4. Discussion
This study systematically evaluated the inhibitory effects of GTE and its primary active components against ZIKV, investigating their mechanisms of action and potential implications for blood safety.
The compositional analysis of GTE revealed that epigallocatechin gallate (EGCG, 10%), epicatechin gallate (ECG, 12%), and epigallocatechin (EGC, 70%) are the major constituents. However, only EGCG and ECG (below 40 μM) exhibited significant inhibition of ZIKV replication; the EGC showed negligible antiviral activity. This disparity likely stems from structural differences: EGCG and ECG possess galloyl groups that enable direct interactions with viral envelope proteins (e.g., ZIKV E protein) or host receptors via hydrogen bonding and hydrophobic forces, whereas EGC lacks this functional moiety. These results align with prior studies highlighting the critical role of galloylation in the antiviral potency of catechins against flaviviruses [27]. Furthermore, the synergistic inhibition of ZIKV NS2B-NS3 protease by EGCG and ECG may enhance their antiviral efficacy [12]. This underscores that GTE’s activity depends not only on total polyphenol content but also on the structural specificity of its components.
A time-course study demonstrated that GTE effectively suppresses ZIKV replication when administered during the pre-treatment, co-treatment, or full-course treatment phases, but not during post-infection. This indicates that GTE primarily targets early stages of the viral lifecycle, such as viral attachment and entry. Binding and entry assays further clarified this mechanism: GTE inhibited ZIKV binding to host cells in a dose-dependent manner, whereas its effect on viral entry was “non-dose-dependent”. These observations suggest two potential modes of action. 1. Competitive binding: EGCG and ECG may block ZIKV E protein from interacting with host receptors (e.g., Axl or TIM-1) by occupying key binding domains (e.g., Asn154, Thr156) [16]. 2. Non-specific entry interference: GTE might alter virus membrane fluidity or form a physical barrier to impede viral internalization. This multi-modal mechanism enables GTE to achieve significant antiviral effects at low concentrations (100 μg/mL), highlighting its practical advantage for therapeutic applications.
A new finding of this study is that GTE and its active components (EGCG/ECG) can inactivate ZIKV in RBC preparations, rendering the virus non-infectious. Our results showed that at concentrations below 300 μg/mL (maximal non-cytotoxic dose), ZIKV was reduced by more than −4 logs. This has critical implications: mitigating transfusion-transmission risks. ZIKV remains viable in RBCs for weeks, posing a threat to blood safety [28]. Conventional PRTs, such as methylene blue, amotosalen, and riboflavin-based photochemical treatments, often target the nucleic acid of pathogens and may compromise RBC functionality [29,30]. The S-303, combined with the glutathione treatment regimen, once showed promise, but during clinical trials, it was discovered that some subjects developed antibodies against the S-303-treated red blood cells. Therefore, this method was also limited [31]. In contrast, GTE inactivates ZIKV while preserving RBC membrane integrity (hemolysis rates remain stable), offering a natural and biocompatible alternative for blood additive solutions [32]. Our data suggest that the synergistic interaction of constituents within GTE is more effective in preserving RBC membrane integrity than any single catechin component tested.
Given the demonstrated efficacy of EGCG/ECG against other transfusion-transmissible viruses (e.g., HBV, HCV, DENV) [12,33], GTE may serve as a universal viral inactivation agent for RBC. However, the in vivo persistence and metabolic fate of GTE following transfusion remain to be fully elucidated. As a potential pharmaceutical, its long-term stability, bioavailability, and sustained activity in the circulation require further characterization. From a public health and sustainable pharmaceutical development perspective, establishing the safety, durability, and scalability of GTE-based viral inactivation is critical to support its future translation into clinical and industrial settings.
Furthermore, preclinical in vivo transfusion studies based on animal models remain an indispensable step toward the application and development of the GTE-based virus reduction strategy. To further validate the translational potential of GTE as a viral inactivation agent for blood products, subsequent comprehensive studies will be focused on two core aspects: first, to systematically evaluate the impacts of GTE on RBC functionality and safety, including detailed detection of critical physiological and functional parameters such as RBC oxygen-carrying capacity, energy metabolism, and membrane deformability during long-term storage; second, to expand the research scope of viral pathogens and verify the virucidal effectiveness of GTE against a variety of clinically important transfusion-transmissible viruses (e.g., HBV, HCV, DENV). These in-depth studies will provide more sufficient experimental evidence for the development of GTE as a safe and effective broad-spectrum viral inactivation strategy in transfusion medicine.
Regarding the clinical safety of GTE-mediated viral inactivation in RBCs, the final concentration of GTE in the transfusion product is 300 μg/mL, corresponding to approximately 150 mg per 2-unit transfusion. After systemic dilution, the estimated peak plasma concentration is approximately 30 μg/mL. Current safety data and acceptable daily intake limits for catechins are derived from oral administration and cannot be directly extrapolated to transfusion, which bypasses hepatic first-pass metabolism and results in higher bioavailability [35,36,37]. Therefore, the safety of this concentration requires further validation. To improve safety, the GTE concentration can be reduced, since our results demonstrated that 100 μg/mL GTE still significantly reduced ZIKV infectivity. In addition, GTE could be removed by filtration after viral inactivation to further enhance transfusion safety.
This study establishes that GTE inhibits ZIKV by targeting viral attachment, with EGCG and ECG identified as the key active components. Crucially, GTE inactivates ZIKV in RBCs without compromising cellular function, offering a novel strategy to enhance blood safety—particularly in resource-limited regions and during emerging arboviral outbreaks. Future work should prioritize in vivo validation, formulation optimization, and expansion to other transfusion-relevant pathogens, accelerating the translation of GTE from bench to bedside.
