White Spot Syndrome Virus (WSSV) Experiment

White Spot Syndrome Virus (WSSV) Experiment swindleWhite espy syndrome virus (WSSV) continues to cause huge economic losings in the aquaculture farms due to rapid spread and broad host range. In this study, we synthesized a clean synthetic compound 3-(1-chloropiperidin-4-yl)-6-fluoro benzisoxazole 2 and were screened for antiviral activity against WSSV using unfermented water pediculosis pubis Paratelphusa hydrodomous (Herbst). In vivo bio-assay was carried out to view the antiviral activity. Reverse transcriptase polymerase chain reaction (RT-PCR) and Histopathology were used for the comp give the axe of bio-assay. Overall result shows that the tonic compound has strong antiviral self-assurance against WSSV.Keywords Paratelphusa hydrodomous, White spot syndrome virus, Synthetic compound, RT-PCR, Histopathology.White spot syndrome virus (WSSV) is a highly pathogenic whispovirus belongs to the family Nimaviridae responsible for causing white spot disease, leads to coulomb % mortality within 3-10 days of infection in farmed shrimp (Sudheer et al., 2012). several(prenominal) antiviral and immunostimulatory compounds be identified from terrestrial plants as well as from the marine origin were tested against WSSV. For example Sulfated galactans isolated from red seaweed (Gracilaria fisheri) exhibited immunostimulant and foe against WSSV in Penaeus monodon (Wongprasert et al., 2014), Aqueous extract of Cynodon dactylon showed strong antiviral activity against WSSV in marine shrimp (Balasubramanian et al., 2007).Synthetic compounds like Piperidines and Benzisoxazoles are important congregation of heterocyclic compounds in the field of medicinal chemistry. These compounds defend significant biological and pharmacological properties like anti-inflammatory, antifungal, antimicrobial, and anticancer activities (Gaba et al., 2014 Ramalingan et al., 2004) (Ramalingan, 2004 21Gaba, 2014 22). some(prenominal) fluorinated, benzisoxazole derivatives are currentl y used in the treatment of diseases (Prasad et al., 2009). In such a way, there is an immediate need for non-toxic drug to treat WSSV disease. gum olibanum the present study was carried out to determine the antiviral activity and protective effect of a novel synthesized compound 3-(1-chloropiperidin-4-yl)-6-fluorobenzisoxazole 2 (Fig. 1) against WSSV infection in fresh water rice-field crab P. hydrodomous, it was highly susceptible to WSSV (Sahul Hameed et al., 2001).A entailment of novel 3-(1-chloropiperidin-4-yl)-6-fluorobenzisoxazole was carried out under mild reaction conditions using 1.2 equivalents of atomic fig 20 hypochlorite. Interestingly, the developed method does not involve any additives like acids or bases and provides 96 % of isolated yields at room temperature. This novel molecule, 3-(1-chloropiperidin-4-yl)-6-fluorobenzisoxazole 2 was still at ambient conditions and stereo chemistry was established the single quartz XRD technique. The materials were purchase d from Sigma-Aldrich, Merck and were used without any additional purification. All reactions were monitored by thin spirit level chromato graphy (TLC). Melting points were recorded on an Elchem digital melting point utensil in open capillaries and are uncorrected. The 1H nuclear magnetic resonance was measured on a Bruker Avance-400 MHz instrument at room temperature. The 1H NMR was measured for 0.03 M solutions in CDCl3 using TMS as internal reference. The accuracy of the 1H shifts is considered to be 0.02 ppm. The twin constants J are in Hertz. volume spectra were obtained using ESI mass spectrometry. 6-fluoro-3-(piperidin-4-yl) benzisoxazole 1 (5 g, 22.7 mmol) was taken into the round bottom flask dissolved in 50 mL of acetonitrile. To this calcium hypochlorite (3.9 g, 27.3mmol) was slowly added over ten to twenty minutes. reception was monitored by TLC. After the reaction completion, reaction mass was filtered and salts was washed with acetonitrile. final result was dried under reduced pressure. Crude solid was purified by tugboat chromatography to give 3-(1-chloropiperidin-4-yl)-6-fluorobenzisoxazole 2 in 96 % (5.54g, 21.8mmol) as pale lily-livered color solid. The structure of the N-chloro benzisoxazole was conrmed from their spectral data from NMR, ES Mass and single crystal XRD.Mp 81-83oC1H NMR (CDCl3, 400 MHz) d (ppm) 7.71-7.08 (m, 3H), 3.65 (d, 2H), 3.22 (t, 3H), 2.36-2.15(m, 4H)13C NMR (CDCl3, degree centigrade MHz) d (ppm) 165.3, 163.9, 162.8, 159.9, 122.6, 122.2, 122.1, 116.9, 112.4, 97.6, 97.4, 32.9 MS (ESI) m/z Calcd 254.1, found 253 (M-1) exclusive crystal crystal data 3-(1-chloropiperidin-4-yl)-6-fluoro benzisoxazole 2Mol. FormulaC12H12ClFN2O CCDC reference number is 878706 Intensity data were collected on an APEX CCD diffract measure equipped with MoKa (l = 0.7107 A) radiation electric cell length a =5.8979(4) Cell length b=10.4965(7) Cell length c=19.1492(12) Cell Angle =90.0 Cell Angle =91.783 Cell Angle =90.0 Cell mickle=1184.9 0(11) The crystallographic data for N-chloro benzisoxazole have been deposited with the Cambridge Crystallographic Data Centre. Copies of this information may be obtained bare of charge from the Director, CCDC, 12 Union Road, Cambridge, CB2 1EZ, UK Fax 44(1223)336033, or http// www.ccdc.cam.ac.uk.The crabs P. hydrodomous (20-25 g automobile trunk weight) were collected from the rice field located at kalavai, Vellore, India. Crabs were transported to the laboratory. A previous method was followed for maintaining the crabs and preparation of WSSV inoculum (Nambi et al., 2012). For in vivo closing of antiviral activity, the healthy crabs were divided into three groups contains 3 crabs per group and each trial was conducted in triplicates. Crabs in the group I were injected with snow l of a mixture of viral good luck and NTE buffer which served as exacting throw. In Group II crabs were injected with NTE buffer alone served as negative control. In Group cardinal crabs were inject ed with viral suspension, novel compound and NTE buffer served as treated. The viral suspensions for all groups were incubated at room temperature for 3 h. Later it was injected into respective observational groups intramuscularly. The data-based animals were examined twice per day for unwashed signs of disease, and the number of deaths was recorded until end of the experiment. Animals in the treated and negative control group were survived without any mortality and sign of WSSV infection until end of the experiment. Whereas the positive control group reached 100 % mortality at 7th day of post injection with gross signs including reduced feed consumption, less active in slow in movement. The observation of this bio-assay was plotted in a cumulative mortality graph (Fig. 2).Hemolymph from all the 3 groups was collected for hematological analysis (Total hemocyte count and clot time). In positive control, there were significant reduction in innate hemocyte counts as well as the he molmph was failed to clot. No significant hematological changes were discover in between the negative and treated groups.For RT-PCR analysis Gill, head-soft create from raw stuff, heart and muscular tissue tissue were excised from each crabs of the experimental group and pooled together for extraction of entire ribonucleic acid using Trizol (Invitrogen, USA) according to the manufacturers instructions. cdesoxyribonucleic acid was synthesized from 1.0g of the total RNA using a One-step Reverse Transcriptase (Invitrogen, USA) as per the kit instructions and used as template for gene scene analysis of WSSV specific primer VP28. -actin served as an internal control for RNA quality and amplification efficiency. The sequences of primers used in this present study were precondition in add-in 1. The cycling conditions are initial denaturation at 95C for 5 min followed by 35 cycles of denaturation at 95C for 30 bit, annealing at 50C for 30 sec and extension at 72C for 30 sec with a final extension at 72C for 10 min. The amplified PCR products were electrophoresed in 1.0 % agarose gel varnished with ethidium bromide and visualized by ultraviolet radiation transilluminator. There were no bands was found for negative control and all the tissue cDNA templates from the treated crabs, a band came at 615 bp for positive control (Fig. 3A). Bands came well for the same templates subjected to -actin PCR (Fig. 3B).For histological analysis, a small portion of branchia and head-soft tissue was taken from all the three experimental groups and was fixed in Davidsons fixative for subsequent histological preparations (Bell and Lightner, 1988) with haematoxylin and eosin according to the standard protocol. The stained sections of gills and head-soft tissue from the control crabs show no histopathological changes (Fig. 4A 4B). Whereas in the positive control cells having hypertrophied nuclei with intranuclear inclusions typical for WSSV infection (Fig. 4C 4D). No signific ant changes were observe in treated group (Fig. 4E 4F) indicates no WSSV infection. In conclusion, a novel compound 3-(1-chloropiperidin-4-yl)-6-fluorobenzisoxazole 2 derivatives showed strong antiviral activity against WSSV in fresh water crabs P. hydrodomous. This works may help to design a novel non-toxic drug to treat WSSV infection.AcknowledgementsReferencesBalasubramanian, G., Sarathi, M., Kumar, S.R., Hameed, A., 2007. Screening the antiviral activity of Indian medicinal plants against white spot syndrome virus in shrimp. Aquaculture 263, 15-19.Bell, T.A., Lightner, D.V., 1988. A enchiridion of normal penaeid shrimp histology. World aquaculture society, Baton Rouge, LA.Gaba, M., Singh, S., Mohan, C., 2014. Benzimidazole An emerging scaffold for analgesic and anti-inflammatory agents. Eur. J. Med. Chem. 76, 494-505.Nambi, K.N., Majeed, S.A., Raj, N.S., Taju, G., Madan, N., Vimal, S., Hameed, A.S., 2012. In vitro white spot syndrome virus (WSSV) replication in explants of th e heart of freshwater crab, Paratelphusa hydrodomous. J. Virol. Methods 183, 186-195.Natividad, K.D.T., Nomura, N., Matsumura, M., 2008. Detection of White spot syndrome virus DNA in pond soil using a 2-step nested PCR. J. Virol. Methods 149, 28-34.Prasad, S.B., Vinaya, K., Kumar, C.A., Swarup, S., Rangappa, K., 2009. price reduction of novel 6-fluoro-3-(4-piperidinyl)-1, 2-benzisoxazole derivatives as antiproliferative agents A structureactivity relationship study. Invest. New Drugs 27, 534-542.Ramalingan, C., Balasubramanian, S., Kabilan, S., Vasudevan, M., 2004. Synthesis and study of antibacterial and antifungal activities of novel 1-2-(benzoxazol-2-yl) ethoxy-2, 6-diarylpiperidin-4-ones. Eur. J. Med. chem. 39, 527-533.Sahul Hameed, A., Yoganandhan, K., Sathish, S., Rasheed, M., Murugan, V., Jayaraman, K., 2001. White spot syndrome virus (WSSV) in two species of freshwater crabs (Paratelphusa hydrodomous and P. pulvinata). Aquaculture 201, 179-186.Sudheer, N., Philip, R., Singh , I.B., 2012. Antiwhite spot syndrome virus activity of Ceriops tagal aqueous extract in giant tiger shrimp Penaeus monodon. Arch Virol. 157, 1665-1675.Wongprasert, K., Rudtanatip, T., Praiboon, J., 2014. Immunostimulatory activity of sulfated galactans isolated from the red seaweed Gracilaria fisheri and development of protection against white spot syndrome virus (WSSV) in shrimp. Fish Shellfish immunol. 36, 52-60.Table 1Primers used for the RT-PCRPrimer nameSequence (5- 3) indurate temperatureProduct sizeVP28-FATG GAT CTT TCT TTC ACVP28-RTTA CTC GGT CTC AGT GC50C615 bp-actin-F-actin-RGTG CCC ATC TAC GAG GGA TAGTG TTG GCG TAC AGG TCC TT55C404 bpFig. 1. champion crystal ORTEP diagram of the 3-(1-chloropiperidin-4-yl)-6-fluoro benzisoxazole 2Fig. 2. Cumulative mortality graph for the experimental groups.Fig. 3. (A) RT-PCR of WSSV envelope protein VP28 in different organs of treated group. Lane 1, 100 bp DNA marker 2, WSSV positive control 3, negative control 4, gill 5, head-soft ti ssue 6, heart 7, muscle tissue. (B) RT-PCR results of the same samples for -actin gene.Fig. 4. Photomicrographs of tissue from crabs of experimental groups 4A gill and 4B head-soft tissue of negative control showing normal cells (Arrow) 4C gill and 4D head-soft tissue of positive control showing hypertrophied nuclei with intranuclear inclusions (Arrow) 4E gill and 4F head-soft tissue of treated group showing uninfected (Arrow). Original magnification grounds X.