V. Evaluation of Cross-Immunity against Type 1 Dengue Fever in Human Subjects Convalescent from Subclinical Natural Japanese Encephalitis Virus Infection and Vaccinated with 17D Strain Yellow Fever Vaccine
Hemorrhagic toxin g (HT-g) was isolated from Crotalus atrox (western diamondback rattlesnake) venom using a five-step purification procedure to obtain ≈5.9 mg of purified HT-g from 2.0 g of crude venom. The purified toxin was homogeneous by disc electrophoresis on polyacrylamide gel at pH 8.3 and 4.3, and by isoelectric focusing. HT-g possessed lethal, hemorrhagic and proteolytic activities. These activities of toxin were inhibited by ethylenediamine-tetraacetic acid (EDTA), 1,10-phenanthroline or ethyleneglycol (β-amino-ethyl) N,N,N′,N′-tetracetic acid (EGTA), but not by cysteine or soybean trypsin inhibitor (SBTI). Its molecular weight was approximately 60,000 and the isoelectric point was 6.8. The toxin contains 516 amino acid residues. HT-g did not coagulate fibrinogen to fibrin; however, the toxin hydrolysed the Aα-chain or Bβ-chain of fibrinogen without cleaving the γ-chain. HT-g produced only local hemorrhage in internal organs such as the intestine, heart and liver.