Isolation and Functional Characterization of Erythrofibrase: An Alfa-Fibrinogenase Enzyme from Trimeresurus erythrurus Venom of North-East India.

Green pit viper bites induce mild toxicity with painful local swelling, blistering, cellulitis, necrosis, ecchymosis and consumptive coagulopathy. Several bite cases of green pit vipers have been reported in several south-east Asian countries including the north-eastern region of India. The present study describes isolation and characterization of a haemostatically active protein from Trimeresurus erythrurus venom responsible for coagulopathy. Using a two-step chromatographic method, a snake venom serine protease erythrofibrase was purified to homogeneity. SDS-PAGE of erythrofibrase showed a single band of ~30 kDa in both reducing and non-reducing conditions. The primary structure of erythrofibrase was determined by ESI LC-MS/MS, and the partial sequence obtained showed 77% sequence similarity with other snake venom thrombin-like enzymes (SVTLEs). The partial sequence obtained had the typical 12 conserved cysteine residues, as well as the active site residues (His57, Asp102 and Ser195). Functionally, erythrofibrase showed direct fibrinogenolytic activity by degrading the Aα chain of bovine fibrinogen at a slow rate, which might be responsible for causing hypofibrinogenemia and incoagulable blood for several days in envenomated patients. Moreover, the inability of Indian polyvalent antivenom (manufactured by Premium Serum Pvt. Ltd., Maharashtra, India) to neutralize the thrombin-like and plasmin-like activity of erythrofibrase can be correlated with the clinical inefficacy of antivenom therapy. This is the first study reporting an α-fibrinogenase enzyme erythrofibrase from T. erythrurus venom, which is crucial for the pathophysiological manifestations observed in envenomated victims.

Multilevel Comparison of Indian Naja Venoms and Their Cross-Reactivity with Indian Polyvalent Antivenoms.

Snake envenoming is caused by many biological species, rather than a single infectious agent, each with a multiplicity of toxins in their venom. Hence, developing effective treatments is challenging, especially in biodiverse and biogeographically complex countries such as India. The present study represents the first genus-wide proteomics analysis of venom composition across Naja species (N. naja, N. oxiana, and N. kaouthia) found in mainland India. Venom proteomes were consistent between individuals from the same localities in terms of the toxin families present, but not in the relative abundance of those in the venom. There appears to be more compositional variation among N. naja from different locations than among N. kaouthia. Immunoblotting and in vitro neutralization assays indicated cross-reactivity with Indian polyvalent antivenom, in which antibodies raised against N. naja are present. However, we observed ineffective neutralization of PLA2 activities of N. naja venoms from locations distant from the source of immunizing venoms. Antivenom immunoprofiling by antivenomics revealed differential antigenicity of venoms from N. kaouthia and N. oxiana, and poor reactivity towards 3FTxs and PLA2s. Moreover, there was considerable variation between antivenoms from different manufacturers. These data indicate that improvements to antivenom manufacturing in India are highly desirable.

Proteome Decomplexation of Trimeresurus erythrurus Venom from Mizoram, India.

Green pit vipers are the largest group of venomous vipers in tropical and subtropical Asia, which are responsible for most of the bite cases across this region. Among the green pit vipers of the Indian subcontinent, Trimeresurus erythrurus is the most prevalent; however, limited knowledge is available about its venomics. Proteome decomplexation of T. erythrurus venom using mass spectrometry revealed a blend of 53 different proteins/peptides belonging to 10 snake venom protein families. Phospholipase A2 and snake venom serine proteases were found to be the major enzymatic families, and Snaclec was the major nonenzymatic family in this venom. These protein families might be responsible for consumptive coagulopathy in victims. Along with these, snake venom metalloproteases, l-amino acid oxidases, disintegrins, and cysteine-rich secretory proteins were also found, which might be responsible for inducing painful edema, tissue necrosis, blistering, and defibrination in patients. Protein belonging to C-type lectins, C-type natriuretic peptides, and glutaminyl-peptide cyclotransfreases were also observed as trace proteins. The crude venom shows platelet aggregation in the absence of any agonist, suggesting their role in alterations in platelet functions. This study is the first proteomic analysis of T. erythrurus venom, contributing an overview of different snake venom proteins/peptides responsible for various pathophysiological disorders obtained in patients. Data are available via ProteomeXchange with the identifier PXD038311.

Its all in the name: Another new Cyrtodactylus Gray (Squamata: Gekkonidae) from northern Mizoram, North-east India.

A new species of Cyrtodactylus is described from Vairengte town, situated in the Kolasib District of Mizoram State, north-eastern India. The new species is found to be a member of Indo-Burman Cyrtodactylus khasiensis clade based on ND2 gene sequences and morphological parameters, such as number of precloacal pores, mid-ventral scale rows, paravertebral tubercles on the trunk, dorsal tubercle rows, subdigital lamellae on pes and subcaudal scalation, making it the sixth endemic Cyrtodactylus from Mizoram and twenty second from north-east India. Moreover, phylogenetic evidence suggests the new species to be sister to the recently described C. aaronbaueri, and morphological analyses also reveal marginal separation between the two species based on the PCA of infralabials, lamellae on fingers and toes, paravertebral tubercles on the trunk, and dorsal tubercle rows.

A new cryptic species of green pit viper of the genus Trimeresurus Lacépède, 1804 (Serpentes, Viperidae) from northeast India.

A new cryptic species of green pit viper is described from northeast India, based on specimens collected from the state of Mizoram and Meghalaya. The new species is a member of the subgenus Viridovipera and is sister to Trimeresurus medoensis based on molecular data for mitochondrial cytochrome b gene, whereas resembles Trimeresurus gumprechti morphologically. A combination of characters helps delimit the new species from its congeners. Description of the new species highlights the need for dedicated surveys across northeast India to document its reptilian diversity, as this represents the third new species of the genus to be described in the past three years.

A new bent-toed gecko (Cyrtodactylus Gray: Squamata: Gekkonidae) from the state of Mizoram, India.

Herein we describe a new species of Cyrtodactylus from Lunglei District in the state of Mizoram, India. Based on morphology and ND2 gene sequences, the species was found to be a member of the Cyrtodactylus khasiensis group. The species can be identified by its moderate size (adult SVL 64.975.1 mm) with rounded, bluntly conical and feebly keeled dorsal tubercles in 2428 longitudinal rows; 3240 paravertebral tubercles between the level of the axilla and the level of the groin; 3743 mid-ventral scale rows; 35 precloacal pores in males and 57 pitted precloacal scales in females; 1618 subdigital lamellae under IV toe; no single row of transversely enlarged subcaudal scales; dorsal markings are dark brown, irregular and distinct; tail with alternating dark and light bands.

Genetic diversity of the Pegu Rice Frog, Microhyla berdmorei (Anura: Microhylidae) based on mitochondrial DNA.

The Pegu Rice Frog, Microhyla berdmorei is distributed across ten Asian countries. However, the DNA barcoding information (COI gene) is restricted to only Southeast Asian countries. Here, we sampled a specimen of M. berdmorei in Mizoram state, northeast India to allow the genetic diversity of the species across its range. We generated both COI and 16S ribosomal RNA sequences of the studied species to check the population genetic diversity. The Bayesian analyses clearly discriminate M. berdmorei from its sister species Microhyla pulchra. The present datasets of M. berdmorei also revealed 11 and 19 haplotypes with high uncorrected pairwise genetic distances in COI (3.8-11.8%) and 16S rRNA (0-4.6%) gene, respectively. Owing to the high intra-species genetic distances and different haplotypes with sufficient mutational steps in both mitochondrial genes, this study affirms the existence of M. berdmorei species complex or cryptic diversity within its range distribution in South and Southeast Asia.

Four new Bent-toed geckos (Cyrtodactylus Gray: Squamata: Gekkonidae) from northeast India.

Based on morphology and ND2 gene sequences, four new species of Cyrtodactylus, two each from the Indian states of Meghalaya and Mizoram are described herein. The new species are a part of the Cyrtodactylus khasiensis group. The species from Meghalaya represent the lowland clade whereas the species from Mizoram represent the highland clade within the south of Brahmaputra clade of Indo-Burmese Cyrtodactylus. The two distinct populations from Meghalaya are sister to one another, differing from each by an uncorrected p-distance 0.065 and collectively are sister to Cyrtodactylus septentrionalis. The species from Mizoram differ from each other by an uncorrected p-distance of 0.0850.121 and collectively are sister to Cyrtodactylus montanus.

DNA barcoding reveals distinct population of Dopasia gracilis (Squamata: Anguidae) in Mizoram, Northeast India.

The DNA barcode data of Asian Glass Lizard, Dopasia gracilis, is limited in the global database, especially from India. The present study aimed to generate a barcode sequence of morphologically identified D. gracilis from the Mizoram state in northeast India and compared with other Anguidae species. The studied species showed monophyletic clustering in the Bayesian analysis (BA) phylogeny with strong posterior probability support and also discriminated sufficient Kimura 2 parameter genetic distances. The barcode data of D. gracilis revealed high intra-species genetic variability and formed two clusters in BA phylogeny. The Templeton, Crandall, and Sing network also depicted four different haplotypes within the barcode sequences of D. gracilis. The DNA sequences generated from northeast India showed 6.5-6.6% and 7.3% genetic distances with the sequences generated from Yunnan Province and Tibetan Plateau, respectively. Considering the high genetic distances, multiple clustering, and distinct haplotypes, the present study assumed the presence of possible cryptic diversity of D. gracilis in the Indochina sub-region and a distinct population in northeast India. We recommended the generation of more DNA information from different localities to elucidate the actual diversity of D. gracilis within the known range distribution.