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American Journal of Infectious Diseases 6 (2): 24-28, 2010
ISSN 1553-6203
© 2010 Science Publications
Corresponding Author: Bhaswati Bandyopadhyay, Department of Microbiology, Virology Unit, School of Tropical Medicine,
Kolkata-700073, India
24
Decreased Intensity of Japanese Encephalitis Virus Infection in Chick
Chorioallantoic Membrane Under Influence of Ultradiluted Belladonna Extract
1
Bhaswati Bandyopadhyay,
2
Satadal Das,
1
Milan Sengupta,
3
Chandan Saha,
4
Kartick Chandra Das,
4
Debabrata Sarkar
and
5
Chaturbhuj Nayak
1
Department of Microbiology, Virology Unit, School of Tropical Medicine, Kolkata-700073, India
2
Department of Pathology and Microbiology, D.N. De H. Medical College,
West Bengal University of Health Sciences, Kolkata-700046, India
3
Department of Clinical and Experimental Pharmacology,
School of Tropical Medicine, Kolkata-700073, India
4
Drug Proving Research Centre, CCRH, Government of India,
Kolkata-700 046, India
5
Department of AYUSH, Ministry of Health, CCRH, Government of India, JLN Anudandhan Bhawan,
61-65 Intitutional Area, Janakpuri, New Delhi 110058
Abstract: Problem statement: No specific antiviral therapy is currently available despite an
emergence and resurgence of Japanese encephalitis in South-East Asian Countries. There are only few
recent studies, which were aimed to treat Japanese encephalitis with newer drugs. There is thus a real
need for study on antiviral agents that can reduce the toll of death and neurological sequelae resulting
from infection with this virus. Approach: Optimum dilution of the JE virus was determined which
could produce significant number of pocks on Chorioallantoic Membrane (CAM). Then ultradiluted
belladonna preparations were used to see their inhibitory action on JE virus infection in CAM.
Results: Ultradiluted belladonna showed significantly decreased pock count in CAM in comparison to
JE virus control. Conclusion: Ultradiluted belladonna could inhibit JE virus infection in CAM, which
may be mediated through glycosidase inhibitory role of calystegines present in belladonna.
Key words: Japanese Encephalitis (JE), Chorioallantoic Membrane (CAM), pock, belladonna
INTRODUCTION
Neglect of the basic requirements of health; poor
political support for health; a weak public health
capacity; centralized programs for control based on
selective interventions and poorly-planned development
projects have created conditions ideal for the outbreak
of JE like diseases (Bhargava and Chatterjee, 2007). A
good community awareness of encephalitis, a prompt
referral system and a good supportive treatment for the
patients and a good surveillance system was found to
help in the reduction in cases, deaths and disabilities
due to this disease (Gupta et al., 2008). Approximately
2 billion people live in countries where JE presents a
significant risk to humans and animals, particularly in
China and India, with at least 700 million potentially
susceptible children (Gould et al., 2008). In Southeast
Asia around 50.000 cases and 10.000 deaths occur per
year affecting essentially children below 10 years of
age. Further threats to humanity are there because the
JE virus has shown a tendency to extend to other
geographic areas. The combined effects of climate
change, altered bird migratory patterns, increasing
movement of humans, animals and goods, increasing
deforestation and development of irrigation projects
will also help this geographic dispersal of the virus
producing an enhanced threat. The disease is also
highly prevalent in animals. In Nepal, sero-
prevalence of JE in pigs, ducks and horses was
48.11, 26.79 and 50.0%, respectively (Pant, 2006).
At present in Nepal JE is seasonally endemic to the
Terai region (Wierzba et al., 2008) and in Kathmandu
valley (Partridge et al., 2007) affecting population of
both lowland plains as well as the hilly regions.
Phylogenetic analysis showed that JE isolates in India
belonged to genogroup 3 (Parida et al., 2005).
Belladonna or Atropa belladonna (deadly
nightshade)-the source of the drug atropine belong to
Am. J. Infect. Dis., 6 (2): 24-28, 2010
25
genus Atropa along with two other species (Hunziker,
2001). Although the phylogenetic affinity of Atropa to
Hyoscyameae has been controversial for over a century
(Hunziker, 2001), however, in a recent study this
affinity was conclusively proved (Yuan et al., 2006).
The plant Atropa belladonna is surrounded by myth,
fear and awe. That this plant contains poison is known
from the ancient Greek and Roman civilization to
medieval witches, professional poisoners, sorcerors and
ultimately in 1830s atropine was isolated from the plant
(Lee, 2007). Atropa belladonna is rich in tropane
alkaloids, primarily atropine and scopolamine
(Talaty et al., 2005).
Roots are the major organs of tropane alkaloid
biosynthesis and after their formation in roots, tropane
alkaloids are transported to the aerial parts of the plant.
Putrescine N-Methyltransferase (PMT) is the pivotal
enzyme for the biosynthesis of tropane alkaloids. The
PMT cDNAs were cloned from A. belladonna and were
found to encode a protein homologous to spermidine
synthases indicating the evolutionary origin of PMT
from spermidine synthase. In A. belladonna, PMT is
located in pericycle and xylem cells of the root. Root
cultures of A. belladonna form the tropine derived
alkaloids hyoscyamine, scopolamine and calystegines.
While hyoscyamine and scopolamine are also found in
other related species to A. belladonna, thus calystegines
appear to be the key agent which is present in high
concentration to A. belladonna only and thus
differentiates biological activities of belladonna from
other related plants. The biosynthetic pathway of
tropane alkaloids is very complex. Putrescine first
converted into N-methyl putrescine by PMT. N-methyl
putrescine yields tropinone. In tropinone I pathway
tropinone is converted to tropine, tropine is converted
to hyoscyamine and finally converted to scopolamine
by the action of hyoscyamine-6-hydroxylase. In the
relatively less investigated tropinone II pathway at first
there is formation of pseudotropine, which is then
converted to calystegine.
Although most human infections are mild or
asymptomatic, about 50% of patients who develop
Japanese encephalitis suffer permanent neurologic
defects and 30% of them die due to the disease
(Babu et al., 2006).
Vaccines for JE have been available for many years
and their use has been effective in reducing the incidence
of JE disease in several countries (Diagana et al., 2007)
but, as disease incidence has decreased, concerns
regarding adverse events following immunisation have
increased (Beasley et al., 2008). Childhood mass
immunization programs with first generation, mouse
brain-derived vaccines showed occurrence of severe
side effects in Japan.
No specific antiviral therapy is currently available
(Gould et al., 2008) despite an emergence and
resurgence of flavivirus-mediated diseases (Ray and
Shi, 2006). There are few recent studies, which were
aimed to treat JE with new drugs. A plant lignan
arctigenin was found to reduce viral load and viral
replication within the brain, neuronal death and
secondary inflammation and oxidative stress resulting
from microglial activation, suggesting its potential for
treating JE, however, unless it is tested in human beings
it can not be used in treatment of JE (Swarup et al.,
2008). Similarly, Interferon alpha-2a was tested in
children with JE, but with negative results. There is
thus a real need for antivirals that can reduce the toll of
death and neurological sequelae resulting from
infection with JE virus (Gould et al., 2008). Therefore,
this study was aimed to see whether ultradiluted
belladonna has a role in this infection.
MATERIALS AND METHODS
Ultradiluted belladonna: For the study we selected
Ultradiluted Belladonna 3,6,30,200. These medicines
were included in this study because they are claimed by
practitioners and researchers of alternative medicine to
have a positive role in the treatment and prevention in
Japanese Encephalitis (JE). All these medicines were
available commercially and were prepared according to
standard procedures advocated by homeopathic
pharmacopoeia of India (Ministry of Health,
Government of India, 1971, 1:1, 7-16, 72).
Procurement of ultradiluted belladonna for this
study: Medicines were reconstituted in sterile pyrogen
free water immediately before their applications after
complete elimination of alcohol.
The aqueous dilution of Belladonna (3, 6, 30, 200)
was prepared and procured from reputed Homeopathic
drug company, Hahnemann Publishing Co. Pvt. Ltd
(HAPCO), Kolkata.
Virus stock: JE virus stock (Nakayama strain), which
is maintained in School of Tropical Medicine, Kolkata,
was used in this study.
Embryonated chick egg inoculations: For preventive
studies one dose (50 µL) of aqueous dilution of each
selected medicine was inoculated in the chorioallantoic
membrane followed by the administration of 50 µL of
the JE viral suspension, 5-10 min later. The chorio
allantoic membrane consists of an outer layer of stratified
epithelium, which constitutes the respiratory surface of
the embryonated egg and an inner layer of endoderm (the
lining of the allantoic cavity).
Dermotropic viruses (poxviruses and some herpes
viruses) and JE viruses grow on this membrane and at
Am. J. Infect. Dis., 6 (2): 24-28, 2010
26
low concentrations, they produce discrete foci of cell
proliferation and necrosis (pocks). The membrane was
therefore used to assay JE viruses in this study.
Different viruses cause pocks of different color and
morphology and this is also of diagnostic value for
distinguishing between different viruses. One-day-old
fertile hen’s (White leghorn) eggs were obtained from
State Poultry Farm of Govt. of West Bengal,
Tollygunge, Kolkata. They were collected from healthy
flocks, which were maintained on a well balanced and
antibiotic free diet. The eggs were incubated at 37°C
within a special egg incubator with 65% humidity.
On the 12th day eggs were candled with the help of
an illuminator in a dark room to check viability,
movements of embryos and the area of the blood vessels
was defined. The air space was marked on the eggshell
with a pencil and a point was selected on CAM avoiding
injury of large blood vessels. The air space was
punctured with a pointed end of hand punch. The shell
was also punctured after clearing with a sterile cotton
swab on the marked spot over the CAM, using the hand
punch with slight rotatory motion, avoiding injury to the
shell membrane. The shell dust was blown away with
capillary pipette. A drop of sterile normal saline was
placed over the inoculation site. The tip of the blunt
instrument was inserted through the drop of saline. This
tore the cell membrane and the drop of fluid was sucked
inside as the CAM fell away creating a new air space.
Slight suction with a rubber teat over the hole at the blunt
end of the egg was applied to have a complete dropping
of the membrane confirmed by candling.
The inoculum was deposited into the CAM with
the help of tuberculin syringe and the inoculated egg
was rotated to facilitate the dispersion of the inoculum.
The hole in the air sac was sealed and the inoculated
eggs were incubated at 37°C for 48 h in a horizontal
position. After 48 hours the shell over the false air sac
was painted with tincture Iodine and the shell
membrane was broken with a blunt forceps for
maximum exposure of the CAM. The membrane was
cut out with a sterile pair of scissors and placed in a
Petri dish for further examination.
Control study: Japanese encephalitis virus (50 µL) in
the same concentration (10
−3
) in bovine albumin
phosphate saline pH 7.20 mixed with equal volume
(50 µL) of sterile pyrogen free distilled water was also
inoculated on chorioallantoic membrane. The pock
count of this control virus study was considered as
baseline data and any deviation from this baseline was
noted after application of different medicines in the test
series. An initial experiment was also done with
different concentrations of viruses to find out the
dilution which gave the maximum number of pocks on
CAM (optimum dilution). If during the study, there was
death of the inoculated eggs or membranes were not
found properly the data of that lot were excluded.
Apart from this virus control experiment, similar
control studies were also done with all the medicines
without the virus in equal dilutions with water. Control
studies were also performed with Bovine albumin in
phosphate saline pH 7.2 and potentised distilled water
and studied similarly as viral dilutions.
Observation of growths on CAM: Inoculated CAMs
were observed after 48 h of inoculation particularly to
see the formation of pocks and other associated changes
on CAM.
RESULTS
Optimum dilution of the JE virus was studied first
which can produce significant number of pocks on
CAM with different concentrations of the virus-Neat
(10% infected brain suspension), 10
−1
, 10
−2
, 10
−3
,10
−4
,
10
−5
, along with control (buffer solution without the
virus). The results showed that 10
−3
dilutions showed
maximum pock count and thus this optimum
concentration was used throughout the experiment.
Results of different experiments with Belladonna 3,
6, 30, 200 are given in Table 1, Fig. 1 and 2. Control
studies with different medicines without virus and
bovine albumin phosphate saline showed no significant
findings on CAM. The results showed significantly
decreased pock count when JE virus infection on CAM
was challenged with belladonna.
Table 1: Changes in pock count on CAM with ultradiluted belladonna
Experiment Pock count on CAM in number (Average ± SD ± SEM) t-value of the difference and its significance
Belladonna 3 (N-500)
Virus control 80.73±31.91±5.32 9.84, P value highly significant at 0.01 level
Virus + Medicine 25.32±11.18±1.86
Belladonna 6 (N-500)
Virus control 90.97±12.87±2.14 28.43, P value highly significant at 0.01 level
Virus + Medicine 20.74±7.43±1.24
Belladonna 30 (N-200)
Virus control 55.92±15.56±2.59 15.61, P value highly significant at 0.01 level
Virus + Medicine 13.31±5.20±0.87
Belladonna 200 (N-300)
Virus control 53.97±28.21±4.70 6.95, P value highly significant at 0.01 level
Virus + Medicine 18.17±12.66±2.11
CAM: Chorioallantoic Membrane; SD: Standard Deviation; SEM: Standard Error of Mean; N: Number of inoculated eggs; Eggs that were dead
or yielded deformed or absent CAM, were not considered for calculation of the results
Am. J. Infect. Dis., 6 (2): 24-28, 2010
27
(a)
(b)
Fig. 1: Pocks on CAM-(a) with JE virus, 10
−3
dilution; (b) inhibition of pocks with JE
virus, 10
−3
dilution and Belladonna 3
Fig. 2: Remarkable decreased pock count on JE virus
infected CAM with Belladonna
DISCUSSION
In this study JE virus infections on CAM and in
mice were challenged with different ultradiluted
Belladonna preparations-Belladonna 3, 6, 30, 200.
Encouraging results were obtained with all ultradiluted
Belladonna 3, 6, 30, 200 preparations used in this study
showing inhibition of viral growth on CAM. Therefore,
this initial study conclusively showed the beneficial
role of these medicines in JE infection. However, it is
not possible to explain this action with our current poor
knowledge about these ultradiluted preparations.
Various mechanisms of these ultradiluted preparations
were postulated by many workers but there is no
confirmation of all these hypothetic research
(Andersson et al., 1997).
If we look into the occurrence of calystegines and
related compounds in A. belladonna and related plants
then it is obvious that these are present in significantly
higher amounts in A. belladonna only. Thus amounts
(microgram per gram fresh mass) of calystegine A
3
in
young leaf, flower, mature leaf and in root of
A. belladonna are 280, 146, 62 and 14 respectively.
Similarly amounts (microgram per gram fresh mass) of
calystegine B
2
in young leaf, flower, mature leaf and in
root of A. belladonna are 380, 263, 70 and 13
respectively (Draeger et al., 1995).
Calystegines are selective glycosidase inhibitors in
contrast to common tropane alkaloids atropine and
scopolamine of A. belladonna, which are mainly
parasympatholytic.
Like most glycosidase inhibitors, calystegines
compete with the substrate for binding to the active site.
There are evidences that N-linked oligosaccharide
processing events in the endoplasmic reticulum are
important for the secretion of some enveloped viruses
(Mehta et al., 1998) characterized by sequential
trimming of the glucose residues on oligosaccharide
precursor. It was found that Dengue virus envelope
glycoprotein processing in cells was strongly affected
by this unique mechanism. Thus it is probable that
these ultradiluted preparations may also act in a similar
way by calystegines.
CONCLUSION
In conclusion, we may claim that ultradiluted
Belladonna 3,6,30,200 have potential role in
diminishing JE virus infection on CAM. The probable
mechanism of action of these ultradilued preparations
appeared to be due to glycosidase inhibitor action of
calystegines present in Belladonna.
ACKNOWLEDGEMENT
This study was done as a part of the project study
(No.17-88/2006-2007/CCRH/Tech/Coll/STM) financed
by CCRH, AYUSH, Govt. of India. We thank Prof.
D.K. Neogi, Ex Professor, Department of Virology,
School of Tropical Medicine, Kolkata; Dr. P.C. Mal,
Dr. N.R. Dey of CCRH, Govt. of India for fruitful
suggestions. We are also thankful to our technical
Am. J. Infect. Dis., 6 (2): 24-28, 2010
28
personnel Surja Kumar Halder, Suniti Bikash Ghosh,
Kajori Mukherjee and Kartick Chandra Roy, for their
dedicated study in the project. Lastly we sincerely
acknowledge the helpful suggestions given by Dr.
Rathin Chakraborty, Member Scientific Advisory
Committee, CCRH, Govt. of India.
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