This research was done to assess the effects Belladona 200 produces in virus replication in Japanese encephalitis cases. The experiment showed that Belladona 200 reduced the severity of Japanese encephalitis by diminishing the viral loads in brain. The study further suggests that pre-treatment of Belladonna 200C may reduce the viral entry, its propagation in different organs particularly in the brain and thus may reduce the pathological sequel of the disease.
- Background: Japanese encephalitis (JE) is highly prevalent in many states of India. Belladonna 200C is widely used in the prevention and treatment of JE. The effect of Belladonna 200C in virus replication inside different tissues has not been studied.
- Objective: To study the effect of Belladonna 200C in virus replication inside different tissues utilising chick embryo model.
- Materials and Methods: Twelve-day-old fertilised eggs of Black Australorp were inoculated with JE via chorioallantoic membrane (CAM) route in different experimental sets: infection, Belladonna 200C treated and vehicle control, keeping matched blank sets. All experimental sets were incubated for 48 hours. After incubation, viable eggs were sacrificed humanly and different tissues were observed and collected for viral load determination by real-time-polymerase chain reaction (PCR).
- Results: The control group showed visible pocks over the CAM; brains were liquefied due to haemorrhagic liquefactive necrosis and white patches were found over the liver. However, the medicine-treated group was apparently normal; there were no visible changes in the brain and the liver was healthy like control. Real-time-PCR results showed high viral load in CAM and brain with absence of viral RNA in liver of the virus-infected group. Pre-treatment with Belladonna 200C significantly reduced the overall load (P < 0.05) in CAM and brain which correlated with the morbid pathological changes of the organs.
- Conclusion: Although Belladonna 200C did not completely inhibit JE viral replication in the brain, it reduced the severity of JE by diminishing the viral loads in this tissue.
In spite of the availability of vaccine against Japanese encephalitis (JE), it is not possible to immunise a large group of population in the endemic area like India due to socioeconomic and other constrains. The mortality rate among the diseased people is approximately 20%–30%, and 20%–50% of survivors develop permanent neurological disorders.1,2,3,4 Some studies on the antiviral role of Belladonna 200C against JE virus (JEV) have already been done, which are based on the Chick Chorioallantoic Membrane (CAM) and mouse model of infection.5 However, the viral replication potency in different organs, particularly in brain, under the influence of Belladonna 200C, has not been studied till now. Considering the importance of the neuropathological sequeal of the JE, in this study, we aimed to find out changes in the viral load in different tissues, particularly in the brain under the influence of Belladonna 200C.
Embryonated chicken egg is an excellent in vivo platform for a range of viral infection study including JE. The highly vascularised environment, with immature immunity, imparts the system as an ideal model for host pathogen interaction that mimics the actual pathogenesis during infection. After application of the virus, it replicates in the outermost layer of CAM, the ectoderm and then evades the mesoderm (the stromal layer).6 The motile blood cells, mainly the macrophages present in this layer, are responsible for the dissemination of the virus and infection to other body parts, especially the brain, by evading the Blood–Brain Barrier (BBB).7,8 The virus then multiplies in the brain and is responsible for the neuropathological outcome. This situation may mimic the pathology with other hosts including human for JE viral infection and confers the embryonated eggs as a potential system for in vivo studies.
The JEV infects both the Central Nervous System (CNS) and non-CNS organs.9 The present study is focussed on the antiviral role of Belladonna 200C against JE by highlighting the altered viral load in different organs of chick embryo mainly in CAM, brain and liver.
Materials and Methods
One-day-old embryonated chicken eggs of Black Australorp were procured from State poultry farm, Tollygunge, Kolkata, India. The eggs were incubated at 37°C with 60% relative humidity. From the 3rd day onwards, the eggs were turned 3–4 times/day till twelfth day and candled to determine the live/dead status of the embryonated chicken eggs.
JEV was procured from the National Institute of Virology (NIV), Pune, India (NIV no. P-20778, M/K no M-52134). The copy number of the virus inoculum was determined (8.23 × 107/ml) by real-time-Polymerase Chain Reaction (PCR) (Path JEV real-time PCR kit, Primer design) according to the manufacturer's instruction.
The study was approved by the Institutional Ethics Committee of Dr. Anjali Chatterjee Regional Research Institute for Homoeopathy, Kolkata.
Lethal dose50 dose was first determined for the inoculums standardisation.10 On the twelfth day, live eggs were divided into four groups, with each group containing 10 eggs. Group I (infection) was challenged with JEV (5.6×107/ml, LD50 dose) through CAM route.11 Briefly, the surface of the eggs was disinfected with 70% alcohol followed by puncturing on the air space and on the lateral side using egg punch with moderate pressure. Slight suction with rubber teat was applied over the hole at the air space (blunt end), which creates a depression over CAM on lateral surface of the egg. 50 μl of the JEV inoculum was applied over the CAM through the lateral hole using tuberculin syringe. Group II (Treatment) and Group III (Alcohol control) eggs were similarly punched and 50 μl of Belladonna 200C (alcoholic extract) and potentised alcohol 200C, respectively, were applied over CAM followed by JEV infection after 15 min. Among the Group IV (control) eggs, 50 μl of bovine albumin phosphate saline was applied. After CAM inoculation, the holes were sealed with wax and the eggs were incubated horizontally at 37°C for 48 h. The eggs were candled on every 12 h up to 48 h to determine the viability and after 48 h the live eggs were humanely killed by chilling on ice for 30 min. CAM and embryo were harvested from all dead or sacrificed eggs after disinfecting the shell, using sterile forceps and scissors. Brain and liver were then studied macroscopically and collected separately for further analysis.
RNA Isolation and Determination of Viral Load
The total RNA from CAM, brain and liver was extracted using trizol reagent following manufacturer's guidelines.12
The viral load in the samples was determined using real-time PCR kit (Genesig, Primer Design, Watchmoor point, Camberley, UK) on CFX96 Real-Time System (Bio-Rad, USA) following manufacturer's instruction.
Each experiment was replicated at least thrice and the experimenters were blinded towards the identity of all groups. The data are represented as mean ± standard deviation. Graph Pad Prism (Version 5, California, USA) was applied for analysis with one-way analysis of variance (ANOVA) and t-test.
JEV-infected CAM showed visible pock lesions [Figure 1] d, with highly congested blood vessels. Similar observation was also found in the alcohol (200C) control group [Figure 1] c. Severe haemorrhages were found all over the body of embryo and in the brain of these two groups [Figure 2]. Moreover, in these two groups, colliquative necrosis with severe blood clots was found in the brain. However, in the Belladonna 200C-treated group, although no such haemorrhages and pock lesions were present, congestions of the blood vessels were observed in few areas of CAM [Figure 1]b. Overall, the CAM was clear and healthy as control [Figure 1] a with intact brains in this medicine-treated group.
Figure 1: Representative photographs of chorioallantoic membrane tissues from
(a) Control and Japanese encephalitis virus-infected groups
(b) pre-treated with Belladonna 200C,
(c) Pre-treated with potentised alcohol (200C),
(d) Only virus infected. Control chorioallantoic membrane shows clear blood vessels.
Belladonna-treated chorioallantoic membrane is apparently healthy. Chorioallantoic membrane from alcohol-treated and virus-infected groups show huge pock lesions (solid arrow) with congested blood vessels (broken arrow)
Figure 2: Morbid pathology of chick embryo infected with virus showing haemorrhages in brain (black solid arrow, a,c), yellowish and enlarged liver (broken arrow, b,d), congested blood vessels (blue arrow, c) and haemorrhages throughout the body
The morbid pathologic observation also correlates well with the viral load as determined by viral RNA copy number among different test groups. There were significant changes of JE viral copy number both in the CAM tissue [P = 0.0033, one-way ANOVA, [Figure 3] a and in the brain tissue [P = 0.0339, one-way ANOVA, [Figure 3]b. There were significantly high viral load in the CAM tissues of infection control (P = 0.0017, one tailed t-test) and alcohol-treated group (P = 0.0052, one-tailed t-test) compared to the Belladonna 200C-treated group. Similar pattern was found in the brain tissue of infection control (P = 0.0086, one-tailed t-test)- and alcohol-treated group (P = 0.0165, one-tailed t-test) compared to the Belladonna 200C-treated group.
Figure 3: Changes in viral load in
(a) Chorioallantoic membrane and
(b) brains of chicken embryo infected with virus and treated with potentised alcohol (200C) and Belladonna (200C).
There were significant changes among these three different infection groups both in chorioallantoic membrane and brain as analysed by one-way analysis of variance
Homoeopathy is the cheapest and preferred way of controlling the JE infection in a country like India where poor adherence, negligence and other socioeconomic constraints prevail. Vaccination does not give 100% protection to JE. Belladonna derived from the plant Atropa Belladonna contains many active components which have effects on central and peripheral nervous systems and have been found effective against JE.
Apart from tannins, flavonoids and coumarines, tropane alkaloids are the main constituents of A. Belladonna. Being a neurotrophic virus, JE evades the CNS via transmigrating phagocytic cells and crosses the BBB.13Belladonna alkaloids can not only cross the BBB, but it may pass through placenta and may be found in the milk.14
In contrast to the previous investigations on Belladonna 200C with adult mouse model,5 chick embryo model is more advantageous due to the immune immaturity. The developed adaptive and innate immunity prevent the adult mice being infected with JEV.15 Moreover, the cost-effectiveness, handling procedure and easier ethical procedures with this in vivo embryonated egg model is one of the preferred models for host pathogen interaction tools.
The virus was inoculated through CAM route. However, the presence of the virus in huge number in the brain indicates the dissemination through the blood via BBB. There is only one study which has demonstrated the replication of JEV in different organs of experimentally infected pigs through intravenous route and highlighted the tissue tropism both in the CNS and non-CNS organs such as liver.9
However, in the present investigation, viral loads were almost similar both in the brain and CAM. Although no JEV was observed in the liver, white patches and yellowish colouration which were observed may be due to reflections of general sickness [Figure 2]b. In this study, with chick embryo, the JEV showed tissue tropism mainly on the brain. The presence of virus in the brain in the medicine-treated group indicates that Belladonna 200C cannot totally restrict the viral entry and its replication in the brain.
However, pre-treatment of the medicine significantly reduced the replication of the virus both in CAM and brain. Very few congested blood vessels in the CAM and absence of haemorrhages with colliquative necrosis in the brain were found in the medicine-treated group, which indicates the role of Belladonna 200C in reducing the complications and severity of the disease.
Belladonna 200C has the antiviral property against JE. Pre-treatment of Belladonna 200C may reduce the viral entry, its propagation in different organs particularly in the brain and thus may reduce the pathological sequel of the disease.
Financial support and sponsorship
The work was supported by CCRH, Ministry of AYUSH, Government of India.
Conflicts of interest
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