Antiviral activity of Turbinaria ornata against white spot syndrome virus in freshwater crab (Paratelphusa hydrodromous)

White spot disease in penaeid shrimp is caused by white spot syndrome virus and causing serious threat to shrimp farming industry. The present study was carried out to determine the antiviral activity of Turbinaria ornata against WSSV in freshwater carbs Paratelphusa hydrodromous. The crabs were injected with acetone extract of T. ornata along with WSSV and the experimental groups were observed for more than 60 days post-infection. The efficacy of the T. ornata acetone extract was confirmed by bioassay, histopathology, and in silico analysis. GC–MS analysis of T. ornata acetone extract identified 16 compounds respectively. Docking studies recommended that pregnane-3,20-dione, 17,21-[(methylborylene)bis(oxy)]-, (5. beta)- had the highest binding energy of − 7.1 kcal/mol and could be used as a potent antiviral compound. The results of the present study confirmed that acetone extract of T. ornata has significant antiviral activity against WSSV and it can be used as a possible prophylactic in shrimp culture for prevention of WSSV infection.


Introduction
White spot syndrome virus (WSSV) is one of the most devastating and threatening viral pathogen, results in high mortality and huge economic losses in the crustacean aquaculture industry (Verbruggen et al. 2016;Santaniello et al. 2017 mortality within 3-10 days and it remains undetected until they produce a significant outbreak in shrimp farms. It consists of double-stranded circular DNA of 300 kbp in size and belongs to the family Nimaviridae, genus Whispovirus (Sundaram et al. 2016). WSSV infects a range of crustacean species including shrimp, crab, lobster, and crayfish. The mode of transmission of WSSV is both horizontal and vertical. The major physiological symptoms of WSSV are the occurrence of white spots inside the epidermis, appendages, and exoskeleton of shrimp (Sánchez-Paz 2010). WSSV infection progresses at various rates in different tissues with the stomach being the most preferred replication tissue in the early stages of infection. The skin, stomach, and gills are the most highly infected tissues causing symptoms such as skin colour changes, reduced food ingestion, and gathering at the water surface leading to hypoxia (Sun et al. 2013). Furthermore, WSSV causes apoptosis and oxidative stress in shrimp (Yuan et al. 2016). During viral infection, envelope proteins play a critical role in attachment to the host cellular receptor. 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).
To tackle the pathogens, the crustaceans depend on innate immunity since they lack an adaptive immune system. Although in the last 20 years, substantial progress has been achieved in host anti-WSSV immune defense (Li et al. 2019), no effective approach has been established. There has been no successful production of commercial vaccine against WSSV, and safety issue has become a major concern for developing vaccines rather than their efficacy (Haq et al. 2012). Even though there are several drugs on the market with anti-WSSV activity, the problem remains unsolved.
Seaweeds are rich source of bioactive compounds and produce secondary metabolites with a varying range of biological functions such as antibacterial, antifungal, antiviral, anti-inflammatory, nematocidal and anticoagulant (Singh and Chaudhary n.d.;Immanuel et al. 2012a;De Corato et al. 2017;Caijiao et al. 2021). Turbinaria ornata is a brown seaweed found in the Indian coast. It has wide range of biological activities. However, there has never been any report on the anti-WSSV activity in T. ornata. Therefore, the aim of the study was to determine the antiviral properties of T. ornata against WSSV by in vivo and in silico studies.

Collection and maintenance of animal
Freshwater crabs P. hydrodromous (150 numbers and body weight 20-25 g) were collected from the rice field located at Brahmapuram (12.9657° 79.1676°) Vellore, India. The animals were transported to the laboratory and kept at room temperature (25℃) in a 100L aquarium. The crabs were fed twice a day with boiled egg white (Karthikeyan et al. 2022). The crabs were maintained in the described condition for one week prior the experiment. Tissue samples including gills, muscle, and head-soft tissue were subjected to polymerase chain reaction to ensure the animals were WSSV-free before the experimental challenge (Sundaram et al. 2016).

Seaweed sample collection
Turbinaria ornata (T. ornata) was collected from Thangchimadam (9.293434 • 79.23951 • ) and was identified by Central Marine Fisheries Research Institute (CMFRI) Mandapam Rameswaram, Tamil Nadu, India. The samples were washed and cleaned with distilled water to remove impurities and salt present on the surface. The samples were then shade dried to avoid direct exposure to sunlight. The samples were then ground into fine powder using dry grinder and were sieved to get uniform particle size. They were then stored in airtight container and were used for extraction (Roohi Fatima et al. 2016).

Preparation of seaweed extract
The powdered T. ornata was extracted with acetone using a modified procedure (Roohi Fatima et al. 2016). Ten grams of the powder were extracted with 100 mL of aqueous acetone, and petroleum ether (1:10 w/v), and kept in a shaker at room temperature for 24 h. The extract was then filtered using Whatman No.1 filter paper. The filtrate was collected, and the solvent was removed by using a rotary evaporator. The extract was lyophilized and stored at 4℃ for further studies.

GC-MS analysis
The analysis of compound in acetone extract of T. ornata was performed using Gas Chromatography (Perkin Elmer GC Clarus 680 system, United States) equipped with Elite-5MS column (30.0 m, 0.25 mm IID, 250 µm df) and 70 eV of ionization energy was employed for GC-MS detection (Dinesh et al. 2017). The carrier gas was helium (99.99%) and was used at a constant flow rate of 1 mL/min and an injection volume of 1IL (split ratio of 10:1). The temperature of the injector was 260℃. The initial oven temperature was set to 60℃ for 2 min followed by an increase of 10℃/min to 300℃, holding for 6 min. Mass spectra were collected at 70 eV, with a scanning interval of 0.5 s and fragments ranging from 50 to 600 Da. The total run time of GC was 32 min. Each component's relative percentage quantity was computed by comparing its average peak area to the overall area. TurboMass Version 5.4.2 was used to handle mass spectra and chromatograms. The retention periods of the matching compounds were compared to those of legitimate compounds, as well as the spectrum data collected from the library database, National Institute of Standards and Technology (NIST) (Eswaraiah et al. 2020).

Preparation of WSSV inoculum
Hemolymph was collected from WSSV-infected shrimp through heart puncture using a 26G syringe. The collected hemolymph was centrifuged at 3000 rpm for 20 min at 4℃. The supernatant was collected and transferred to a fresh tube and it was centrifuged at 8000 rpm for 30 min at 4℃ the supernatant was filtered with a 0.45 µm filter and stored at -20℃ for future studies (Yoganandhan et al. 2003). The presence of WSSV in hemolymph was confirmed by performing PCR. The structural gene VP28 was amplified using a 20 μl standard PCR reaction containing 10 μl of 2 × master mix, 5 pmol vp28 primer 1 3 (Forward-ATG GAT CTT TCT TTCAC and Reverse-TTA CTC GGT CTC AGTGC), 3 μl PCR grade water and 1 μl template DNA (Sundaram et al. 2016). Viral titer used in the present study was 1.6 × 10 6 copies µL −1 as reported in our earlier study (Natarajan et al. 2017).

Bioassay analysis
The prepared T. ornata acetone extract was investigated for antiviral activity efficacy against WSSV in freshwater crab, Paratelphusa hydrodomous. This animal model is reported as one of the best suitable model for WSSV bioassay studies (Sundaram et al. 2014(Sundaram et al. , 2016Karthikeyan et al. 2022). The dose-dependent concentration of 0.5, 1, and 2 mg was primarily studied to confirm the therapeutic dose against WSSV and observed 1 mg shows a better effect with no mortality on crabs, thereby a dose of 1 mg was chosen for the following studies. The experimental animal was divided into three experimental groups each group containing three crabs per group and the experiment was performed in triplicate (Sundaram et al. 2016). The viral inoculum mixture was incubated for 3 h before injection. The viral inoculum mixture was injected into the respective group after 3 h of incubation. All the crabs were injected intramuscularly at the second abdominal segment. Briefly, healthy crabs received the following injection: in group I, the crabs were injected with 100µL NTE buffer which served as a negative control, and in group II, the crabs were injected with 95 µl NTE buffer and 5 µl viral inoculum which served as a positive control. In group III the animals were injected with 85 µl NTE buffer, 5 µl viral inoculum, and 10 µl acetone extract (1 mg). The crabs were fed twice a day and observed regularly for the gross sign of disease. The experiment was carried out up to 60 days post-infection with WSSV and mortality was recorded to plot cumulative mortality graph.

Macromolecule and small molecule preparation
The 3D structure of the VP28 (2ED6) envelope protein of WSSV was obtained from Protein Data Bank (Berman et al. 2000). Using the AutoDock 4.2 software, the protein structure was prepared for docking by eliminating any non-amino acid moieties (Morris et al. 1998). The simplified molecular input line entry system (SMILES) is a line notation standard that represents the connectivity and chirality of a molecule. The canonical SMILES of the compound were obtained from PubChem and the PDB format of the compound was generated using the online SMILES Translator (https:// cactus. nci. nih. gov/ trans late/) (Manimaran et al. 2018).

Molecular docking
Molecular docking investigations were carried out using Autodock-4.2 to study the interaction between T. oranta compounds and envelope protein VP28 (Sudharsana et al. 2016) (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 viewed using Discovery studio visualizer v20.1.0.19295, Dassault Systems Bio via corp (Thirumal Kumar et al. 2019).

Histopathology
Experimental crabs injected with acetone 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 tissue was rehydrated and stained with haematoxylin and eosin (H&E) and histological changes were then evaluated under a compound microscope (Carl Zeiss, India) (Lightner and Redman 1998).

Bioassay
Freshwater crabs (P. hydrodromous) were used as a model organism for the experiment to determine the antiviral activity of T. ornata acetone extract. The result proved that T. oranta acetone extract has strong antiviral activity against WSSV. In group III, crabs injected with T. oranta acetone extract and viral inoculum showed 100% survival until the end of the experiment (Fig. 1). In group II, the crabs injected with viral inoculum recorded 100% mortality within 7 days of post-infection. In group I, the crabs injected with NTE alone survived without any sign of WSSV infection and showed zero mortality until the

Molecular docking studies
Docking of the different compounds identified from T. ornata acetone extract with the target protein of WSSV VP28 showed the least binding energy of − 7.1 kcal/mol respectively ( Table 2). The amino acid residues involved in the interaction with VP28 envelope protein were visualized using the Discovery Studio visualizer. The lead compound pregnane-3, 20-dione, 17, 21-[(methylborylene)bis(oxy)]-, (5. beta.)-formed 2 hydrogen bonds (PRO145 & LYS147) with VP28 during interaction (Fig. 3). The strongest interactions responsible for the binding are hydrogen bonds. The amino acid residues involved in the interaction are PROD:145 LYSD:147, ALAE:107, and VALE:172. The interaction involved includes van der Walls, hydrogen bonds, and alkyl interactions which are depicted in Fig. 3. The possible ligand binding site of VP28 contains all of the amino acids involved in the interaction.

Discussion
WSSV infection in shrimp is still a major problem for shrimp farmers. There is no proper treatment to control the WSSV infection in shrimp. It is important to develop antiviral drugs against WSSV. Several investigations have been carried out in search of seaweed extract with substantial anti-WSSV activity that can protect cultured shrimp against this virus. Seaweed (Rouphael et al. 2016) extracts possess antiviral as well as immunostimulant characteristics (Güroy et al. 2022). Fucoidans extracted from different seaweeds reported to inhibit the replication of several enveloped virus which includes HIV, HSV, and human cytomegalovirus (Thuy et al. 2015;Garrido et al. 2017  illustrated biological activity such as antibacterial, antioxidant anti-inflammatory activity, and wound healing properties (Shaibi et al. 2021). Earlier, several attempts were done to find anti-WSSV properties from seaweeds and plants (Citarasu et al. 2006;Balasubramanian et al. 2007;Peraza-Gómez et al. 2014;Sundaram et al. 2014Sundaram et al. , 2016. In this present study, T. ornata acetone extract was tested for antiviral activity against WSSV in freshwater crab P. hydrodomous. In this experiment, we found that the acetone extract of T. ornata showed strong activity against WSSV with 100% survival. The positive control showed 100% mortality. A similar study was done with methanolic extract of Hypane spinella that showed antiviral activity against WSSV in freshwater crabs (Sundaram et al. 2014) The WSSV inoculum incubated with T. ornata extract effectively inactivated the virus replication by the interaction between the extract and the envelope proteins of the WSSV virus. Furthermore, the effect of the extract on WSSV replication prevents virus multiplication in host organisms. It is assumed that the immune mechanism is triggered by T. ornata extract which tries to combat against WSSV in the crab. Sudheer et al.(2011) reported that aqueous extract of Ceriops tagal when administered as feed to P. monodon showed 74% survival rate against WSSV. The shrimp feed with ethanolic leaf extract of Pongamia pinnata showed 80% survival rate against WSSV (Rameshthangam and Ramasamy 2007). Fucoidan isolated from Sargassum wightii z fed to P. monodon enhanced the immunity and resistance against WSSV (Immanuel et al. 2012b). Similarly sulfated galactans isolated from Gracilaria fisheri exhibited antiviral activity against WSSV in P.monodon and the mortality rate was lower (Wongprasert et al. 2014). It was reported that the hot water extract of Ulva instestinalis when supplemented as feed to Litopenaeus vannamei upregulated the immune response and inhibited WSSV (Klongklaew et al. 2021). Dupuy et al. (2004) reported that pre-incubation of WSSV with Mytillin for 3 h at room temperature showed anti-WSSV activity by decreasing the mortality in shrimp. In this study, the acetone extract of T. ornata administered along with WSSV was incubated for 3 h at room temperature and showed virucidal activity suggesting the presence of molecules in the extract that could render the virus inactive.
A chemically synthesized compound methyl 1-chloro-7-methyl-2-propyl-1 h-benzo[d] imidazole-5-carboxylate was tested against WSSV target protein VP28 by in silico docking analysis (Karthikeyan et al. 2022). They also reported that the compound interacted with METB139 of VP28 envelope protein. According to previous reports VP28 binds to shrimp cells as an attachment protein, allowing the virus to enter the cytoplasm (Yi et al. 2004). As a result, blocking of this protein will prevent the entry of WSSV in the host. Similarly, docking of VP28 with A. marina derived phytochemicals exhibited the potential to block VP28 protein (Sahu et al. 2012). In this study, in silico analysis revealed that pregnane-3,20-dione, 17,21-[(methylborylene)bis(oxy)]-, (5. beta)-interacts with PROD:145 LYSD:147, ALAE:107, and VALE:172. of VP28 protein and it also showed highest binding affinity to the protein VP28 which can inhibit the replication of WSSV. It is proposed that the interaction between pregnane-3,20-dione, 17,21-[(methylborylene)bis(oxy)]-, (5. beta)-with VP28 can prevent the formation of PmRab-VP28 complex. There have been very few efforts to purify components responsible for anti-WSSV activity and all these studies focused primarily on the crude extract from single seaweed or a combination of both Velmurugan et al. 2015).

Conclusion
It is concluded from the present study that acetone extract of T. ornata showed significant antiviral activity against WSSV. The presence of antiviral compound in T. ornata was also confirmed from this study. The acetone extract of T. ornata could be effectively used as prophylactic and both in silico and in vivo approaches reveal the potential activity of T. ornata to control WSSV infection in shrimp farms. The computational analysis reveals that the phytocompound pregnane-3,20-dione, 17,21-[(methylborylene)bis(oxy)]-, (5. beta)-from T. ornata could be the potential molecule for the treatment of WSSV in shrimp culture.