Toxicon, Vol. 24, No.5, pp. 510-513, 1986.
Printed in Great Britain.
0041-0101186 $3.00+ .00
Pergamon Journals Ltd.
PRELIMINARY STUDIES ON THE VENOM OF THE CHINESE
SNAKE AZEMIOPS FEAE, BOULENGER (FEA'S VIPER)
DARWIN K. VEST
Department of Zoology, Washington State University, Pullman, WA 99164-4220, U.S.A.
(Accepted jor publication 11 December 1985)
D. K. VEST. Preliminary studies on the venom of the Chinese snake Azemiops jeae, Boulenger
(Fea's viper). Toxicon 24, 510- 513, 1986. - Fea's viper (Azemiops jeae) produces a venom
which is highly toxic to mice when injected by the s.c. or Lv. routes. The Lv. LDso of Azemiops
venom for Swiss-Webster laboratory mice is 0.52 mg/kg. Azemiops venom produces no
hemorrhagic activity in mice or rabbits. Immunodiffusion indicates that some fractions of
Azemiops venom are antigenically related to viperid, elapid and crotalid venoms. SDS-PAGE
electrophoresis reveals that this venom contains as many as 22 proteinaceous components.
THE FEA'S VIPER (Azemiops jeae) is a small viperine snake distributed in the Chinese
provinces of Yunan, Guizhou, Sichuan, Guangxi, Fujian, Jiangxi and Zhejiang (ZHAO
and ZHAO, 1981) and also occurs in Burma. Due to the rarity of this serpent, virtually
nothing is known concerning its venom. Histological investigation of Azemiops venom
glands by KOCHVA and GANS (1965, 1966) and KOCHVA et ale (1967) show that Azemiops
venom glands exhibit the general pattern of taxonomic characteristics of all viperine
Seven adult specimens of Azemiopsjeae were obtained from a commercial source (J. J.
Vandenbrink, Jabria B.V.) and were subjected to venom extraction. The venom was
collected in a snlall plastic 'auto-analysis' cup (Sardstedt no. 73.641), immediately frozen,
then lyophilized and weighed. Lyophilized venom was reconstituted in 0.9070 physiological
saline. Twenty-five healthy Swiss-Webster laboratory mice weighing 18 - 22 g were used
for the lethality screen. The Lv. LD so was calculated according to the method of
LITCHFIELD and WILCOXON (1949). All mice were closely observed for 12 hr post
challenge. Respiratory rates were measured by counting respirations against a lap timer.
Thirteen Swiss-Webster mice were depilated and challenged s.c. with venom
concentrations of 5 - 50 JAg in 100 JAI 0.9070 physiological saline. Additionally, a California
giant laboratory rabbit was dorsally depilated and challenged intracutaneously with six
concentrations of (5 - 50 JAg) Azemiops venom reconstituted as described above. Mice
surviving s.c. challenge, as well as the rabbit, were killed at 24 hr post-injection and
examined for hemorrhagic and local responses. Immunodiffusion of Azemiops venom
against several commercial monovalent and polyvalent antivenoms, as well as active
monovalent antisera of rabbit origin (Arizona State University), was performed on
Ouchterlony plates and micro-immunodiffusion slides using 2070 agar noble in the
diffusion medium. Antigen wells were loaded with venom solutions containing 5 mg/ml
Azemiops venom, and were developed at room temperature. SDS-Polyacrylamide gel
electrophoresis (slab format) was performed at pH 6.8 according to the method of
LAEMMLI (1970). Wells were loaded in duplicate with venom quantities of 50, 75, 100, 125
and 150 IJg. All samples were reduced with (3-mercaptoethanol and run at 10 rnA per gel
through a 3 mm thick, 5070 stacking gel, then a 15070 separating gel.
Five of the Azemiops specimens subjected to extraction delivered only trace amounts of
venom, despite vigorous biting. The other two specimens delivered significant,
approximately equivalent volumes of venom ( rv l.75 mg/snake). The venom appeared as
a clear, golden-yellow liquid, similar to that of many other viperine snakes. Its viscosity,
likewise, was similar to that delivered by most viperines.
The i.v. LD so of Azemiops feae venom in Swiss-Webster mice was 0.52 mg/kg. Mice
challenged with doses of 0.50 - 0.60 mg/kg usually exhibited a transitory, minor-to
moderate vasodilatation of ear vessels within 10 min of injection. These mice became
torpid within 30 ± 10 min and respirations began to decrease, generally followed by clonic
convulsions. Paralysis became virtually complete 90 - 110 min post-injection, although
mice retained a very slight ability to move the legs and were able to right themselves until
the very terminal stage of poisoning. A paroxysm occurred 86 -150 min post-injection, in
which respirations reached a critical minimum (less than 35 per min). This paroxysm
terminated in death for mice challenged by a lethal dose; all mice that tolerated
envenomation for 180 min survived. All mice receiving s.c. challenges of between 0.50
and 0.60 mg/kg died, while those receiving 0.40 mg/kg or less survived.
Local tissue responses and hemorrhagic activity in laboratory mice were only slight. No
indications of frank hemorrhagic activity nor local tissue degradation were evident.
Intracutaneous injection of Azemiops venom into rabbit skin produced no local
manifestations or evidence of hemorrhagic phenomenon.
Ouchterlony and micro-immunodiffusion of Azemiops venom vs. commercial
antivenins resulted in the formation of precipitation lines between wells containing
Azemiops venom and several commercial antivenins (Fig. 1). Significant precipitation
lines developed against the antisera to tiger-snake, death adder, mamba and Iran cobra,
while weak reactions were seen against Thai cobra, and no reaction developed against
king cobra. Strong, multiple precipitation lines developed against Serum Europe, Serum
North Africa, Fitzsimmon's Polyvalent and Wyeth Polyvalent, with weaker lines visible
against Haffkine Polyvalent and Serum Near and Middle East. No precipitation reaction
was observed against monovalent Agkistrodon acutus antivenene or Malayan pit viper
antivenene, nor against monovalent Agkistrodon bilineatus, Agkistrodon piscivorus,
Crotalus atrox, Crotalus scutulatus or Heloderma suspectum antisera.
SDS-PAGE of fJ-mercaptoethanol-reduced Azemiops venom revealed the presence of
22 visible proteinaceous bands with molecular weights ranging from approximately 10,000
to 80,000. When 100 - 150 IJg of venom were applied, 20 - 22 bands were visible,
including one smaller component (mol. wt 9000 - 11,000) not well visualized when lesser
amounts of venom were applied.
Fea's viper (Azemiops feae) produces a venom which causes marked flaccid paralysis,
respiratory depression and clonic convulsions in mice, while eliciting only negligable local
tissue responses. There was no apparent hemorrhagic phenomenon, either local or
systemic. Generally, convulsions signaled the onset of a paroxysm in which respiratory
rates fell below 30 per min and finally ceased altogether, terminating in the death of the
mouse. Superficially, the progressive respiratory depression, which closely paralleled the
progression of flaccid paralysis, resembles poisoning by those elapid snakes whose
venoms contain a post-synaptic, peptide neurotoxin.
The present study using mice indicates that Azemiops envenomation may be expected to
produce visual, respiratory and paralytic effects in humans, but does not suggest that this
venom is capable of inducing local or hemorrhagic phenomenon, as reported for humans
(ZHAO and ZHAO, 1981).
Immunodiffusion of Azemiops venom against multi-genus polyvalent antivenins
demonstrate the presence of antigenically related components between Azemiops and all
major groups of terrestrial venomous snakes. Precipitation lines between Azemiops
venom and monovalent Australian antivenenes, as well as genus-specific mamba
antivenene, indicate a definite antigenic relationship between these venoms and
Azemiops, but it should be noted that these sera react with an exceptionally large number
of heterologous venoms, including colubrid Duvernoy's secretions (MINTON, 1979;
Minton, personal communication). Wyeth Polyvalent Antivenin proved to be the only
crotalid preparation reacting against Azemiops venom, with no reaction seen against
monovalent Agkistrodon or Crotalus antisera.
Azemiops jeae may be considered a member of the Viperidae which possesses a venom
with immunological and electrophoretic characteristics similar to those of other viperine
.-- • .~ •
o • -'"¢J 0
FIG. I. MACRO OUCHTERLONY IMMUNODIFFUSION OF SOME ANTIVENINS ACTIVELY PRECIPITATING
Photographed 72 hr post-loading: (A) Azemiops /eae venom, 5 mg/ml; (D) death adder
antivenom; (F) Fitzsimmon's Polyvalent; (H) Haffkine Polyvalent; (I) Iran cobra antivenom; (M)
mamba Polyvalent; (T) tiger snake antivenom; (NA) Serum North Africa; (NM) Serum Near and
Middle East; (TC) Thai cobra antivenom. Comparable, but more sharply defined precipitations
were observed using micro-immunodiffusion slides.
snakes. Its toxicity characteristics, however, are not typical of the majority of the better
known, clinically significant Viperidae, whose venoms induce changes in local tissues,
hemorrhage and hematological discrasias. Azemiops venom appears to be one of the
'atypical' viperid venoms, which, like the berg adder Bitis atropos (CHRISTENSEN, 1955,
1968), elicits pronounced paralytic phenomenon in mice, without evidence of local and/or
Acknowledgement - For comments on the manuscript, I thank SHERMAN A. MINTON, EDWARD K. JOHNSON
and K. V. KARDONG. For technical assistance, I thank TERRY ELTON, K. GASSER, H. L. HOSICK, J. D. HUBER, R.
REEVES, P. C. SCHROEDER and L. KIRSCHNER. This work was supported in part by grant BNS 7817465,
Psychobiology Program, National Science Foundation, to K. V. KARDONG.
CHRISTENSEN, P. A. (1955) South African Snake Venoms and Antivenoms. Johannesburg: South African
Institute of Medical Research.
CHRISTENSEN, P. A. (1968) Venoms of Central and South African snakes. In: Venomous Animals and their
Venoms, Vol. 1, p. 447 (HOCHERL, W., BUCKLEY, E. and DEULOFEU, V., Eds). New York: Academic Press.
KOCHVA, E. and GANS, C. (1965) The venom gland of Vipera palaestinae with comments on the glands of some
other Viperinae. Acta Anat. 62, 365.
KOCHVA, E. and GANS, C. (1966) Histology and histochemistry of the venom gland of some crotaline snakes.
Copeia 3, 506.
KOCHVA, E., SHAYER-WOLLBERG, M. and SOBOL, .R. (1967) The special pattern of the venom gland in
Atractaspis and its bearing on the taxonomic status of the genus. Copeia 4,·763.
LAEMMLI, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
LITCHFIELD, J. T. JR and WILCOXON, I. (1949) A simplified method of evaluating dose-effect experiments. J.
Pharmac. expo Ther. 96, 99.
MINTON, S. A. (1979) Common antigens in snake venoms. In: Handbook ofExperimental Pharmacology, Vol.
52, Snake Venoms, p. 847 (Lee, C.-Y., Ed.). New York: Springer-Verlag.
ZHAO, E. and ZHAO, G. (1981) Notes on Fea's viper (Azemiopsfeae Boulenger) from China. Acta herpetol. sin.