In-silico identification of stigmasta-4,22-dien-3-one as a potential White spot syndrome virus (WSSV) inhibitor via ligand-receptor interaction analysis

doi:10.21203/rs.3.rs-2854608/v1

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In-silico identification of stigmasta-4,22-dien-3-one as a potential White spot syndrome virus (WSSV) inhibitor via ligand-receptor interaction analysis

https://doi.org/10.21203/rs.3.rs-2854608/v1

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White spot syndrome, a viral disease caused by the white spot syndrome virus in penaeid shrimp, is causing significant economic losses in the shrimp farming industry. Envelope structural proteins are considered to be the first molecules to interact with the host cell upon viral attachment. Thus, these envelope proteins are identified as promising molecular targets for drug development. In the present study, the anti-viral activity of Sargassum wightii was determined by both in-vitro and in-silico analysis. Crabs were injected with petroleum ether extract of S. wightii along with WSSV for the experimental challenge and observed 30 days post-infection. The anti-viral activity of S. wightii was confirmed by bio-assay, histopathology and in-silico analysis. GC–MS analysis of S. wightii identified 15 compounds, respectively. An in-silico molecular docking of the envelope protein VP28, VP26 and VP24 with ligand stigmasta-4,22-dien-3-one exhibited high binding energy. Molecular simulation and dynamics were done to validate the stability protein-ligand binding. Therefore, the results of the present study confirmed that S. wightii can be used for treatment of WSSV.

WSSV

Sargassum wightii

Anti-viral activity

PCR

Shrimp culture is affected by an array of viral, bacterial, and fungal pathogens as well as protozoan and metazoan parasites (Lightner et al. 2012). The white spot disease (WSD) is caused by white spot syndrome virus (WSSV), which is a serious disease-causing pathogen and causes significant economic losses from time to time (Huang et al. 2020). WSSV belongs to the family Nimaviride consisting of the genus Whispovirus. WSSV is an ovaloid DNA virus with a flagellum-like tail and a helical nucleocapsid (Huang et al. 2015; Verbruggen et al. 2016). It infects a wide range of crustaceans, leading to 100% mortality within 3–10 days of infection. Infected animals exhibit lethargy, decreased feeding, anorexia, a loose cuticle, and a change in body color. White spots appear in the exoskeleton as a result of calcium deposits, particularly in the carapaces and last abdominal segments (Sánchez-Paz 2010). The envelope proteins of WSSV are thought to play critical roles in virus infection, such as recognition and attachment to receptors on the host cell surface.

The initial line of infection in shrimp is mediated by a multiprotein complex constituted of major structural proteins VP28, VP26, VP24 and VP19 (Hasan et al. 2022). In WSSV, VP28 is the major envelope protein that has been shown to exert systemic infection in shrimp and also interacts with several host proteins (Sivakumar et al. 2016). Conversely the tegument protein VP26, has an actin binding domain that aids in the adhesion of the virion to the shrimp cells (Tsai et al. 2004; Liao et al. 2021). VP24 interacts with other proteins and is essential for virus assembly and infection (Sun and Wu 2016).

Crustaceans lack adaptive immunity and rely on their innate immune system to fight against microbe’s invasion; as a result, developing vaccine against pathogens to prevent diseases in crustaceans is not possible. However, on a laboratory scale, a few of them found that administration of plant extracts, probiotics, DNA vaccines, or antibodies raised against WSSV envelope proteins delayed WSSV-related mortality (Syed Musthaq and Kwang 2015). The use of natural products to suppress viral infection in aquaculture is a potential means that generated interest in researchers in the recent past.

The aim of the study was to determine the antiviral properties of Sargassum wightii against WSSV through in-vivo and in-silico studies

2.1 Collection and maintenance of experimental animals

Freshwater crabs P. hydrodromous (20–25 g body weight) were collected from the rice field located at Padavedu (12.631007° 79.052885°) Vellore, India. Their phenotypical characteristics served as confirmation for species identification. The animals were transported to the laboratory and kept at room temperature (27℃) in a 100L aquarium. Boiled egg whites were given to the crabs twice a day as feed (Karthikeyan et al. 2022). Prior to the experimental challenge tissue samples such as head-soft tissue, gills, and muscles were evaluated for WSSV using a polymerase chain reaction to make sure the animals are free of WSSV infection (Sundaram et al. 2016).

2.2 Seaweed collection and extract preparation

Sargassum wightii was collected from Thangchimadam (9.293434⁰79.23951⁰) and was identified by Central Marine Fisheries Research Institute (CMFRI) in Mandapam Rameswaram, Tamil Nadu, India. The impurities and salt present on the sample surface were washed and cleaned using distilled water. The samples were then shade-dried to avoid direct exposure to sunlight, then powdered using a dry grinder and sieved to ensure uniform particle size (Raja et al. 2023). Further stored in an air-tight container for utilising in extraction process. Powdered S. wightii was extracted using petroleum ether using a modified protocol (Fatima et al. 2022). 100 grams of powder were extracted with 1litre of petroleum ether and kept in a shaker at room temperature for 24 h. The extract was then filtered through Whatman No.1 filter paper. The solvent was removed from the filtered extract using a rotary evaporator and the extract was lyophilized and stored at 4℃ for further studies.

2.3 GC-MS analysis

Petroleum ether extract of S. wightii was analyzed for the presence of compounds using Gas Chromatography (Perkin Elmer GC Clarus 680 system, United States) in a column equipped with Elite-5MS (30.0 m, 0.25 mm IID, 250m df) and 70eV of ionization energy was used detection. Helium (99.99%) was used as the carrier gas, with a constant flow rate of 1mL/min and an injection volume of 1IL (split ratio 10:1) and the injector temperature was 260°C. The oven temperature was initially set to 60°C for 2 min and then it was increased by 10°C/min to 300°C holding for 6 minutes. At 70eV, mass spectra were collected with a scanning interval of 0.5s and fragments ranging from 50 to 600 Da. The GC’s total run time was 32 min. The relative percentage quantity of each component was calculated by comparing its average peak area to the total area. To handle mass spectra and chromatograms, TurboMass version 5.4.2 was used. The retention time of the matching compounds was contrasted to those of legitimate compounds, as well as the spectrum result obtained from the National Institute of Standards and Technology (Eswaraiah et al. 2020).

2.4 Preparation of viral inoculum:

As mentioned, in our previous reports (Natarajan et al. 2017) the haemolymph was collected from WSSV-infected shrimp using a 26G syringe by heart puncture. The collected hemolymph was centrifuged for 20 minutes at 4℃ at 3000rpm. The supernatant was collected and it was centrifuged for 30 minutes at 4℃ and then it was filtered using 0.45µm and stored at -20℃ for further studies. The hemolymph was subjected to PCR to validate the presence of WSSV. A standard 20 µl PCR reaction containing 10 µl of 2× master mix, 5pmol WSSV-VP28 primer (Forward-ATGGATCTTTCTTTCAC and Reverse-TTACTCGGTCTCAGTGC), 3 µl PCR grade water and 1 µl template DNA was used to amplify the structural gene VP28 (Sundaram et al. 2016). The viral titre quantified as understood by real-time PCR is 1.6 × 10⁶ µl^− 1 was used as stock in this study.

2.5 Bio-assay analysis

The anti-viral activity of S. wightii petroleum ether extract was evaluated against WSSV in freshwater crab Paratelphusa hydrodomous. This animal model has been selected as it’s susceptible for WSSV infection. The dose-dependent concentrations of 0.5, 1 and 1.5 mg were examined to confirm the therapeutic dose against WSSV and it was observed that 1mg had a positive impact with no mortality in crabs, therefore a dosage of 1mg was chosen for the subsequent studies. The experimental animal was divided into three groups with ten crabs per group and the experiments performed in triplicates. Prior to injection the viral inoculum was incubated with S. wightii petroleum ether extract for 3 hours and injected. All the crabs received an intramuscular injection at the second abdominal segment. Concisely, healthy crabs were administered with the following in Group 1 the crabs were injected with 100µL NTE buffer which served as a negative control, and in Group 2 the crabs were injected with 95µl NTE buffer and 5µl viral inoculum which served as a positive control. In group 3 the animals were injected with 85µl NTE buffer, 5µl viral inoculum, and 10 µl petroleum ether extract (1 mg). The crabs were fed twice a day and were regularly observed for the sign of disease and mortality. The study was carried out up to 30 days post-infection and mortality was recorded to generate a cumulative mortality graph (Sundaram et al. 2016; Karthikeyan et al. 2022).

2.6 Preparation of small molecule

The SMILES (simplified molecular input line entry server system) of the compounds was obtained from PubChem and the PDB format of the compounds was generated using the online SMILES Translator (https://cactus.nci.nih.gov/translate/).

2.7 Preparation of macromolecule

The 3D structure of WSSV envelope protein VP28 (PDB ID: 2ED6) and VP26 (PDB ID: 2EDM) and VP24 (5HLJ) was retrieved from Protein Data Bank (Sudharsana et al. 2016; Dinesh et al. 2017; Narasimman and Ramachandran 2023). Using Auto dock 4.2 software the protein structure was prepared for docking by eliminating non-amino acid moieties.

2.8 Molecular docking

The target protein used in this study was VP28, VP26 and VP24 well-known envelope protein of WSSV. The docking of compounds and envelop proteins (VP28, VP26 and VP24) was carried out using Autodockv4.2 (Dinesh et al. 2017; Daniel and Devi 2019). Blind docking was used to allow the ligand to select their preferred binding sites. The partial charges for protein and ligand were calculated using Gasteiger-Marsili and Kollman charges. The grid box was set over the entire protein since blind docking was performed and the grid space was set to 0.375A. Using the Lamarckian genetic algorithm, the conformers of the ligand bound to the protein were generated. The number of docking runs was set to 10 leaving the other settings to default values. Results obtained from Autodock-4.2 were analyzed using Discovery Studio visualizer v20.1.0.19295, Dassault Systems Bio via corp.

2.9 Molecular dynamics and simulation

The stability of the interaction was investigated using molecular dynamic simulation (MD) analysis on the protein-ligand conformation of VP28, VP26 and VP24 for 50ns, using the Desmond software Release 2019-2 for academic licensing (Schrödinger, LLC, New York, NY, USA) (Sudharsana et al. 2016; Ren and Liu 2021). To mimic the physiological ionic concentration, a TIP3P water model and 0.15 M NaCl were used in the simulation. Energy minimization was carried out for 100ps. Simulations were run for 50ns at 300 K and 1.01 bars in NPT conditions. At 1.2 ps intervals, the energy was recorded. To balance the net charge system in the MD-simulated model, the charge of the protein-ligand complex was neutralized by the addition of Na + or Cl- ions. To maintain the temperature of all MD systems at 300 K and 1.01 bar pressure the Nosé-Hoover chain and Martyna-Tobias-Klein algorithms were used. With periodic boundary conditions and the OPLS 2005 force filed parameter all well-minimized and equilibrated systems were subjected to a 50-ns MD run in the NPT assembly (Das et al., 2022).

2.10 Haematoxylin and eosin stain

Experimental crabs injected with petroleum ether extract and WSSV along with negative and positive control were dissected and organs such as gills and head soft tissue were rinsed in PBS buffer and fixative with 10% formalin, which was then dehydrated with various concentrations of alcohol, washed and embedded with paraffin wax. The embedded tissues were sectioned using microtome precisely at 5 µm thickness. The sectioned tissue was fixed with a glass slide containing bovine serum albumin. Then the tissues were rehydrated and stained with Haematoxylin and Eosin (H&E) and histological changes was then evaluated under a compound microscope (Carl Zeiss, India)(Lightner and Redman 1998).

3.1 Bioassay

In this experiment freshwater crabs (P. hydrodromous) were used as a model organism to determine the anti-viral activity of S. wightii petroleum ether extract. The study revealed that S. wightii petroleum ether extract has potent antiviral activity against WSSV. Crabs in group 1 injected with NTE alone survived without showing any signs of WSSV infection and showed zero mortality until the end of the experiment. Crabs in group 3 injected with S. wightii petroleum ether extract and viral inoculum resulted in 100% survival until the end of the experiment (Fig. 1). Crabs in group 2 injected with viral inoculum showed 100% mortality within 7 days of infection. In WSSV-infected crabs, a decrease in feed consumption and slow movement was observed. The crabs in the treated groups survived for more than 30 days with no WSSV symptoms as observed until the study was concluded.

3.2 GC-MS analysis

The presence of bioactive compounds in the petroleum ether extract of S. wightii was revealed by GC-MS analysis (Fig. 2). Table 1 shows the molecular weight (MW), molecular formula (MF), and retention time (RT) for the bioactive compound found in the extract based on the search. There were fifteen compounds identified in the extract. The prevailing compounds were Dihomocubane, Oxalic acid, monoamide, n-propyl, tridecyl ester, Oxalic acid, allyl octyl ester, Oxazolidine A, 4-Methyl-3-heptanone, 1-Undecyne, ethylsulfolane, ethylsulfolane, 2,2,4,6,6-pentamethylheptane-3,5-dione, Hexadecane, Sulfurous acid, octyl 2-propyl ester, Heptacosane, 1-Iodo-2-methylnonane, 2-Ethylbutanol and Stigmasta-4,22-dien-3-one.

Table 1

Compounds detected in petroleum ether extract of *Sargassum wightii* through GC-MS analysis
S.no	Retention Time	Compound	Molecular Weight	Molecular Formula	PubChem ID	Area %	mg/g
1	6.575	Dihomocubane	132.20	C₁₀H₁₂	548716	2.400	24
2	13.693	Oxalic acid, allyl octyl ester	242.31	C₁₃H₂₂O₄	6420247	2.833	28.33
3	14.578	Oxazolidine A	101.15	C₅H₁₁NO	62010	2.382	23.82
4	15.594	4-Methyl-3-heptanone	128.21	C₈H₁₆O	22512	5.487	54.87
5	16.069	Oxalic acid, monoamide, n-propyl, tridecyl ester	313.5	C₁₈H₃₅NO₃	6420322	3.339	33.39
6	17.064	1-Undecyne	152.28	C₁₁H₂₀	75249	5.287	52.87
7	17.504	ethylsulfolane	148.23	C₆H₁₂O₂S	544727	2.223	22.23
8	17.784	4-Bromomethylheptane	193.12	C₈H₁₇Br	545964	4.510	45.10
9	17.829	2,2,4,6,6-pentamethylheptane-3,5-dione	198.30	C₁₂H₂₂O₂	231586	1.842	18.42
10	19.465	Hexadecane	226.44	C₁₆H₃₄	11006	1.403	14.03
11	19.765	Sulfurous acid, octyl 2-propyl ester	236.37	C₁₁H₂₄O₃S	6420352	5.346	53.46
12	20.140	Heptacosane	380.7	C₂₇H₅₆	11636	2.744	27.44
13	21.571	1-Iodo-2-methylnonane	268.18	C₁₀H₂₁I	537317	1.871	18.71
14	23.222	2-Ethylbutanol	102.17	C₆H₁₄O	7358	4.842	48.42
15	29.354	Stigmasta-4,22-dien-3-one	410.7	C₂₉H₄₆O	5364563	45.813	458.13

3.3 Molecular docking studies

The fifteen compounds were identified from the S. wightii petroleum ether extract and were docked with the envelope proteins VP28, VP26 and VP24. Table 2 summarizes the binding energy of the different compounds and also the amino acid residues involved in the interaction with the envelope proteins. The compounds bind to the envelope proteins to a varying degree. The docking of different compounds with envelope protein VP28 was investigated and the compound stigmasta-4,22-dien-3-one exhibited the lowest binding energy of -8.6 kcal/mol. The binding pattern analysis showed that the compound formed one hydrogen bond with GLY H:136 during interaction (Fig. 3). The ligand also forms two hydrophobic bonds HIS I:195 (Pi-sigma) and TYR G:183 (Pi-alkyl). The docking of different compounds with envelope protein VP26 was investigated and the compound stigmasta-4,22-dien-3-one exhibited the highest binding energy of -6.6 kcal/mol. The binding pattern analysis showed that the compound formed one hydrogen bond ASN A:193 (Fig. 4). The docking of different compounds with envelope protein VP24 was investigated and the compound stigmasta-4,22-dien-3-one exhibited the highest binding energy of -6.0 kcal/mol. The binding pattern analysis showed that the compound formed one hydrophobic bond TYR A:159 (Fig. 5). The result of the molecular docking of 15 compounds against 3 envelope proteins of WSSV revealed that the phytochemical stigmasta-4,22-dien-3-one had the highest efficacy and binding interaction against all three proteins. The result of this study also exhibited that stigmasta-4,22-dien-3-one present in S. wightii petroleum ether extract can be used as a potential anti-WSSV compound against the envelope protein of WSSV.

Table 2

*In-silico* docking investigation of *Sargassum wightii* compounds interactions with WSSV target proteins VP28, VP26 and VP24.
S.no	Compound	VP28 (Binding Affinity)	VP26 (Binding Affinity)	VP24 (Binding Affinity)
1	Dihomocubane	-7.7	-5.8	-5.1
2	Oxalic acid, allyl octyl ester	-5.1	-4.2	-3.5
3	Oxazolidine A	-4.9	-3.7	-3.1
4	4-Methyl-3-heptanone	-4.5	-3.3	-3.6
5	Oxalic acid, monoamide, n-propyl, tridecyl ester	-3.7	-4.2	-3.0
6	1-Undecyne	-3.6	-3.7	-3.4
7	ethylsulfolane	-4.5	-3.8	-3.8
8	4-Bromomethylheptane	-3.6	-3.4	-3.2
9	2,2,4,6,6-pentamethylheptane-3,5-dione	-5.5	-4.4	-4.2
10	Hexadecane	-4.4	-3.5	-2.7
11	Sulfurous acid, octyl 2-propyl ester	-3.5	-3.8	-3.1
12	Heptacosane	-3.6	-2.7	-3.1
13	1-Iodo-2-methylnonane	-4.0	-3.9	-2.5
14	2-Ethylbutanol	-3.9	-3.5	-4.1
15	Stigmasta-4,22-dien-3-one	-8.6	-6.6	-6.0

Table 3

Amino acid residues of proteins (VP28, VP26 and VP24) involved in interaction with Stigmasta-4,22-dien-3-one
Protein	Amino acid residues
VP28	PHE I:158, GLY I:136, THR I:135, TYR G:193, VAL G:194, MET G:137, GLN G:138, HIS G:195, GLY G:136, ASN H:51, HIS H:195, GLY H:136, THR H:135, PHE H:158, TYR I:193, GLN I:138, ASN I:51, GLN H:138, HIS I:195 and ASN G:51
VP26	ASN A:45, ASP A:47, PRO A:72, TYR A:46, VAL A:126, ASN A:193, SER A:191 and ILE A:195
VP24	THR A:178, VAL A:157, TYR A:159, PHE A:160, LEU A:158, ASN A:110, GLU A:118, GLY A:176, VAL A:111 and LEU A:109

3.4 Molecular dynamic simulation analysis

The stability of binding ligands on free protein and the protein-ligand complex was analyzed using molecular dynamics by comparing the 50 ns MD trajectories.

3.4.1 Root Mean Square Deviation (RMSD)

The root-mean-square-deviation (RMSD) values provide deviation at Å level with time duration nanosecond (ns) level for protein and ligand, indicating the complex (compound) stability as a whole. The protein RMSD was relatively stable throughout the simulation, with insubstantial fluctuations ranging from 0.5 Å- 1 Å, which indicates that VP28 has not undergone significant conformational changes. The RMSD plot (Fig. 6a) indicates that the stigmasta-4,22-dien-3-one -VP28 (2ED6) complex stabilized at 10 ns with respect to the reference time as 0 ns after commencing simulation. The fluctuations were observed at 45 ns and was stabilized. The variations were in the order of 1 Å indicating the complex has not undergone conformational changes. The overall RMSD plot of stigmasta-4,22-dien-3-one -VP28 complex suggest that ligand is bound stably to VP28 and has not diffused away from the protein. The RMSD plot (Fig. 6b) indicates that stigmasta-4,22-dien-3-one -VP26 (2EDM) complex stabilized at 20 ns with respect to the reference time as 0 ns after commencing simulation. The fluctuation in RMSD values for protein remain within 3.0 Å during the simulation period. The ligand fit to proteins oscillate within 1.5 Å up to 40 ns. This indicates that the complex is very much stable. The RMSD plot (Fig. 6c) indicates that stigmasta-4,22-dien-3-one -VP24 (5HLJ) complex and the protein converged at 15 ns and the RMSD values remain within 3 Å and diverged at 35 ns. The complex stabilized at 38 ns and variations were in the order of 2.5 Å indicating the complex has not undergone conformational changes. The overall RMSD plot of stigmasta-4,22-dien-3-one -VP24 complex suggest that ligand is bound stably to VP24 and has not diffused away from the protein.

3.4.2 Root Mean Square Fluctuation (RMSF)

The root mean square fluctuation (RMSF) represents the fluctuation of the protein and ligand at the atomic and residual levels. In this study, we have only analysed the protein fluctuation at the residual level in terms of residue number and interaction with stigmasta-4,22-dien-3-one. The residues of VP28 have performed steadily that only a few residues have fluctuated beyond 2 Å and those residues are THR32, VAL33, THR34, LYS35 GLY200 and THR201. The ligand molecule interacted with the protein 15 times involving VAL140, TYR193, ASN51, THR135, VAL140, PHE158, TYR193, HIS195, ASN51, LEU52, VAL140, PHE158, TYR193, VAL194 and HIS195 (Fig. 7a). The VP26 has performed stable during the simulation period and the ligand atoms interacted with the protein 18 times involving VAL24, ALA44, TYR46, PRO72, LEU73, THR124, GL125, VAL126, ASP127, ASN193, VAL194, ILE195, ASP196, ILE197, LYS198, ASP199, GLU200 and ILE201 (Fig. 7b). The VP24 residues fluctuated slightly more than 2 Å and those residues are PHE203, LYS204, VA205, GLY206, GLU207 and LYS208. The ligand molecule interacted with the protein 10 times THR108, LEU109, ASN110, VAL111, ASN112, GLU118, ASP136, VAL138, PHE156, VAL157, LEU158, TYR159, PHE160, LYS161 and THR178 (Fig. 7c). In the overall protein-RMSF analysis some residues showed slight fluctuations and the interactions were not observed to be specific to the range of fluctuated amino acids and the width of the line (green colour) indicates the duration of the interaction’s duration. The stigmasta-4,22-dien-3-one fit appropriately in the pocket of the proteins and destabilizes its core functionalities to inhibit the viral growth.

3.5 Histopathology

The crabs in the negative control group (NTE buffer alone) had no histological changes (Fig. 8a & b). H&E-stained sections of gills and head soft tissue from WSSV infected animals revealed histopathological changes such as condensed nuclei and cells with nuclear hypertrophy with intracellular inclusions (Fig. 8c & d). In contrast, no histological changes were observed in the treated group (Fig. 8e & f).

White spot syndrome virus has been recognized as a global threat to shrimp culture industry causing immense shrimp losses and resulting in huge economic losses. There is no effective treatment to control the spread of WSSV infection in shrimp farms due to a variety of known and unknown vectors (Hameed et al. 2005; Sivakumar et al. 2016).Seaweeds extracts have antiviral characteristics and they have also shown promise in protecting shrimp against WSSV (Sundaram et al. 2016; Schleder et al. 2020; Klongklaew et al. 2021). S. wightii is easily available and a well-known seaweed with wide range of pharmacological activities such as antioxidant, anti-inflammatory, antidiabetic, antihypertensive, etc. (Maneesh et al. 2016). Fucoidans are polysaccharides isolated from S. wightii has proved to have inhibit WSSV activity (Sivagnanavelmurugan et al. 2012). Since the crude extract of S. wightii demonstrated antiviral activity against WSSV, we wanted to know about the active phytochemicals responsible for the antiviral activity.

In the present study S. wightii petroleum ether extract was studied for its antiviral activity against WSSV. This experiment concluded that S. wightii petroleum ether had strong activity against WSSV, with 100% survival in the treatment group. It was concluded in this experiment that S. wightii petroleum ether showed strong activity against WSSV with 100% survival in treatment group. It was reported Artemia nauplii enriched with hot water extract of S. wightii fed to P. monodon showed resistance against WSSV and the survival rate was 65% (Immanuel et al. 2010). Similarly, fucoidan isolated from S. wightii can improve the resistance against WSSV in P. monodon. But the mean survival percentage of WSSV challenged fucoidan group was 51–72% (Immanuel et al. 2012). Sulfated galactans isolated from Gracilaria fisheri inhibited WSSV infection in P. monodon and the mortality rate was decreased compared to control (Wongprasert et al. 2014).

Incubating the WSSV inoculum with S. wightii inhibited virus replication by interacting with the envelope proteins. A similar study was done with Acetone extract of Turbinaria ornata that showed antiviral activity against WSSV in freshwater crabs (Raja et al. 2023). It was reported that Mytillin pre-incubate with WSSV for 3 hours at room temperature demonstrated anti-WSSV activity by reducing shrimp morality (Dupuy et al. 2004). In this study incubating WSSV inoculum with extract showed anti-viral activity suggesting the presence of molecules in the extract that could inactivate the virus. In addition, the extract prevents WSSV multiplication in host organisms. The immune mechanism is presumed to be activated by S. wightii extract attempts to combat WSSV infection in crabs.

GC-MS finding indicate that S. wightii petroleum ether extract contains bioactive molecules which are presumed to have anti-WSSV activity. Stigmasta-4,22-dien-3-one was recorded highest in the petroleum ether extract of S. wightii and this molecule can be assumed responsible for WSSV suppression.

WSSV structural proteins (VP28, VP26 and VP24) are essential for viral attachment and are regarded as the first molecule to interact with the host cell during replication (Xie and Yang 2006; Lightner et al. 2012). The inhibition of these proteins prevents WSSV entry into the host. These viral proteins have evolved into a promising molecular target for drug development The docking analysis of phytocompound stigmasta-4,22-dien-3-one exhibited highest binding energy to the envelope protein VP28, VP26 and VP24. The docking score of the ligand with all the proteins individually varies from − 8.6 to -6.0 kcal/mol. It is also hypothesized that interaction between stigmasta-4,22-dien-3-one and structural proteins (VP28, VP26 and VP24) can inhibit the virus assembly and infection. Overall, the RMSD and RMSF were less than 2 Å and the biological systems provide up to this level of flexibility since the complex systems are virtually heated with multiple parameters causing minor deviation. Similarly, the 2H-1-benzopyran-6-ol, 3,4-dihydro-2,5,7,8-tetramethyl- 2-(4,8, 12-trimethyltridecyl)-acetate identified from Phyllanthus amarus extract showed exhibited highest binding energy to VP28 and VP26 (Dinesh et al. 2017). Hence Stigmasta-4,22-dien-3-one can be considered for further studies.

The current study concluded that S. wightii petroleum ether extract exhibited significant antiviral activity against WSSV. The presence of antiviral compound in S. wightii was also confirmed from this study. Both in-silico and in-vivo approaches reveal potential activity of S. wightii to control WSSV infection shrimp farms. The computational analysis reveals that the phytocompound stigmasta-4,22-dien-3-one from S. wightii could be the potential molecule for the treatment of WSSV in shrimp culture. These findings could contribute to the development of antiviral drugs against WSSV thus preventing both rapid mortality and massive economic losses to the aquaculture industry.

Acknowledgment

The authors are grateful to the management of the Vellore Institute of Technology for providing the necessary support to write this article. The authors acknowledge Dr. Sudhakaran R of Vellore Institute of Technology – aquaculture laboratory for his guidance in writing this review article.

Author Contribution

Raja Bharath- This is his PhD project and executed the complete experiment. Vidya Radhakrishnan- Took part in designing, monitoring of project execution and manuscript writing.

Conflict of interest

The authors declare that there were no competing interests either financial or non-financial.

Funding

The authors confirm that this research article was not written under the financial sponsorship of any funding agency

Ethics approval

Not applicable

Code availability

Not applicable

Availability of data and material

The authors confirm that the data supporting the findings of this study are available within the article

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In-silico identification of stigmasta-4,22-dien-3-one as a potential White spot syndrome virus (WSSV) inhibitor via ligand-receptor interaction analysis

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Abstract

Figures

1. Introduction

2. Materials and methods

2.1 Collection and maintenance of experimental animals

2.2 Seaweed collection and extract preparation

2.3 GC-MS analysis

2.4 Preparation of viral inoculum:

2.5 Bio-assay analysis

2.6 Preparation of small molecule

2.7 Preparation of macromolecule

2.8 Molecular docking

2.9 Molecular dynamics and simulation

2.10 Haematoxylin and eosin stain

3. Results

3.1 Bioassay

3.2 GC-MS analysis

3.3 Molecular docking studies

3.4 Molecular dynamic simulation analysis

3.4.1 Root Mean Square Deviation (RMSD)

3.4.2 Root Mean Square Fluctuation (RMSF)

3.5 Histopathology

4. Discussion

5. Conclusion

Declarations

References

Additional Declarations

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