In vitro immunoreactivity and in vivo neutralization of Trimeresurus gracilis venom with antivenoms targeting four pit viper species.

Snakebite envenomation is a significant global health issue that requires specific antivenom treatments. In Taiwan, available antivenoms target a variety of snakes, but none specifically target Trimeresurus gracilis, an endemic and protected species found in the high mountain areas of Taiwan. This study evaluated the effectiveness of existing antivenoms against T. gracilis venom, focusing on a bivalent antivenom developed for Trimeresurus stejnegeri and Protobothrops mucrosquamatus (TsPmAV), as well as monovalent antivenoms for Deinagkistrodon acutus (DaAV) and Gloydius brevicaudus (GbAV). Our research involved in vivo toxicity testing in mice and in vitro immunobinding experiments using (chaotropic) enzyme-linked immunosorbent assays, comparing venoms from four pit viper species (T. gracilis, T. stejnegeri, P. mucrosquamatus, and D. acutus) with three types of antivenoms. These findings indicate that TsPmAV partially neutralized T. gracilis venom, marginally surpassing the efficacy of DaAV. In vitro tests revealed that GbAV displayed higher binding capacities toward T. gracilis venom than TsPmAV or DaAV. Comparisons of electrophoretic profiles also reveal that T. gracilis venom has fewer snake venom C-type lectin like proteins than D. acutus, and has more P-I snake venom metalloproteases or fewer phospholipase A2 than G. brevicaudus, T. stejnegeri, or P. mucrosquamatus. This study highlights the need for antivenoms that specifically target T. gracilis, as current treatments using TsPmAV show limited effectiveness in neutralizing local effects in patients. These findings provide crucial insights into clinical treatment protocols and contribute to the understanding of the evolutionary adaptation of snake venom, aiding in the development of more effective antivenoms for human health.

Comparative analysis of Deinagkistrodon acutus venom from Taiwan and China utilizing chromatographic, electrophoretic, and bioinformatic approaches, along with ELISA employing a monospecific antivenom.

Deinagkistrodon acutus is a medically important pitviper inhabiting mainly South China and Taiwan. The hemorrhagic effects of its envenoming are compatible to its venom, which is abundant in metalloproteases (svMPs) and C-type lectin-like proteins. In this study, we investigated geographic variations in the venom of D. acutus collected from Taiwan and four Mainland Chinese provinces: Fujian, Jiangxi, Anhui, and Hunan. The variations were assessed through high-performance liquid chromatography, non-metric multidimensional scaling analysis, gel electrophoresis, and enzyme-linked immunosorbent assay (ELISA) with a monospecific antivenom (DaMAV) generated against the Taiwanese D. acutus venom, and discussed based on venom-protein sequences in databases and literature related to D. acutus venom. Additionally, the cross-reactivity of DaMAV against Crotalus horridus and Calloselasma rhodostoma venoms was investigated. We noted differential abundances of D. acutus venom metalloproteases, C-type lectin-like proteins, and phospholipase A2, along with point mutations and selective expression of serine protease isoforms. The ELISA results revealed that the venom from Taiwan was more reactive toward Taiwanese DaMAV than the four Mainland Chinese venoms, consistent with chromatographic profile differences, whereas C. horridus venom presented moderate cross-reactivity with DaMAV. The observed immunoreactivities of these venom with DaMAV can be attributed to the high prevalence of their PIII-svMPs, which are the dominant antigens, and the conservation of PIII-svMP epitopes.

Venom Proteomics of Trimeresurus gracilis, a Taiwan-Endemic Pitviper, and Comparison of Its Venom Proteome and VEGF and CRISP Sequences with Those of the Most Related Species.

Trimeresurus gracilis is an endemic alpine pitviper in Taiwan with controversial phylogeny, and its venom proteome remains unknown. In this study, we conducted a proteomic analysis of T. gracilis venom using high-performance liquid chromatography-tandem mass spectrometry and identified 155 toxin proteoforms that belong to 13 viperid venom toxin families. By searching the sequences of trypsin-digested peptides of the separated HPLC fractions against the NCBI database, T. gracilis venom was found to contain 40.3% metalloproteases (SVMPs), 15.3% serine proteases, 6.6% phospholipases A2, 5.0% L-amino acid oxidase, 4.6% Cys-rich secretory proteins (CRISPs), 3.2% disintegrins, 2.9% vascular endothelial growth factors (VEGFs), 1.9% C-type lectin-like proteins, and 20.2% of minor toxins, nontoxins, and unidentified peptides or compounds. Sixteen of these proteoforms matched the toxins whose full amino-acid sequences have been deduced from T. gracilis venom gland cDNA sequences. The hemorrhagic venom of T. gracilis appears to be especially rich in PI-class SVMPs and lacks basic phospholipase A2. We also cloned and sequenced the cDNAs encoding two CRISP and three VEGF variants from T. gracilis venom glands. Sequence alignments and comparison revealed that the PI-SVMP, kallikrein-like proteases, CRISPs, and VEGF-F of T. gracilis and Ovophis okinavensis are structurally most similar, consistent with their close phylogenetic relationship. However, the expression levels of some of their toxins were rather different, possibly due to their distinct ecological and prey conditions.

Case Report: Symptoms and Prognosis of Trimeresurus gracilis Envenomation.

Trimeresurus gracilis is a pit viper inhabiting the high mountains of Taiwan. No specific antivenom against T. gracilis is available, and we did not find any prior published case reports on its clinical envenomation. We present two patients with T. gracilis envenomation who were both bitten on the index finger and were administered bivalent hemotoxic antivenom against Trimeresurus stejnegeri and Protobothrops mucrosquamatus. In case 1, the patient was administered seven vials of antivenom within 2 days of envenomation. She received surgical intervention on day 26 and recovered from the wound 3 months after envenomation, but her left index finger was slightly and irreversibly bent. In case 2, the patient was administered 12 vials of antivenom within 10 hours after envenomation, received surgical intervention on day 1, and underwent debridement and reconstruction surgery on day 7 after envenomation. The wounds healed 1.5 months after envenomation, and no deformity occurred. Laboratory data showed elevated D-dimer levels and prothrombin times. The cross-neutralizing ability of bivalent hemotoxic antivenom against T. stejnegeri and P. mucrosquamatus appeared insufficient to treat the local effects of T. gracilis envenomation. The deformity of the finger of the patient in case 1 might have been caused by the lower dose, later administration of antivenins, and a delay in the necessary surgery.

Species Identification of Fragmented or Faded Shed Snake Skins by Light Microscopy.

Few convenient/expeditious methods for identifying the species of shed snake skins in specific areas have been developed. The scales on shed snake skins are permeable to light and can be examined by light microscopy (LM), which is of higher availability-especially for wild animal researchers and citizen scientists-than conventional approach which examines the scale microstructures by scanning electron microscopy. We collected and examined a total of 801 shed samples or scale specimens from 53 snake species in Taiwan and adjacent islands, and developed the first guide to identify the fragmented or faded shed skins of most snake species by LM. Morphological characters of scales can be examined by LM include the apical notch, apical pits, apical lobes, keels, scale symmetry, unpigmented spots (mechanoreceptor-like organs), interscale follicles, cross/longitudinal micro-ridge, oberhautchen cells, rows of spines, light/tiny dots, and other microstructures. The microstructures on the scale specimens prepared by the stripped method and the impression method were similar to those on shed skins when examined by LM. We investigated the variations of scale morphology associated with ontogeny, body region, and position on scales, discussed the character evolution of snake scale morphology, and certified that the interscale follicles and the unpigmented spots could also be useful characters for shed skin identification. The methods and results of this study could be applied to identify squamate skins/sloughs and even fecal remnants.

Newspaper reporting of suicides in Hong Kong, Taiwan and Guangzhou: compliance with WHO media guidelines and epidemiological comparisons.

BACKGROUND: Media guidelines for suicide reporting are available in many countries. However, to what extent the mass media comply with the guidelines is unknown. Few studies are available that investigate systematically whether the mass media reflect the epidemiological reality of suicide deaths in their articles. METHODS: Based on the WHO media guidelines, this study investigated the characteristics of newspaper articles of suicides in three Chinese communities, namely Hong Kong, Taiwan and Guangzhou. Epidemiological comparisons were conducted to identify the age and gender differences between the suicide victims as reported in the newspapers and the official records of suicide deaths in all three places. RESULTS: The results found that one media characteristic complied with the WHO media guidelines (ie, only about 2% of the articles were printed on the front page), but there were a number of instances of non-compliance (ie, only 4-14% provided sources for help-seeking and 27-90% printed with photos). The epidemiological comparisons revealed an over-representation of younger suicides and an under-representation of late-life suicides in the newspapers of all three places. Furthermore, female suicides were found to be under-reported in Taiwan and Guangzhou newspapers, but not in Hong Kong papers. CONCLUSION: Non-compliant suicide articles are prevalent in the newspapers of these three Chinese settings. The observed media misrepresentations may potentially mislead the public and the policy makers about the actual risk for suicide in some demographic groups.