Viral infections are a major threat to the aquaculture industry throughout the world. Betanodavirus is one of the most infectious viruses that cause the highest mortality in larval and juvenile stages of Latescalcarifer commonly known as Barramundi. It is a single-stranded positive- sense RNA virus and causes viral nervous necrosis(VNN).VNN is caused by a RNA virus that gets transmitted both horizontally and vertically so the most effective method against this virus is to vaccinate the fish, however, vaccination becomes difficult since the disease is associated with outbreaks in larval and juvenile stages which are not that much immunocompetent. In our previous in silico studies, we proved the stability of camphene as a better phytochemical agent. In continuation to prove the authenticity of camphene as a potential antiviral agent against betanodavirus, its in vitro validation was performed. Sea bass kidney cell line (SISK) was selected for carrying out the in vitro studies and cytotoxicity studies of camphene in the SISK were done by MTT assay. Based on the analysis of the MTT assay, different dosages of camphene were selected viz.,0.2, 0.5, 1, 1.5, 2, 2.5, 5, 10, 20, 30 μg/ml. The SISK cells were infected with a virus inoculum(200μl). Further, the antiviral activity of camphene on infected SISK cells by Betanodaviruswas elucidated with the help of quantitative Real-time PCR(qPCR) on the 3rd and 5th day of infection. Analysis of results depicted that the dose of camphene 2 to 10 μg/ml is the safest dose against Betanodavirus. Hence this is aptly revealed that camphene can be used as a potential antiviral agent against Betanodavirus.

Among many relevant issues dealing with fish farming, microbial infections are a major problem. There are different viral infections, which are continuously creating problems in fish farming and among these viral infections Betanoda viral infection is a foremost problem. The Betanodavirus is an important, emerging group of viruses known to infect around 40 species worldwide. The major target of this virus is the central nervous system and retina of fishes especially in Barramundi species. Viral Nervous Necrosis (VNN) is now a serious problem for different fish species which is yet to be resolved through strong antiviral compounds. The In-silico screening of potential phytochemicals as a drug molecule with low or no side effects against viral nervous necrosis in barramundi is the major objective of the study. The present study discusses the molecular interaction studies carried out between virtually screened phytochemicals and MX protein of barramundi fish. Findings based on virtual screening, calculation of molecular properties and bioactivity score showed that among 101 compounds, the hypogallic acid, cineole, eugenol, linalool, camphene, oligonol, azulene, caravacrol, pistol and squalene are the active phytochemicals against the selected MX protein. Further intense screening showed that Camphene is the best screened phytochemical with the lowest binding energy in complex with MX protein of Barramundi. Further molecular dynamic simulation study at 100ns (Nano seconds) proved the importance, stability and establishment of camphene as better natural prophylactic and therapeutic approaches to overcome or reduce the problem of viral nervous necrosis in barramundi.

Betanoda virus is one of the most important and emerging groups of viruses known to infect around 40 species found to be worldwide in distribution. The most common and virulent target of infection for this virus is (Lates Calcarifer) (barramundi). It is found that the expression of MX protein is found to be the more susceptible reason for this viral infection. Considering this current study including characterization to structure prediction revolves around the MX protein as a target. The progression of this study describes the amino acid sequence of MX protein was retrieved from UniProt database in Fasta format and further primary structure analysis and characterization including nature of amino acids, instability index reading, GRAVY, determination of phosphorylation as well as signal peptide cleavage sites was done with the help of various tools. Secondary structure prediction has proceeded through SOPMA server analysis revealed that MX protein has mixed secondary structure, i.e., mostly alpha-helix and beta-turn. The progression of this work prediction of a 3D structure along with functional site prediction of MX protein of Fish (Lates Calcarifer) is done through standard modeling tools. The 3D structure of this protein of (Lates Calcarifer) as documented in this study may provide a valuable aid for designing an inhibitor or better ligand against viral nervous necrosis disease and could play a vital role in drug design.