Ethics statements

Institutional Review Board (IRB): The study protocol, informed consent forms, and participant information sheets were reviewed and approved by the Institutional Review Board of Tri-Service General Hospital (IRB No. 2-107-05-039). The principal investigator, Cheng-Hsuan Ho, was responsible for reporting all serious adverse events (SAEs) and unanticipated problems in compliance with applicable regulatory requirements. The IRB operates in strict accordance with Good Clinical Practice (GCP) guidelines and relevant national laws.

Informed Consent: Written informed consent for the collection and publication of clinical data was obtained from all participants or their legally authorized representatives prior to inclusion in this study.

Animal Ethics (IACUC): All animal experimental procedures were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of the National Defense Medical University, Taiwan (Protocol No. IACUC-24–130).

Clinical patients

Snakebite patients who were admitted to the emergency department of Tri-Service General Hospital from 2017 to 2025 were included in this study, following approval obtained from the Institutional Review Board of Tri-Service General Hospital (IRB No. 2-107-05-039). Of the 81 patients enrolled, 50 were identified as individuals envenomated by P. mucrosquamatus on the basis of the clinical identification of the snake carcass or digital photographs provided by the patient. This study employed a retrospective analytical design to evaluate the clinical predictors of recovery in these 50 patients envenomated by P. mucrosquamatus. To identify factors influencing the duration of local edema remission, a multiple linear regression model incorporating 12 independent clinical variables was constructed using the Enter method. Demographic variables included sex and age, whereas pre-existing comorbidities were defined as the presence of diabetes mellitus or chronic kidney disease (CKD) stages 1–5 according to the KDIGO 2026 Clinical Practice Guideline [27]. Clinical presentation was characterized by the bite-to-emergency department (ED) time in hours and the anatomical bite site. Edema severity was quantified using the Blaylock classification at two critical time points: the initial Blaylock level upon arrival and the final Blaylock level, which represents the peak of swelling progression, ranging from minimal to gross edema [28]. The Blaylock classification defines clinical severity as minimal for localized edema, mild for swelling restricted to the hand or foot, moderate for edema extending from the hand to the shoulder or from the foot to the thigh, severe for progression from the hand to the chest or from the foot to the groin, and gross for swelling extending from the foot to the trunk or from the hand to the contralateral chest [28]. Treatment and hospitalization factors were defined as the total dosage of FHAV administered, the duration of ED stay in hours, the need for surgical intervention, and the duration of hospital admission in days. The dependent variable for this predictive model was remission duration, defined as the total time in days required for the complete resolution of venom-induced localized edema, which was documented through patient recall during telephone follow-up interviews or routine outpatient clinical visits.

Reconstitution and chromatographic fractionation of venom reagents

Lyophilized crude P. mucrosquamatus venom was provided by the Department of Biology and Anatomy at National Defense Medical University (Taipei, Taiwan). Upon receipt, the lyophilized venom was divided into single-use aliquots and stored at −20°C until use. This procedure was adopted to standardize sample handling and minimize potential variability associated with prolonged storage and repeated freeze‒thaw cycles [29,30]. Immediately before each experiment, an individual aliquot was reconstituted in sterile phosphate-buffered saline (PBS; Gibco [Thermo Fisher Scientific, Waltham, MA, USA]) to the required concentration. To remove particulate matter, the solution was gently vortexed for 10 s and centrifuged at 10,000 × g for 5 min at 4°C using an Eppendorf 5424 R centrifuge (Eppendorf AG, Hamburg, Germany). The clarified supernatant was filtered through a PVDF syringe filter (Millipore, Burlington, MA, USA) and divided into single-use aliquots, ensuring that each venom aliquot was subjected to only one use to avoid freeze-thaw degradation or loss of enzymatic activity. The protein concentration was quantified using a bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA) with bovine serum albumin (BSA; Sigma‒Aldrich, St. Louis, MO, USA) as a standard, and calibration curves were constructed using serial dilutions of BSA ranging from 0–2,000 µg/mL. Unless otherwise specified, all the reagents used were of analytical or HPLC grade, and all the injectable and chromatographic solutions were prepared using ultrapure water generated from a Direct-Q water purification system (Millipore, Burlington, MA, USA) and passed through 0.22 µm PVDF syringe filters prior to use. For subsequent protein isolation, the clarified crude P. mucrosquamatus venom was fractionated on the basis of molecular mass using a Superdex 200 Increase 10/300 GL column connected to an ÄKTA Pure chromatography system (Cytiva, Uppsala, Sweden). The column was equilibrated and eluted with 50 mM phosphate buffer containing 150 mM NaCl (pH 7.4) at a flow rate of 0.5 mL/min, and the elution profile was monitored at 280 nm. Fractions corresponding to the same peaks were pooled and tentatively assigned to the peptide-enriched fraction (< 10 kDa), the PLA₂-enriched fraction (approximately 14 kDa), the SVSP-enriched fraction (approximately 30 kDa), and a high-molecular-weight SVMP/CTL-enriched fraction (P-II or P-III; > 50 kDa). Because snake C-type lectin (Snaclec/CTL) is often present in a multimeric form with a molecular weight of approximately 121 kDa, it was not specifically isolated in this experiment but was instead cofractionated with snake venom metalloproteinase, and this fraction was labeled SVMP/CTL. Each pooled fraction was then lyophilized using a FreeZone 2.5 L freeze dryer (Labconco, Kansas City, MO, USA) and stored at −20°C with desiccant until further use. To confirm the composition and hydrophobicity profiles, each size-exclusion chromatography (SEC)-derived fraction was reconstituted in 0.1% trifluoroacetic acid (TFA) and analyzed using an Agilent 1260 Infinity II HPLC system (Agilent Technologies, Santa Clara, CA, USA) equipped with a Jupiter C18 column (250 × 4.6 mm, 5 μm particle size, 300 Å pore size; Phenomenex, Torrance, CA, USA). Mobile phase A consisted of water containing 0.1% TFA, and mobile phase B consisted of HPLC-grade acetonitrile containing 0.1% TFA. The proteins were eluted using a linear gradient from 5–70% mobile phase B over 85 min at a flow rate of 1.0 mL/min with monitoring at 215 nm. The fraction assignments in this study were established on the basis of a previous study [6] in which the major snake venom protein families, including PLA₂, SVSP, SVMP, and CTL, were thoroughly mapped using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and characterized under identical chromatographic conditions. Although referencing these established profiles provided a robust baseline for defining elution windows, the specific pooled fractions utilized in the present experiments were not independently reconfirmed by LC-MS/MS, SDS-PAGE, or toxin-family-specific enzymatic assays.

Oversight, ethical approval for the animal study and animal housing

All procedures involving animals were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of the National Defense Medical University, Taiwan (protocol number IACUC-24–130) and were conducted in strict accordance with institutional guidelines and national regulations governing the ethical use of laboratory animals. Every effort was made to minimize animal suffering and reduce the total number of animals used in the study. Because acute inflammatory edema served as the primary experimental endpoint, no preemptive analgesics were administered prior to paw thickness measurements to prevent pharmacological interference with the release of inflammatory mediators or changes in vascular permeability. Male ICR/CD-1 mice between 6–8 weeks of age weighing 20 ± 2 g obtained from BioLASCO Taiwan Co., Ltd. (Taipei, Taiwan) were utilized for all experiments. These animals were housed under standard facility conditions, including a 12-h light/dark cycle at an ambient temperature of 22 ± 2°C and a relative humidity between 40% and 60%. The mice were acclimatized for at least 7 d with ad libitum access to standard laboratory chow and water and housed in groups of 3–5 per cage with corncob bedding. Before venom or its isolated fractions was administered, the mice were briefly anesthetized with 2%–3% isoflurane (Forane; Baxter Healthcare Corporation, Deerfield, IL, USA) delivered in oxygen to minimize handling stress. All the animals recovered immediately following the subplantar injection procedure and were monitored closely for any signs of abnormal behavior or local tissue damage. This intensive postinjection monitoring ensured rapid recovery and the total absence of distress in strict compliance with Institutional Animal Care and Use Committee regulations, and humane endpoints were predefined and monitored throughout the entire experimental period.

Establishment of the paw edema model and characterization of venom- and fraction-induced local edema

The local inflammatory response to P. mucrosquamatus venom and its isolated components was evaluated using a modified subplantar paw edema assay in mice (n = 6 per group). These group sizes were determined on the basis of the initial variability in the paw thickness measurements, which was deemed sufficient to characterize dose-dependent and treatment-related effects while strictly minimizing animal usage in accordance with ethical guidelines, although a formal a priori power calculation was not performed. Baseline paw thickness was measured at the central plantar pad using digital calipers (Mitutoyo Digimatic [0.01 mm resolution]; Mitutoyo Corp., Kawasaki, Japan) prior to treatment. Under brief isoflurane anesthesia, the animals received a 50 µL subplantar injection of either crude venom (CV) at various concentrations (10, 50, 100, 250, 500, or 1,000 µg/mL, equivalent to 0.5–50 µg per paw) or protein mass-matched doses of isolated toxin fractions. These fractions included PLA₂ at 50 or 100 µg/mL, SVSP at 250 or 500 µg/mL, and SVMP/CTL at 250 or 500 µg/mL, with saline serving as the vehicle control. Paw thickness was recorded 3 h postinjection at the same anatomical location, a time point corresponding to maximal swelling as determined by preliminary kinetic observations. All measurements were performed in triplicate for each animal to calculate a mean value for analysis. The experimental results are expressed as the mean ± SD.

Neutralization of venom-induced edema by antivenom and small-molecule inhibitors in vivo

The neutralizing efficacy of freeze-dried hemorrhagic antivenom (FHAV; Taiwan Centers for Disease Control, Taipei, Taiwan; lot number 60-06-0020) and small-molecule inhibitors against venom-induced edema was evaluated in a murine model. To minimize potential batch-to-batch variability, all the experiments were conducted using a single lot of antivenom, which was stored at 4°C and prepared immediately before use according to the manufacturer’s instructions. Mixtures containing CV and FHAV were preincubated at 37°C for 30 min with gentle agitation at 300 rpm using a ThermoMixer C (Eppendorf AG, Hamburg, Germany) prior to administration. Each 50 µL mixture was administered via subplantar injection into the left hind paw of mice under brief isoflurane anesthesia (n = 6 per group). The control groups received either antivenom alone or CV at a concentration of 100 µg/mL. The small-molecule inhibitors (SMI) utilized in this study included marimastat (Cat. No. SI-M2699; Selleck Chemicals, Houston, TX, USA), batimastat (Cat. No. S7036; Sigma‒Aldrich, St. Louis, MO, USA), and varespladib (Cat. No. HY-13402; MedChemExpress, Monmouth Junction, NJ, USA). All stock solutions were prepared according to the manufacturers’ instructions and diluted to the target concentrations immediately before the experiment. For the inhibitor assays, the three SMIs were prepared at a concentration of 10 µM, mixed with 100 or 250 µg/mL CV, and preincubated under the same temperature and agitation conditions as the pure antivenom (n = 3 per group). Paw thickness was recorded at baseline and 3 h postinjection using digital calipers (Mitutoyo Corp., Kawasaki, Japan).

Characterization of the antivenom binding profiles via an indirect enzyme-linked immunosorbent assay (ELISA)

Venom samples, including CV and purified fractions such as PLA₂, SVSP, and SVMP/CTL, were diluted in carbonate–bicarbonate buffer (0.05 M, pH 9.6) to a final concentration of 10 µg/mL. Each well of a flat-bottom 96-well ELISA plate (Nunc MaxiSorp; Thermo Fisher Scientific, Waltham, MA, USA) was coated with 100 µL of the antigens and incubated overnight at 4°C. After the wells were washed with phosphate-buffered saline containing 0.05% Tween-20 (PBST), the wells were blocked with 5% bovine serum albumin (BSA) in PBS for 1 h at room temperature. Reconstituted FHAV was serially diluted 1:500, 1:5,000, and 1:50,000 in blocking buffer, and 100 µL was added to the wells in triplicate, followed by incubation at 37°C for 1 h. After the wells were washed, bound equine antibodies were detected using horseradish peroxidase (HRP)-conjugated goat anti-horse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) diluted 1:10,000 for 1 h at 37°C. The wells were subsequently washed, and the signal was developed using 3,3,’5,5’-tetramethylbenzidine (TMB) substrate (Thermo Fisher Scientific, Waltham, MA, USA) for 10 min in the dark. The reaction was terminated by the addition of 1 N H₂SO₄ (Sigma‒Aldrich, St. Louis, MO, USA), and the absorbance was measured at 450 nm using a Synergy H1 microplate reader (BioTek Instruments, Winooski, VT, USA). Background wells without FHAV were included for normalization, and the results are presented as background-subtracted optical density (OD) values at 450 nm.

Statistical methods

Statistical analyses were performed using IBM SPSS Statistics version 23 (IBM Corp., Armonk, NY, USA) and GraphPad Prism version 11.0.1 (GraphPad Software, San Diego, CA, USA). Descriptive statistical data are summarized as the mean ± standard deviation (SD) or frequency for all the variables. Clinical predictors were evaluated using Pearson’s correlation and multiple linear regression models. A mixed-effects model was employed to analyze the edematogenic responses to CV and isolated toxin families, including PLA₂, SVSP, and SVMP/CTLs, as well as the results of neutralization assays involving FHAV or SMIs. The experimental design utilized n = 6 mice per group for CV titration and FHAV neutralization, whereas n = 3 mice per group were used for SMI testing. Although cohort sizes were selected pragmatically without formal a priori power calculations, the group size of six mice balanced the ethical reduction in animal usage with the statistical reliability required to accommodate observed interanimal biological variability in paw thickness measurements, providing consistent and statistically detectable responses. To compare ELISA results across multiple dilution factors, two-way ANOVA was performed. Tukey’s post hoc test was used for multiple comparisons to analyze significant differences between treatment groups, time points, and antigens. All ELISA experiments were conducted in triplicate, with saline or designated negative controls serving as references. For all analyses, statistical significance was defined as P < 0.05.

Clinical predictors and multivariate analysis of the duration of remission in patients with Protobothrops mucrosquamatus envenomation

The descriptive statistics for the study cohort of 50 patients envenomated by P. mucrosquamatus who presented to the ED of Tri-Service General Hospital from 2017 to 2025 are summarized in Table 1. The study population (n = 50) had a mean age of 53.24 ± 19.6 years (range: 5–88) and was predominantly male (n = 34, 68%). Comorbidities were present in a minority of the patients, including diabetes mellitus (n = 2, 8%) and chronic kidney disease (n = 28, 56% for stage 1; n = 13, 26% for stage 2; n = 9, 18% for stage 3). The mean time from bite to ED arrival was 3.17 ± 5.47 h (range: 0–29 h, with 0 hours representing an arrival within 30 minutes of envenomation), with the foot being the most frequent bite site (n = 23, 46%). The initial Blaylock severity scores were categorized as minimal in 40% of the patients, mild in 48%, and moderate in 12%, which progressed to peak severity categorized as minimal in 12% of the patients, mild in 54%, moderate in 32%, and severe in 2% during the clinical course. Treatment involved a mean FHAV dosage of 6.52 ± 4.24 vials (range: 1–22) and a mean ED stay of 19.69 ± 11.63 h, with the mean duration for complete local edema remission being 10.80 ± 10.88 d. Univariate analysis using Pearson’s correlation identified total FHAV dosage (r = 0.743, P < 0.001), ED stay duration (r = 0.544, P < 0.001), and final Blaylock level (r = 0.375, P = 0.004) as factors significantly associated with recovery time. To identify independent predictors, a multiple linear regression analysis was conducted, yielding a highly significant model (F_{12, 37} = 7.128, P < 0.001) with an R² of 0.698. Within this model, total FHAV dosage emerged as the strongest independent predictor of the remission timeline (β = 0.658, T = 5.167, P < 0.001), followed by hospital admission duration (β = 0.355, T = 3.274, P = 0.002) and initial Blaylock level (β = - 0.283, T = - 2.039, P = 0.049), whereas age (P = 0.434), bite-to-ED time (P = 0.316), and comorbid diabetes mellitus (P = 0.067) were not independent predictors. Diagnostic tests confirmed the stability of the model, with all variance inflation factors remaining below 2.4. Taken together, these findings demonstrate that the total FHAV dosage serves as the primary clinical indicator of the duration of venom-induced localized edema.

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Table 1. Demographic and clinical characteristics of 50 patients envenomated by Protobothrops mucrosquamatus.

https://doi.org/10.1371/journal.pntd.0014689.t001

Characterization and validation of the dose-dependent edematogenic potential of Protobothrops mucrosquamatus crude venom in a mouse model

Based on clinical findings, a murine paw edema model was established to simulate the progressive localized swelling observed in envenomated patients. Animals received subplantar injections of graded concentrations of CV into the hind paw, and paw thickness was subsequently recorded at 3 h post-injection at the identical anatomical location (Fig 1A). A mixed-effects model analysis revealed a highly significant interaction between intervention and time (F_{6, 35} = 137.7, P < 0.0001) and a predominant main effect of time (F_{1, 35} = 3,490, P < 0.0001). While the saline control group exhibited no significant change in paw thickness from baseline (7.467 mm to 7.533 mm; q = 1.278, P = 0.3724), subplantar injection of CV at all tested concentrations (ranging from 10 μg/mL to 1,000 μg/mL) induced profound and statistically significant localized edema. The magnitude of swelling was strictly dose dependent; specifically, the mean paw thickness of the 10 μg/mL CV group increased by 1.000 mm (q = 21.08, P < 0.0001), whereas that of the 1,000 μg/mL CV group exhibited a substantially greater increase of 2.567 mm (q = 49.19, P < 0.0001). At 3 h postinjection, concentrations of 50 μg/mL or more resulted in a paw thickness that was significantly greater than that in the saline control group (saline vs. 50 μg/mL CV, q = 7.242, P < 0.0001; saline vs. 1,000 μg/mL CV, q = 10.83, P < 0.0001). These findings demonstrate that P. mucrosquamatus CV triggers rapid, high-magnitude localized tissue swelling that scales precisely with venom concentration, thereby providing a robust and sensitive model for evaluating the edematogenic potency of specific toxin families.

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Fig 1. Dose-dependent edematogenic profile and chromatographic toxin fractionation of Protobothrops mucrosquamatus crude venom (CV).

(A) Representative gross morphological changes and quantitative evaluation of the murine paw edema models following subplantar injection of crude venom (CV; 10–1,000 μg/mL), demonstrating rapid, concentration-dependent localized swelling at 3 h postinjection compared with saline controls. (B) Chromatographic fractionation of CV using reversed-phase high-performance liquid chromatography (RP-HPLC), with the proteome resolved into three primary toxin-enriched fractions on the basis of elution time: phospholipase A₂ (PLA₂-enriched; 27.5–46 min), snake venom serine protease (SVSP-enriched; 46–55 min), and snake venom metalloproteinase with C-type lectin-like proteins (SVMP/CTL-enriched; 59–85 min). (C) Edematogenic activity of the PLA₂-enriched fraction, which was identified as the main driver of edema because a low dose (100 μg/mL) elicited a response statistically indistinguishable from that of 100 μg/mL CV (P = 0.9767). (D and E) Edematogenic characterization of the SVSP-enriched and SVMP/CTL-enriched fractions, respectively, showing that while both elicit significant edema (P < 0.0001), they are both less potent than the PLA₂-enriched fraction is and require a fivefold higher concentration (500 μg/mL) to match the swelling magnitude of either the PLA₂ fraction or CV (P = 0.2236). Baseline measurements are indicated by blue circles, and 3 h postinjection values are indicated by red circles, with quantitative data presented as individual points representing the mean ± standard deviation (SD). All the statistical comparisons were performed using a mixed-effects model followed by Tukey’s post hoc test (n = 6 per group; * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 versus baseline).

https://doi.org/10.1371/journal.pntd.0014689.g001

Comparative edematogenic potency of isolated PLA₂-enriched, SVSP-enriched, and SVMP/CTL-enriched fractions from Protobothrops mucrosquamatus venom

To characterize the venom constituents, reversed-phase high-performance liquid chromatography (RP-HPLC) was performed to resolve the major toxin families of P. mucrosquamatus venom, yielding PLA₂-enriched, SVSP-enriched, and SVMP/CTL-enriched fractions on the basis of established chromatographic profiles [6]. As illustrated in the chromatographic profile (Fig 1B), PLA₂-enriched variants eluted in the first major region (approximately 27.5 min to 46 min), followed by the SVSP-enriched fraction from 46 min to 55 min. The most hydrophobic components, identified as the SVMP/CTL-enriched fraction, eluted in the final phase from 59 min to 85 min. The localized edematogenic activities of these three major toxin fractions were evaluated using a mouse paw edema model (Fig 1C–1E). For the PLA₂-enriched fraction (Fig 1C), a mixed-effects model revealed a highly significant interaction between treatment and time (F_{3, 20} = 43.18, P < 0.0001). Post hoc analysis indicated that both the 50 μg/mL and 100 μg/mL doses induced marked swelling relative to baseline, with mean differences in volume of 0.983 mm and 1.583 mm, respectively (P < 0.0001 for both). Similarly, analyses of the SVSP-enriched and SVMP/CTL-enriched fractions revealed a significant interaction between treatment and time (F_{3, 20} = 37.52, P < 0.0001 for both), with doses of 250 μg/mL and 500 μg/mL resulting in substantial increases in paw thickness (P < 0.0001 for all). Collectively, these results indicate that while all three toxin fractions strongly induce localized edema, their relative potencies differ significantly. The PLA₂-enriched fraction emerged as the most potent component; a dose of only 100 μg/mL elicited an edematous response (mean difference: 1.583 mm) that was statistically indistinguishable from that of the 100 μg/mL CV control (P = 0.9767). In contrast, 5-fold greater concentrations of the SVMP/CTL-enriched and SVSP-enriched fractions (500 μg/mL) were needed to produce comparable swelling (mean difference: 1.367 mm) and were statistically similar to the CV control (P = 0.2236 for both). These findings suggest that multiple toxin classes contribute to the local tissue damage characteristic of P. mucrosquamatus envenomation, with the PLA₂ family serving as the primary driver of rapid, high-magnitude edema at relatively low protein concentrations.

Neutralizing efficacy of freeze-dried hemorrhagic antivenom against Protobothrops mucrosquamatus venom-induced edema in a murine model

To translate clinical observations to a preclinical setting, the neutralizing efficacy of FHAV against P. mucrosquamatus CV induced edema was evaluated using a murine paw model established to simulate the progressive localized swelling seen in envenomated patients. A mixed-effects statistical analysis revealed a highly significant interaction between intervention (venom concentration and antivenom coadministration) and time (F_{6, 35} = 19.40, P < 0.0001), demonstrating that the presence of FHAV fundamentally altered the temporal trajectory of venom-induced swelling (Fig 2A). CV alone induced rapid and significant increases in paw volume across all tested concentrations, with mean differences in volume before and after exposure measuring 1.467 mm at 50 μg/mL, 1.900 mm at 100 μg/mL, and 1.900 mm at 250 μg/mL (P < 0.0001 for all). Preincubation of venom and FHAV significantly attenuated the swelling induced by low to moderate venom loads, reducing the difference in mean swelling from 1.467 mm to 0.4167 mm (P = 0.0101) at 50 μg/mL and from 1.900 mm to 0.4333 mm (P = 0.0077) at 100 μg/mL. However, at the highest venom concentration (250 μg/mL), antivenom neutralization plateaued; however, although the difference in swelling was partially reduced to 0.767 mm (P < 0.0001), the final absolute paw volume in the neutralized group (mean = 8.917 mm) was not significantly different from that in the unneutralized control receiving 250 μg/mL venom alone (mean = 9.167 mm, P = 0.9902). These findings demonstrate that while FHAV has a substantial ability to neutralize venom-induced edema, this protective effect is strictly dose dependent. As the concentration of P. mucrosquamatus venom increases, the neutralizing demand of a fixed dose of FHAV increases until its protective effect is overwhelmed, statistically confirming a clear dose‒response relationship and suggesting that clinical scenarios involving heavy venom burdens or progressive swelling require increased FHAV doses to achieve complete therapeutic neutralization.

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Fig 2. Neutralization of Protobothrops mucrosquamatus venom-induced edema with FHAV and small-molecule inhibitors and characterization of antibody immunoreactivity.

(A) Neutralizing efficacy of FHAV following preincubation with crude venom (CV; 50, 100, and 250 μg/mL) at 37°C for 30 minutes prior to subplantar injection. A mixed-effects model revealed a highly significant interaction between intervention and time (F_{6, 35} = 19.40, P < 0.0001), demonstrating that while FHAV significantly attenuated edema at lower venom loads (reducing the response to 100 μg/mL by a mean difference of 0.433 mm; P = 0.0077), its neutralization reached a functional plateau at 250 μg/mL, where swelling remained statistically indistinguishable from that of the venom-only control (P = 0.9902). (B) Evaluation of small-molecule inhibitors (SMIs) revealed that at a fixed concentration of 10 μM, varespladib, marimastat, and batimastat failed to prevent edema induced by 100 or 250 μg/mL CV; despite a significant interaction (F_{5, 12} = 4.787, P = 0.0123), all inhibitor treatment groups exhibited severe swelling (P < 0.0001 versus baseline) that was statistically indistinguishable from that of unneutralized venom. (C) Antigenic immunoreactivity of serial dilutions of FHAV determined via indirect ELISAs (1:500–1:50,000), revealing profound immunological mismatch. The antigen factor accounted for 81.49% of the total variance (F_{3, 24} = 1,119, P < 0.0001), with the antibody sequestered predominantly by the SVMP/CTL-enriched fraction, whereas the reactivity toward the primary edematogenic driver, PLA₂, remained significantly lower across all tested dilutions (P < 0.0001). The data are presented as the mean ± standard deviation (n = 6 for the CV/FHAV groups; n = 3 for the SMI groups).

https://doi.org/10.1371/journal.pntd.0014689.g002

Limited efficacy of small-molecule inhibitors in neutralizing Protobothrops mucrosquamatus venom-induced edema in a murine model

Following the evaluation of swelling induced by individual toxins in the paw edema model, SMIs were used to target specific toxin families: varespladib was used to inhibit PLA₂, and marimastat or batimastat was used to inhibit SVMP. The three SMIs were prepared at a fixed concentration of 10 μM, preincubated with CV at 100 or 250 μg/mL, and administered via subplantar injection into the hind paw of the mice, after which the paw thickness was measured at 3 h postinjection for comparison with baseline values. The mixed-effects model analysis demonstrated a highly significant main effect of time on paw thickness (F_{1, 12} = 1,071, P < 0.0001) and a significant interaction between intervention and time (F_{5, 12} = 4.787, P = 0.0123). Despite the administration of the SMIs at a concentration of 10 μM, subplantar injection of CV at 100 or 250 μg/mL induced intense and statistically significant local edema (Fig 2B). In the 100 μg/mL CV groups, the mean paw thickness increased significantly from baseline in the varespladib group (8.567 mm to 9.900 mm; q = 13.46, P < 0.0001), the marimastat group (8.000 mm to 10.010 mm; q = 21.20, P < 0.0001), and the batimastat group (7.533 mm to 9.733 mm; q = 22.10, P < 0.0001). Comparisons among the different SMIs during the postinjection phase revealed no statistically significant differences in neutralizing efficacy. After injection of 100 μg/mL CV, there were no significant differences between varespladib and marimastat (q = 1.155, P = 0.9616) or between marimastat and batimastat (q = 2.117, P = 0.6694). Similarly, at the higher CV dose of 250 μg/mL, no inhibitor demonstrated superiority with no significant difference detected between marimastat and batimastat (q = 1.540, P = 0.8811) or between varespladib and batimastat (q = 0.7698, P = 0.9936). These results indicate that at the tested concentration of 10 μM, none of these three SMIs exhibited significant neutralizing efficacy against the edematogenic activity of CV. This suggests that a single concentration of these monotargeted inhibitors may be insufficient to disrupt the complex toxic pathways in P. mucrosquamatus venom or that completely arresting the progressive localized swelling may require higher inhibitor dosages or multimodal targeting rather than single toxin family intervention under these specific experimental conditions.

Evaluation of the relative immunoreactivity of freeze-dried hemorrhagic antivenom toward crude venom and isolated toxin fractions

An indirect enzyme-linked immunosorbent assay (ELISA) was used to determine the relative immunoreactivity of FHAV toward the CV and purified fractions, including the PLA₂, SVSP, and SVMP/CTL fractions (Fig 2C). These specific toxin antigens were diluted to a final concentration of 10 µg/mL, various dilutions of FHAV were applied, and the optical density (OD) was subsequently measured to evaluate the binding affinity of FHAV for the different antigens, including CV, , SVSP, and SVMP/CTL. The immunoreactivity of FHAV for P. mucrosquamatus CV and its isolated toxin fractions were assessed using two-way ANOVA. The analysis revealed that antigen type, dilution factor, and their interaction strongly affected antibody binding, with a significant interaction (F_{6, 24} = 85.81; P < 0.0001), a main effect of dilution (F_{2, 24} = 112.0; P < 0.0001), and a main effect of the antigen (F_{3, 24} = 1,119; P < 0.0001). Notably, the antigen accounted for 81.49% of the total variance, indicating a pronounced preference in the FHAV binding profile. Tukey’s post hoc multiple comparisons test indicated that at the lowest dilution of 1:500, the binding of FHAV to CV was significantly greater than its binding to any purified fraction, including SVMP/CTL (mean difference: 0.2900, q = 9.667, P < 0.0001), PLA₂ (mean difference: 1.097, q = 36.56, P < 0.0001), and SVSP (mean difference: 1.247, q = 41.56, P < 0.0001). However, at a greater dilution of 1:50,000, the optical density of CV decreased to a level statistically indistinguishable from that of the SVMP/CTL fraction (mean difference: 0.1167; q = 3.889, P = 0.0509). Furthermore, the binding of FHAV to SVMP/CTL remained significantly greater than its binding to PLA₂ and SVSP across all the tested dilutions; for example, at a dilution of 1:50,000, the comparison between SVMP/CTL and PLA₂ yielded a mean difference of 0.8733 (q = 29.11, P < 0.0001). Taken together, these findings indicate that while FHAV exhibits immunoreactivity with multiple venom components, its primary immunoreactivity is directed toward the SVMP/CTL fraction. At high dilutions, the recognition of SVMP/CTL is comparable to that of CV, whereas FHAV binding to PLA₂ and SVSP remains consistently and significantly lower. These results suggest that proteins within the SVMP/CTL family are the predominant antigens recognized by FHAV.

Limitations

Several methodological limitations inherent to the design of this investigation warrant consideration across its sequential stages. Regarding the clinical cohort, the retrospective sample size was relatively small (N = 50) and consisted primarily of mild-to-moderate envenomations categorized via the Blaylock classification [28] rather than the Snakebite Severity Score [42] to prioritize localized edema, with resolution timelines relying on patient self-reporting subject to recall bias in the absence of objective sonographic or functional measurements. Regarding venom sampling and dosing, CV derived from a single source failed to capture regional toxicological diversity across Taiwan, while fixed experimental doses (2.5 μg and 5 μg for PLA₂; 12.5 μg and 25 μg for SVSP and SVMP/CTL in 50 μL) evaluated intrinsic potency on an equal protein mass basis rather than mirroring natural proteomic proportions (PLA₂ constituting 15.9%-25% of CV), thus complicating direct translational extrapolation [31]. Regarding the animal model, although the rodent paw assay demonstrated high statistical sensitivity (F_{1, 35} = 3,490; P < 0.0001), mice remain an imperfect proxy for human subcutaneous circulation kinetics, and the exclusive use of male ICR/CD-1 mice (n = 6 per group) to eliminate estrous variability left the study unpowered to assess sex-specific differences despite female victims accounting for 32% of the clinical cohort. Regarding therapeutic evaluations, antivenom neutralization was assessed using a single lot of FHAV (lot no. 60-06-0020; Taiwan Centers for Disease Control, Taipei, Taiwan), precluding evaluation of lot-to-lot variability. Finally, pooled fractions were assigned based on established chromatographic profiles [6] without independent reconfirmation by LC-MS/MS, SDS-PAGE, or enzymatic assays, indicating that these preparations represent toxin family-enriched fractions that may contain coeluting proteins.