map connecting conventional (CT) and advanced wastewater-treatment (AT) domains to nature-based options (constructed wetlands [CW]; hybrid CW [HCW]; phytoremediation [Phyto]) and target contaminants (heavy metals [HM], pathogen [Patho], other pollutants [OP]). Phyto is shown as bridging both domains because it is reported both as a CT low-tech approach in some applications and as an AT polishing/hybrid component in integrated treatment trains. “CT” and “AT” here denote bibliometric research domains rather than strict engineering classifications of individual unit processes. Patho types: bacteria (Bac), viruses (Vir), parasitic protozoa (Parapro), and helminths (Helm). Grey shaded bands represent bibliometric domains within the wastewater-treatment literature (CT, CW, HCW, AT, and their overlap), while green-highlighted connections denote phytoremediation links to general pathogen and virus research clusters, which support the antiviral phytoremediation theme. Data source: Dimensions Research Database (https://app.dimensions.ai), accessed on 31 March 2025.

Figure 3. Conceptual antiviral phytoremediation of viral pathogens in wastewater. Letters denote: (A) Influent containing waterborne contaminants and viruses passes through the rhizosphere (plant–substrate–microbe continuum) to produce treated effluent. (B) Key phytoremediation processes include rhizofiltration (physical interception and sorption to roots/media), phytostabilization (immobilization and reduced mobility), rhizodegradation (microbially mediated transformation supported by root exudates and biofilms), and phytodegradation (plant enzymatic transformation and sequestration in tissues); phytoextraction and limited phytovolatilization are also indicated (for viruses, attenuation is via internalization and intracellular degradation rather than true volatilization). (C) Proposed virus-specific mechanisms include sorption–filtration, rhizosphere-mediated chemical and enzymatic inactivation (e.g., reactive oxygen species and enzymes), viral internalization via apoplastic or endocytosis-like pathways, and intracellular antiviral degradation pathways. Color coding: red icons indicate infectious virions; broken red icons indicate inactivated virions; purple layers show biofilms/microbes; blue elements show water/flow; brown elements show substrate/media; green structures show plant roots/tissues and root exudates.

Figure 4. Hydraulics and contact optimization for virus attenuation in vertical subsurface flow (VSSF) beds and free-water surface (FWS) cells. (A) VSSF plan and cross-section showing perforated influent distribution to promote a uniform wetting front, increase contact with filter media/biofilms/roots, and reduce short-circuiting. (B) FWS plan view showing baffles and staged vegetated islands that straighten flow paths, suppress wind-driven recirculation, and direct flow through root-zone “hotspots” for enhanced sorption/filtration and rhizosphere attenuation. Tracer testing and traffic-light diagnostics indicate declining effective HRT and maintenance triggers; stress conditions may require added HRT safety margin or compact pretreatment (e.g., coagulation/roughing filtration. Colors: solid blue arrows denote main water flow paths, green zones indicate active interfaces (roots, biofilm, media), and red dashed arrows indicate bypass or short-circuit flow paths.

Figure 5. Hybrid antiviral plant system for virus attenuation. System components include bed architecture, active interfaces (roots, porous media, biofilms), and staged removal via primary sorption/filtration (HRT target ~1–2 log₁₀) followed by rhizosphere attenuation (additional bed-volume HRT). Performance indicators include effective HRT, RTD (residence time distribution) variance, clogging/ponding, and channelized flow; operational controls and remedial actions (alternating dosing, inlet rotation, tracer testing, hydro-flushing, media scarification, partial media replacement) limit bypass and maintain predictable log removal. Colors: Solid blue arrows show the main water flow path from influent through pretreatment, the planted bed, and polishing stage to effluent, whereas the colored compartments represent charge-stabilization pretreatment, rhizosphere-based attenuation in the planted bed, and disinfection/filtration processes in the polishing stage.

Figure 6. Schematic overview of major constructed wetland (CW) configurations and a hybrid treatment train. (A) Free water surface (FWS) wetland showing attenuation via sedimentation, sorption/filtration on biofilms and root surfaces, modest rhizosphere chemistry effects, and sunlight-driven photolysis/UV exposure. (B) Horizontal subsurface flow (HSSF) wetland highlighting steady filtration through a gravel/media bed, adsorption, and suboxic–anoxic redox conditions along the flow path. (C) Vertical subsurface flow (VSSF) wetland with intermittent dosing, unsaturated flow and aerobic microzones that enhance filtration and rhizosphere oxidation, supporting downstream internalization and degradation processes. (D) Example sequential hybrid CW train combining VSSF and FWS stages with polishing (e.g., compact UV) to meet reuse-oriented effluent standards, with seasonal operation adjustments indicated (e.g., winter HRT increase).

Figure 7. Conceptual modular hybrid antiviral planted treatment systems enabling scalable deployment and reuse-oriented performance. (A) Standardized “minimum viable hybridization” treatment train coupling compact charge-stabilization/roughing (coagulation or electrocoagulation) with a compact planted unit (intensified VSSF bed or baffled FWS cell; barriers 1–2: sorption–filtration and rhizosphere inactivation; target ~2–4 log₁₀ reduction) and downstream polishing (barrier 4: UV or GAC; optimized for residual load) to achieve an overall target of ~3–7 log₁₀ reduction. (B) Compact urban integration concepts (rooftop/podium wetland; linear green-corridor cells) emphasizing staged contact zones, pulsed/alternating dosing, and minimal land-use conflict. (C) Mobile and adaptive containerized units with interchangeable cartridges, rule-based/event-driven control, and rapid pathogen analytics to manage variable influent conditions. (D) Future enhancements using advanced materials and biotechnology (e.g., 3D-printed scaffolds, redox-active fillers/micronutrients, engineered plant–microbe consortia, and biosensing) to increase reactive surface area and real-time performance feedback. Color: blue arrows show the main wastewater flow path across standardized treatment modules; green elements highlight planted or green-infrastructure units; orange and purple modules indicate compact urban, mobile/containerized, and advanced materials/biotechnology options.

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