Embryonated Chick Eggs
The embryonated eggs (one day old) have been procured form a poultry farm situated at Arambagh in the district of South 24 Parganas. Embryonated chick eggs from Black Australorp (Gallus gallus) has been used in our study. After proper disinfection of the shells using 70% ethanol, the eggs were kept in an incubator by maintaining physiological conditions like 37ᵒC temperature and 60% relative humidity (RH). The eggs were maintained in the laboratory using the candling procedure from the third day onwards and were rotated 3–4 times per day to observe certain changes such as vascularization, viability and also germ development until the 11th day as reported in other studies .
The prototype strain of JEV (SH1 strain; GenBank ID: MH753128.1) was used for our study. The virus was obtained in a lyophilized form from National Institute of Virology (NIV), Pune, India and was cultured in Biosafety Level 2 (BSL2) facility as prescribed by the Department of Biotechnology, Government of India. There is no requirement of explicit ethical approval for the same. The lyophilized form was resuspended in BAPS [phosphate-buffered saline (PBS, pH 7.4) (Thermo Fischer Scientific, Waltham, MA, USA) supplemented with 20% bovine serum albumin (BSA), Thermo Fischer Scientific, Waltham, MA, USA)] and the same was used as a stock which was inoculated into the Vero cell line ((ATCC® CCL-81™) to increase the viral copy number. Standardization of the final copy number of the viral inoculum was done by determining the LD50 dose as per standardized protocol described elsewhere . The viral inoculum was prepared by resuspending the isolated virus in BAPS and was subsequently filtered using a 0.22-µm syringe filter before using for treatments as illustrated in the “Experimental Design” section.
Hyoscyamine sulphate was purchased from Sigma-Aldrich (Catalogue No. H1600000), St. Louis, MO, USA. Different concentrations of the same were tested for their ability to reduce the viral load in Vero cells (ATCC® CCL-81™) at 24 h post-infection. On the basis of standardization experiment, 1.48 mM of hyoscyamine sulphate was selected as the optimal dose for further experimental analysis.
Molecular Docking Analysis
The crystal structure of the non-structural protein of JEV (PDB ID: 4K6M) and the ligand (Hyoscyamine: Pubchem ID: 154417 ) was taken from RCSB PDB (Research Collaboratory for Structural Bioinformatics Protein Data Bank) and PubChem servers respectively. The structure of the ligand and the protein were optimized using Chimera and Avogadro respectively. The PDB files of the protein and the ligand were converted into PDBQT [Protein Data Bank, Partial Charge (Q), and Atom type (T)] using Autodock MGL Tools . A three-dimensional grid box has been generated to define binding pocket and the active site was chosen on the basis of the internal ligand position found in the crystal structure of the protein. The ligand and the receptor were kept flexible and rigid respectively and molecular docking was performed using the Autodock command line tool on the basis of gradient optimization algorithm. The best fit docked complex was visualised using Pymol and the 2D interactions were analysed using Biovia Discovery Studio [27, 28].
Interaction of the ligand and the receptor was further analysed by molecular dynamics (MD) simulation using Schrodinger Maestro version 11.9.011 by applying thermodynamic conditions such as pressure, temperature, density and volume. The OPLS2005 force field was used to prepare the protein-ligand complex . Furthermore, the system was built using the pre-defined SPC water model applying orthorhombic periodic boundary conditions at a distance of 10 Å units. Importantly, prior to MD simulation, the charge of the complexes was electrically neutralized by balancing with 0.015M of K+/Cl− ions, and the energy minimization was done through heating and equilibrium processes. The system’s ultimate production phase lasted 100 ns at 300 K and 1 Atm pressure, employing Nose-Hoover method [30, 31] with NPT ensemble. Based on the interactions and dynamical properties of the complexes, the optimal conformations were selected. To assess the structural changes, the trajectories of each protein-ligand complex were analysed using specific parameters like RMSD (root mean square deviation), RMSF (root mean square fluctuation), intermolecular interactions.
Treatment of embryonated eggs with JEV
The experiments were designed using different volumes of JEV that were inoculated into embryonated eggs and LD50 dose was determined according to the protocol of Reed and Muench . The LD50 was found to be 30µl (4.6 × 107 copy number). The eggs were divided into four groups containing 4 eggs in each group for our experimental study. At first, the egg shell surfaces were sterilized with 70% ethanol and were then carefully punctured by applying moderate pressure. A depression was created on the lateral surface of the egg by applying slight suction that was applied over the hole at the air space . The details of treatment in different experimental groups has been clearly summarised in Table 1. Following inoculation, the holes over the egg surface were sealed with molten wax and were kept horizontally in an incubator at a temperature of 37ᵒC for 48 hours. The eggs were subjected to candling after every 12 hours to check their viability and the eggs that were dead within 24 hours post inoculation were sorted out and were discarded. After 48 hours, the eggs were sacrificed by chilling on ice and after proper disinfection, CAM, Brain and Amniotic fluid were harvested and collected.
Different experimental groups along with the details of the treatment
Group I (Control)
40 µl of BAPS was used
Group II (Infection group)
30 µl of viral inoculum along with 10 µl of BAPS was used.
Group III (Pre-treatment group)
10 µl of hyoscyamine sulphate was used followed by 30 µl of viral inoculum.
Group IV (Medicine control group)
10 µl of hyoscyamine sulphate along with 30 µl of BAPS.
|**30 µl of viral stock with 4.6 × 107/ml copy number was used.
|**1.48mM of hyoscyamine sulphate stock was used in our study.
Determination of viral load using by One-Step qRT-PCR
The viral load in CAM, Brain and Amniotic fluid among the experimental sets was determined using Japanese Encephalitis Virus Kit genesig® Standard Kit (Primedesign Ltd., UK) as per instructions of the manufacturer. 20 µl of the reaction mix was prepared which contains 14 µl of Oasig One Step 2X qRT-PCR Mastermix and 1 µl of primer/probe mix. 6 µl of Nuclease free water was used in case of negative control. The reaction was carried out using CFX96 Real time instrument (Bio-Rad, USA) where the cyclical conditions are as follows: Reverse transcription at 55ᵒC for 10 mins, Enzyme inactivation at 95ᵒC for 2 mins, Denaturation at 95ᵒC for 10 second and data collection at 60ᵒC for 60 second. The viral copy number was determined on the basis of linear regression analysis of the standard curve from the positive control RNA and the efficiency of the PCR reaction was determined.
Total RNA was isolated from Brain, CAM and Amniotic fluid from all the experimental sets using Trizol reagent (Thermo fisher, USA) as per standardized protocol as described elsewhere . In our process, one hundred milligrams per one hundred microliter of the sample was used for extraction of RNA using Trizol. DNase I (Thermo Scientific, USA) was used to avoid any genomic DNA contamination in the reaction. The RNA pellet was finally eluted in 40 µl DEPC water. The purity was checked using a Nanodrop Spectrophotometer (Eppendorf, Germany) and those with a A260/A280 ratio ranging between 2.0 to 2.2 were used for further experimental analysis . The RNA samples were stored at -80ᵒC until further use.
Gene Expression Study
The expression levels of different genes such as TLR-3, TLR-4, TLR-7, TLR-8, IFNα, IFNβ, IRF-3 and IRF-7 were studied to assess the pathogenicity of viral infection and the role of Hyoscyamine in reducing the viral load in different experimental groups. RNA was isolated as described above and cDNA was synthesized from 1µg of the total RNA using One Step cDNA Synthesis Kit (Takara, Japan) as per standardised protocol. The expression levels were checked through Semiquantative Real time PCR using SsoAdvanced™ Universal SYBR® Green Supermix (Bio-Rad, USA) in CFX96 Real-Time instrument (Bio-Rad, USA) under amplification conditions as described elsewhere. GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) gene was used as a housekeeping gene for data normalization. 2−ΔΔCt method  was used to estimate the relative fold change in gene expression levels. The primer sequences for the above mentioned genes for chick has been provided elsewhere [36–39].
The experiments were performed in triplicates (n = 3), and the data are represented as + SD. To check and analyse the differences between different experimental groups One-way and two-way ANOVA (analysis of variance) and t-test were used using GraphPad Prism (Version 5) . Pearson’s correlation (Pearson’s Coefficient) was used for correlation study and a p < 0.001 was considered as statistically significant in our study.