Insight into molecular interaction between shrimp and white spot syndrome virus through MjsvCL-VP28 complex: an in-silico approach

Abstract White Spot disease is a devastating disease of shrimps caused by White Spot Syndrome Virus in multifarious shrimp species. At present there is no absolute medication to suppress the disease hence, there is an urgent need for development of drug against the virus. Molecular interaction between viral envelope protein VP28 and shrimp receptor protein especially chitins play a pivotal role in ingression of WSSV. In the present study, we have tried to shed light on structural aspects of lectin protein in Marsupenaeus japonicus (MjsvCL). A structural insight to the CTLD-domain of MjsvCL has facilitated the understanding of the binding mechanism between the two proteins that is responsible for entry of WSSV into shrimps. Further, incorporation of molecular dynamics simulation and MMPBSA studies revealed the affinity of binding and certain hotspot residues, which are critical for association of both the proteins. For the first time we have proposed that these amino acids are quintessential for formation of VP28-MjsvCL complex and play crucial role in entry of WSSV into shrimps. Targeting the interaction between VP28 and CTLD of MjsvCL may possibly serve as a potential drug target. The current study provides information for better understanding the interaction between VP28 and MjsvCL that could be a plausible site for future inhibitors against WSSV in shrimps. Communicated by Ramaswamy H. Sarma


Introduction
White Spot Disease (WSD) has been a major cause for paramount devastation of shrimp aquaculture practices globally since the last few decades. The disease is caused by White Spot Syndrome Virus (WSSV) which adversely affects a multifarious range of host shrimp species commonly including Litopenaeus vannamei, Penaeus monodon, Marsupenaeus japonicus, and Fenneropenaeus indicus. WSD has been reported to occur in China, East and Southeast Asia, America, India, Middle East and Europe (Lightner, 2012;Mohan et al., 1998;Stentiford et al., 2009Stentiford et al., , 2012Stentiford & Lightner, 2011;Wang et al., 2000). Since its emergence, the total economic damage caused by WSD to the shrimp aquaculture industry has been evaluated to be $8-$15 billion (Lightner, 2012). The economic loss has been reported to increase by $1 billion on annual basis (Stentiford et al., 2012). These yearly losses have been estimated to equate to approximately one tenth of global shrimp production (Stentiford et al., 2012). Till date an efficient drug/medicine/antiviral is unknown for the disease mitigation process. The major reason behind unavailability of an absolute antiviral against the disease is lack of comprehensive information pertaining to molecular mechanisms of disease propagation within the shrimp. Furthermore, molecular interactions occurring between host and pathogen proteins during the cascade of events involved in the disease mechanism is a new domain yet to be explored. The disease is disseminated within the shrimp population in a very aggressive manner and in its deadliest form the disease can cause up to 100% mortality in a week span (Lightner, 2012).
Shrimps are cannibalistic in nature hence, the disease gets triggered when a healthy shrimp engulfs any WSSV diseased shrimp (Lotz & Soto, 2002;Wu et al., 2001). The shrimp alimentary canal is the primary site of infection and is composed of oesophagus, stomach, midgut and hindgut. Among all these organs, the stomach is considered to be contaminated with the disease to the maximum extent (Verma et al., 2017). Once the diseased shrimp enters the alimentary canal of healthy shrimp, the WSSV virions get dispersed within the alimentary canal cavity where, the individual virus comes in contact with the membrane of the shrimp digestive tract. The viral envelope and its constituent structural proteins are the most important components that interact with shrimp alimentary canal. The basal lining of the stomach is lined by several receptors that offer a receptive surface to the virus so that virus can attach to the gut passage (Yadav et al., 2021;Verma et al., 2017). The receptor proteins that line the alimentary canal lumen offer proper receptive surface for the viral envelope proteins to get anchored, thereby causing instigation of WSSV disease in the shrimps (Verbruggen et al., 2016;Verma et al., 2017).
Thus recognizing shrimp receptor proteins that facilitate ingression of virus during early viral entry stage would provide great insight into viral-shrimp interactions. C-type lectins (CLs) are typical receptor proteins that line the shrimp alimentary canal lumen and serve as a doorway for the virus particles to enter the shrimp body. Such proteins have distinctive C-type carbohydrate recognition domains (CRDs) that bind to an extensive range of ligands such as protein, lipids, glycans etc (Zelensky & Gready, 2005). These CLs are programmed to recognize the virions and assist in hijacking of these viral particles in order to commence viral ingression into the shrimp body. A very important CL receptor protein present in Marsupenaeus japonicus is MjsvCL (GenBank accession no. KF712277). MjsvCL contains a (i) signal peptide (ii) CTLD-domain and (iii) a Q/N (glutamines/asparagines) rich region that is present between the signal peptide and the CTLD region. Among all these three, CTLD domain is considered of extreme importance due to its binding properties for the viral envelope proteins specifically VP28 (Wang et al., 2014).
WSSV is a DNA virus. The most important protein of WSSV is VP28 that is most extensively studied and its structure is available in Protein Data Bank (PDB) under the ID 2ED6. Functionally, VP28 depicts a mushroom shaped structure and its functional part is trimeric in nature. Each trimer is made up of three monomers that are made up of N terminal a-helix and C terminal b-barrel. The b-barrel of VP28 is typically made up of nine-strands most of which are antiparallel b-strands. The pore formed by the b-barrel is hydrophobic in nature and is composed of amino acids Phe, Ile, Leu, Met, and Val residues. The N terminal a-helix is made up of 15 amino acids and protrudes almost 20 Å above from the surface of the b barrel. It is linked to the b-barrel via a 2 amino acids coil. This N-terminal a-helix region is presumed to be embedded inside the viral envelope making the b barrel structure protrude outside from the envelope and thereby making the b barrel available for the interaction with the host proteins (Tang et al., 2007).
The CTLD-domain of MjsvCL is responsible for interacting with VP28 through protein-protein interactions (Wang et al., 2014). But the exact amino acid interactions that are involved in this protein-protein interaction event are still elusive. Hence, in the present study we try to understand the overall structure of MjsvCL with special focus on CTLDdomain. We propose the 3-dimensional (3 D) structure of CTLD domain of MjsvCL by using molecular modeling approaches. Further, binding mechanism, affinity and perresidue decomposition between CTLD and VP28 are studied by employing molecular docking, molecular dynamic (MD) simulation and MMPBSA approaches. This study provides comprehensive information regarding the hotspot amino acids of VP28 and CTLD-domain, which together associate to form a stable complex.

Sequence analysis of MjsvCL
The nucleotide sequence for shrimp receptor protein MjsvCL is KF712277 (Wang et al., 2014) and the protein corresponding to this has protein_id="AHA83582.1". The sequence of MjsvCL protein was downloaded as FASTA format and further used as input for primary, secondary and tertiary structure prediction studies. The primary structure specifications like number of amino acids, molecular weight, instability index, aliphatic index and grand average of hydropathicity (GRAVY) of MjsvCL protein were computed using ProtParam tool (Wilkins et al., 1999). In order to have more in-depth analysis of MjsvCL, the protein sequence was further searched in Uniprot (The UniProt Consortium, 2017) and Pfam databases (Finn et al., 2014). The secondary structure prediction of MjsvCL was performed using PSIPRED protein analysis workbench (Buchan & Jones, 2019) and JPred server (Drozdetskiy et al., 2015). Multiple sequence alignment of the shrimp lectin proteins was performed using Clustal Omega (Sievers & Higgins, 2018) in order to understand the conserved patterns across the lectin proteins in shrimps.

Three-dimensional (3 D) structure of CTLD domain of MjsvCL and its validation
With the aim to understand the binding mechanism of the CTLD domain of MjsvCL with VP28, we attempted to build a 3 D model of the CTLD domain. A novel tool named 'AlphaFold2' has emerged recently as a powerful and accurate tool to predict the 3 D structure of protein molecule close to experimental methods. It incorporates novel neural network architectures and training procedures based on evolutionary, physical and geometric constraints of molecule structures (Jumper et al., 2021). Hence, here AlphaFold2 has been used as a model generation tool to build the 3 D structure of the CTLD domain of MjsvCL protein. The obtained structure was further validated using certain web-interface like: Prosa-web (Wiederstein & Sippl, 2007), Verify3D (Luthy et al., 1997) and Molprobity (Williams et al., 2018).

Protein-protein docking of VP28 and CTLD domain
In order to obtain the docked complex between the VP28 protein and CTLD domain of MjsvCL, the functional unit (trimer) of the VP28 (PDB-ID: 2ED6) was docked to the modeled CTLD domain. The protein-protein docking was performed using HADDOCK 2.4 server (https://wenmr.science.uu.nl/had-dock2.4/), which takes a data-driven approach, along with support from a wide range of experimental data (van Zundert et al., 2016). The server also calculates Van der waals (vdW), electrostatic, desolvation and restraint violation energies along with RMSD from the lowest-energy structure and solvent accessible surface area (SASA). The output of HADDOCK includes Haddock score and Z-score on the basis of which we select the best docked pose (Williams et al., 2018).

Protein-protein interface analysis
A freely available web server, 'PRODIGY' (PROtein binDIng enerGY prediction) (https://wenmr.science.uu.nl/prodigy/) was deployed to estimate the binding affinity along with interfacial contacts between the VP28-CTLD docked complex (Williams et al., 2018). It incorporates a simple and robust descriptor of binding affinity based only on structural properties of protein-protein complex and is based on contact-based approach. It has also been proved to predict the interfacial contacts at protein-protein interface close to experimental values (Xue et al., 2016).

Molecular dynamics simulation of VP28-CTLD complex
All-atom MD simulation was performed on the VP28-CTLD complex using GROMACS 5.5.1 (http://www.gromacs.org/) using ff99SB-ILDN force-field and TIP3P water model (Van Der Spoel et al., 2005). The 3 D structure of the VP28-CTLD complex was immersed in a cubic box of 1.0 nm and periodic boundary conditions were applied using 'editconf' tool followed by addition of 42,915 SPC water molecules. System was made electrically neutral by adding 27 Na þ ions using the 'genion' tool. First of all the system was energy minimized using energy minimization tolerance (emtol)1000 kJ mol À 1 nm À 1 via steepest descent and conjugate gradient methods with the aim to remove excessive strain. A short constant number of particles, volume, and temperature (NVT) simulation was performed for 100 ps at 300 K temperature, followed by 100 ps of equilibration at constant number of particles, pressure, and temperature (NPT) ensemble possessing atmospheric pressure (1 bar) and position restraints on the macromolecule. Once assured that the system is well equilibrated, it was then subjected to MD simulations for 100 ns. The timestep was kept constant (2 fs) throughout the MD. The 'V-rescale' thermostat and 'Parrinello-Rahman' algorithm was deployed as temperature and pressure coupling methods. The production trajectories were written every 10 ps and gromacs inbuilt tools like 'rms', 'rmsf', 'gyrate' and 'hbond' were used to analyze the data. A plotting tool Qtgrace is used to generate all the graphs (https://sourceforge.net/projects/qtgrace/).

MMPBSA calculations
Integrated with MD simulation, binding free-energy computation methods have turned out to be robust mechanisms in contributing quantifiable estimation for protein-ligand/protein-protein interactions. A gromacs based tool, 'g_mmpbsa' (Kumari et al., 2014) was used to compute the binding free energy (DG bind ) between the receptor (VP28) and the ligand (CTLD domain of MjsvCL) of the complex on a well equilibrated trajectories. The equation DG bind ¼ G complex -G receptor -G ligand is used to estimate binding free energy. The free energy of each of these entity was predicted as a sum of molecular mechanics energy (E MM ; sum of the internal energy of the molecule, the electrostatics and vdW interactions in vacuo), polar contribution to the solvation energy of the molecule (G psolv ) and nonpolar solvation free energy (G npsolv ) as shown in the equation, G ¼ (E MM ) þ (G psol ) þ (G npsolv ). G psolv was calculated by solving the Poisson-Boltzman (PB) and Generalized-Born (GB) equations for MMPBSA and MMGBSA methods, while G npsolv was calculated using the equation where SASA is the solvent-accessible surface area calculated using the linear combinations of pairwise overlaps or Molsurf methods. As, the net charge on both the proteins are highly negative (-27), the dielectric of protein molecule (pdie) was taken as 20 for MM and polar calculations (Baker et al., 2001). All the energy components were calculated on 70 to 100 ns data at the time interval of 500 ps. The per residue decomposition analysis was performed to estimate the energetic contribution of the amino acids involved in protein-protein binding. Binding free energy decomposition was done using g_mmpbsa tool that decomposes the overall binding energy of two entities elucidating an individual amino acid contribution in binding.  (Finn et al., 2014) the whole sequence is divided into three domains: i) signal peptide (1-19 amino acids), ii) Q/N rich region (20-133 amino acids), and iii) CTLD domain (133-250 amino acids). Out of the three portions, Q/N rich is responsible to get associated with calreticulin (CRT) while the CTLD domain is responsible for recognition and attachment to the viral proteins (Wang et al., 2014). Secondary structure prediction servers such as PSIPRED and JPred both revealed the presence of two long loops in the CTLD portion: (i) the 'whole domain loop' ( 143 GSRWNAISIET 153 ) and (ii) the 'long loop region' ( 187 GPGA GAFQGLNWGLTGGFGQPQPDNRAGDEN 217 ) (Supplementary Figure 1). Furthermore, 4 cysteine residues were confirmed at positions Cys-139, Cys-218, Cys-238 and Cys-246. Due to the presence of long loop the CTLD domain experiences large conformational flexibility, which might be a possible reason why the CTLD domain has not been crystallized till date (Costa et al., 2011). The top three hits obtained by PSI-BLAST analysis of the MjsvCL protein are ACJ06430.1 (Penaeus chinensis-67.05% identity), XP_027212003.1 (Penaeus vannamei-64.73% identity), and XP_037796898.1 (Penaeus monodon-64.54% identity). The multiple sequence alignment (MSA) of all the above sequences depicts that 4 cysteine residues and CDRs region remain conserved across various shrimps species (Figure 1). The CRDs region is carbohydrate binding region and binds to various forms of sugars which constitute either of the motifs: EPN, QPD, EPA, EPD (Zelensky & Gready, 2005).

Three-dimensional (3D) structure of CTLD domain of MjsvCL and its validation
The nucleotide as well the protein sequence of MjsvCL is well characterized but little is known about the structural aspects of the protein. In our study, we propose a 3 D structure of CTLD-domain of MjsvCL. The CTLD-domain of MjsvCL has been generated by AlphaFold2 and is shown in Figure 2. It consists of 117 amino acids (133-250) and is made up of two a-helixes, five b-sheets and a characteristic 'loop-in-aloop' structure. The a-helices have been depicted with wheat color and named a-1 and a-2. The b sheets are b-1, b-2, b-3, b-4, and b-5 (yellow color). The 'loop-in-a-loop' structure has been clearly depicted as: (i) 'whole domain loop' ( 143 GSRWNAISIET 153 : blue color) and (ii) 'long loop region' ( 187 GPGAGAFQGLNWGLTGGFGQPQPDNRAGDEN 217 : red color). The second loop, called the long loop region, is structurally and evolutionary flexible and is involved in Ca 2þ dependent carbohydrate binding and interaction with other ligands. Another important feature that could be noticed in the structure of CTLD domain is presence of QPD motif. The QPD motif lying in the long loop region is depicted in green stick representation. This motif is responsible for recognizing sugar moieties such as mannose or galactose (Zelensky & Gready, 2005). Four cysteine residues were detected at positions Cys139, Cys218, Cys246 and Cys238 forms two pairs of disulphide bonds (Cys139-Cys246 and Cys218-Cys238) are shown in cyan color (stick representation). The disulphide bonds are present at base of the loops and act as a bridge between the a1&b5 and b3&b5 thus, providing stability to the modeled CTLD domain. As per the Verify3D, 82.20% of the residues have averaged 3 D-1D score > ¼ 0.2, and ProSA yielded the Z-Score (-3.46) which states that the modeled structure is of good confidence (Supplementary Figure 2). To further estimate the quality of the model, Ramachandran plot was generated by Molprobity, which states 96.6% residues are in favored regions, 100% in allowed regions with no reported outliers (Supplementary   (Zelensky & Gready, 2005). Since all these structural features are very well depicted in the 3 D structure of proposed CTLD (MjsvCL) (Figure 2), hence we consider our structure to be quite close to the actually existing structures and can be carried forward for docking with VP28 receptor protein.

Protein-protein docking of VP28 and CTLD domain
Experimental evidence indicates that the functional unit of VP28 is a trimer (Tang et al., 2007). This trimeric form exists like a mushroom structure that anchors to the viral envelope. Each trimer is made up of 3 monomers which are further composed of a b-barrel and an a-helix. The a-helix protrudes out from the b-barrel and helps in anchoring VP28 to the viral envelope (Tang et al., 2007). Some studies depict that a-helix might be involved in interaction with shrimp receptor proteins (Lan et al., 2013). Other studies suggest that since the a-helix is involved in anchoring b-barrel to the viral envelope hence it is unavailable to interact with shrimp receptor proteins (Jamalpure et al., 2020). Further, some other studies have reported that while envisioning the WSSV envelop structure, the viral envelop protein VP28 is closely associated with other envelop proteins (Sun & Wu, 2016) especially VP24 (Li et al., 2015). Thus the sheer density of proteins distributed on the surface of viral envelope might sterically hinder the host receptor proteins to laterally bind with VP28 mushroom. Looking into such a scenario, it is quite unlikely that shrimp receptor proteins could approach VP28 laterally specifically through the a-helix (Jamalpure et al., 2020). This in turn leaves the top region of the b-barrel to be the most probable site for interaction with host proteins. Same approach of docking the host protein/ligand through the top portion of the b-barrel has further been supported by other studies too (Sudharsana et al., 2016).
Thus, the top region including the b-barrel and its lumen provides an ideal interface for interaction with host proteins hence we restricted docking only to the top region of the VP28 b-barrel. The docking was performed using HADDOCK 2.4 server which predicts several complexes along various clusters. All the poses from the top two clusters were analyzed using Pymol (https://pymol.org/2/) and revealed similar binding conformation, hence, the pose with the lowest Haddock score À 98.7 þ/-2.3 (RMSD: 0.4 þ/-0.3) was considered as the best docked complex. The VP28-CTLD docked complex is depicted in Figure 3A, where the loop portion of CTLD interacts with b-barrel of VP28.

Protein-protein interface analysis
As per the PRODIGY server, VP28 and CTLD both interact with each other via 83 pairs of intermolecular interactions: 1 pair of charge-charge contact, 10 pairs of charged-polar contacts, 12 pairs of charged-apolar contacts, 4 polar-polar contacts, 22 apolar-polar contacts, and 34 apolar-apolar contacts, contributing to total binding affinity (-11.1 kcal/mol). The mapping of the interfacial residues via PRODIGY script (.pml) on the 3 D structure of the docked complex shows the interacting amino acids (stick representation) deciphering the binding pattern among VP28 and CTLD domain of MjsvCL ( Figure 3A) and the zoomed view of the major interactions are shown in Figure 3B. Detailed investigations of the amino acids that contribute to formation of CTLD-VP28 complex have been shown in Figure 4. The flower diagram (Figure 4) represents the major interactions between the CTLD and VP28 (chain A, chain B, chain C) proteins, from which the amino acids of CTLD that interact with VP28A, VP28B, VP28C individually can be deduced. Since both the proteins favor each other by binding with amicable energetic hence the complex can further be considered for MD simulations.

Molecular dynamics simulation of the VP28-CTLD complex
A 100 ns atomistic simulation of the docked complex of VP28-CTLD was performed with the aim to understand the compatibility as well as the binding mechanism of both the proteins. The backbone root mean square deviation (RMSD), backbone root mean square fluctuation (RMSF), and radius of gyration (R g ) were analyzed throughout the trajectories in order to explore the dynamic nature and the stability of the complex. Figure 5A depicts the backbone RMSD of: VP28-CTLD-complex (black), VP28 (red), CTLD-domain (green). Since, VP28 is a crystal structure; its RMSD is least and is converged at �0.2 nm (red). The RMSD of the modeled structure of CTLD domain increases around 32 ns and exhibits fluctuations till 70 ns thereafter which it converges at �0.23 nm (green). Similarly, the RMSD owing to the VP28-CTLD complex also exhibits fluctuations and later converges to �0.3 nm from 70 to 100 ns (black). The well converged RMSD profile of the VP28-CTLD complex infers favorable association of the two with no steric clash. Hence, it can be deduced that complex formation between receptor (VP28) and the ligand (CTLD-domain) is energetically stable with the involvement of complimentary interactions among the two. The RMSF plot of the VP28-CTLD complex is shown in Figure 5B, where none of the amino acids of three chains of VP28 trimer are having higher fluctuations except the terminal ones. However, the RMSF profile of the CTLD domain indicates presence of some amino acid residues with higher fluctuation. Detailed investigation into such high fluctuation regions reveal that such amino acids are either part of CTLD domain terminus or part of those loop regions that have no direct interaction with the VP28 binding site. Moreover, it is also observed that the CTLD region involved in the binding to VP28 exhibits lower mobility, which confirms the firmness at the interface junction between VP28 and CTLD domain ( Figure 5B).
Additionally, radius of gyration (Rg) is the RMSD between the center of gravity of a macromolecule and its end. It is used to measure the stability and compactness of the system and tends to change over time on protein folding/unfolding states. The gyration plot of the VP28-CTLD complex fluctuate around 2.60 to 2.65 nm without any major drift throughout the trajectory ( Figure 5C). This result indicates that no unfolding has occurred in both the proteins; rather the VP28-CTLD complex achieved more compactness together forming a stable complex, unveiling the mystery of their binding pattern.
In order to identify the key interactions at the interface of VP28 and CTLD-domain, H-bond analysis was performed and the occupancies of these H-bonds were also monitored ( Figure 6A and B). Figure 6A depicts that around 8 to 9 Hbonds existed between the CTLD and VP28 from 70 to 100 ns data, but their occurrence is not constant. The major interactions that exhibit high occupancy (� 20%) are shown    in Figure 6B. These amino acids of the CTLD-domain can be considered as crucial and mutating them can disrupt the association of the complex.

Binding free energy and per residue decomposition analysis
As VP28 and CTLD together form a stable complex, the extent of their binding is still a major concern. For this, 'g_mmpbsa' tool is deployed to compute the binding free energy between both the proteins. It gives binding free energy (DG bind ), which is an estimate of the extent of association between the two molecules. It also yield the per residue contribution at the interface region of the protein-protein complex, probing to binding pattern and stability. Table 1 represents various energy components contributing to the binding free energy (DG bind ). From the data it is evident that the binding free energy is significantly lower suggesting the stability of the complex with higher compatibility. The per residue decomposition analysis revealed that apart from charged amino acids there are many other residues which contribute to binding energetics favoring the complex formation (Figure 7). From the above figure it can be inferred that the amino acids from the Bchain and C-chain of the VP28 contributes majorly to the complex formation as compared to A-chain. The major residues of VP28 that contributes to the binding are: Thr111, Gln114, Leu-116, Ala-148, Ser-152, Ala-179, Pro-180, Thr-184 and Ala185 along with CTLD-domain: Tyr-138, Asp167, Val169, Ser-170, Tyr-171, Phe-225, Tyr-226, Leu-241, Lys-242 and Pro-243. As the individual energetic of the above amino acids confers stability and compactness to the complex formation, hence, these could be the probable hotspot residues which render the interactions among the VP28 and CTLDdomain. These residues can be considered quintessential for interaction between VP28 and CTLD. Hence, they play critical role in entry of WSSV into shrimps. They can be further hindered to design a potential inhibitor against WSSV in shrimps.

Conclusion
When a healthy shrimp engulfs a WSSV infected shrimp, the viral structural proteins come in direct contact with gut receptor proteins of the shrimp. MjsvCL is an important shrimp receptor protein that interacts with VP28 trimer in order to attach the virus to the gut lining of the shrimp. During this attachment process, the amino acids Thr111, Gln114, Leu-116, Ala-148, Ser-152, Ala-179, Pro-180, Thr-184 and Ala185 of VP28 interact with Tyr-138, Asp 167, Asn-168, Val169, Ser-170, Tyr-171, Phe-225, Tyr-226, Leu-241, and Pro-243 amino acids of CTLD domain of MjsvCL. Due to such amino acid interaction, the b-barrel of VP28 trimer gets tightly associated with the CTLD domain of MjsvCL to form a complex. Since MjsvCL is a secretory protein, it assists in percolation of WSSV across the basal membrane of the alimentary canal of the shrimp. Once the virus reaches the plasma of the shrimp it directly attacks the target organs v.i.z antennae, eye stalk, gonads. Thus, the CTLD portions anchors the virus tightly and simultaneously the Q/N rich region of MjsvCL gets involved in caveolae mediated endocytosis hence, assisting the virus to penetrate into individual host target cell (Figure 8). Many protein-protein interactions have been held responsible for assisting WSSV release the viral genome into the host nucleus (Verma et al., 2013). Once the virus enters the target host cell, the virus releases its genome machinery to replicate and thereafter, proliferate inside the host body eventually causing death of the host shrimp. Due to this amino acid analysis, for the first time we propose the manner in which shrimp lectin protein MjsvCL interacts with the incoming WSSV particle. Such a study can shed light on the protein cascade events that are involved in WSSV entry into the shrimp. Understanding the disease mechanism of WSSV into shrimps can facilitate the structure based drug designing process against WSD in the shrimps.

Disclosure statement
No potential conflict of interest was reported by the authors.

Funding
The author(s) reported there is no funding associated with the work featured in this article. (C) MjsvCL is a secretory protein, hence while getting attached to WSSV, it assists the WSSV cross the basal membrane of the stomach to enter the hemal sinus; (D) WSSV travels within the hemal sinus till it reaches target organs; (E) While WSSV travels within the hemal sinus, VP28 remains firmly attached to CTLD domain of MjsvCL and Q/ N rich region of MjsvCL gets associated with calreticulin to assist in entry of WSSV into target cell by caveolae mediated endicytosis; (F) WSSV enters into the target cell and releases its genome into the nucleus of target cell where the replication of WSSV genome occurs followed by further proliferation and dissemination of WSSV.