White Spot Syndrome Virus (WSSV) Experiment

White Spot Syndrome Virus (WSSV) Experiment Abstract White spot syndrome virus (WSSV) continues to cause huge economic losses in the aquaculture farms due to rapid spread and broad host range. In this study, we synthesized a novel synthetic compound 3-(1-chloropiperidin-4-yl)-6-fluoro benzisoxazole 2 and were screened for antiviral activity against WSSV using fresh water crabs Paratelphusa hydrodomous (Herbst). In vivo bio-assay was carried out to determine the antiviral activity. Reverse transcriptase polymerase chain reaction (RT-PCR) and Histopathology were used for the analysis of bio-assay. Overall result shows that the novel compound has strong antiviral potency 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 100 % mortality within 3-10 days of infection in farmed shrimp (Sudheer et al., 2012). Several antiviral and immunostimulatory compounds are 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 resistance 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 group of heterocyclic compounds in the field of medicinal chemistry. These compounds have significant biological and pharmacological properties like anti-inflammatory, antifungal, antimicrobial, and anticancer activities (Gaba et al., 2014; Ramalingan et al., 2004) (Ramalingan, 2004 #21;Gaba, 2014 #22). Many fluorinated, benzisoxazole derivatives are currently 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. Thus 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 synthesis of novel 3-(1-chloropiperidin-4-yl)-6-fluorobenzisoxazole was carried out under mild reaction conditions using 1.2 equivalents of calcium 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 stable at ambient conditions and stereo chemistry was established the single crystal XRD technique. The materials were purchased from Sigma-Aldrich, Merck and were used without any additional purification. All reactions were monitored by thin layer chromatography (TLC). Melting points were recorded on an Elchem digital melting point apparatus in open capillaries and are uncorrected. The 1H NMR 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 p pm. The coupling constants J are in Hertz. Mass 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. Reaction was monitored by TLC. After the reaction completion, reaction mass was filtered and salts was washed with acetonitrile. Solvent was dried under reduced pressure. Crude solid was purified by column chromatography to give 3-(1-chloropiperidin-4-yl)-6-fluorobenzisoxazole 2 in 96 % (5.54g, 21.8mmol) as pale yellow color solid. The structure of the N-chloro benzisoxazole was conà ¯Ã‚ ¬Ã‚ rmed from their spectral data from NMR, ES Mass and single crystal XRD. Mp: 81-83oC; 1H 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, 100 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); Single crystal crystal data 3-(1-chloropiperidin-4-yl)-6-fluoro benzisoxazole 2 Mol. Formula:C12H12ClFN2O; CCDC reference number is 878706; Intensity data were collected on an APEX CCD diffract meter equipped with Mo–Ka (l = 0.7107 A °) radiation; Cell length a =5.8979(4); Cell length b=10.4965(7); Cell length c=19.1492(12); Cell Angle ÃŽ ±=90.0; Cell Angle ÃŽ ²=91.783; Cell Angle ÃŽ ³=90.0; Cell Volume=1184.90(11); The crystallographic data for N-chloro benzisoxazole have been deposited with the Cambridge Crystallographic Data Centre. Copies of this information may be obtained free 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 body 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 determination 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 100  µl of a mixture of viral suspension and NTE buffer which served as positive control. In Group II crabs were injected with NTE buffer alone served as negative control. In Group III crabs were injected 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 experimental groups intramuscularly. The experimental animals were examined twice per day for gross 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 clotting time). In positive control, there were significant reduction in total hemocyte counts as well as the hemolmph was failed to clot. No significant hematological changes were observed in between the negative and treated groups. For RT-PCR analysis; Gill, head-soft tissue, heart and muscle tissue were excised from each crabs of the experimental group and pooled together for extraction of total RNA using Trizol (Invitrogen, USA) according to the manufacturers instructions. cDNA was synthesized from 1.0ÃŽ ¼g of the total RNA using a One-step Reverse Transcriptase (Invitrogen, USA) as per the kit instructions and used as template for gene expression analysis of WSSV specific primer VP28. ÃŽ ²-actin served as an internal control for RNA quality and amplification efficiency. The sequences of primers used in this present study were given in Table 1. The cycling conditions are initial denaturation at 95 °C for 5 min followed by 35 cycles of denaturation at 95 °C for 30 sec, annealing at 50 °C for 30 sec and extension at 72 °C for 30 sec with a final extension at 72 °C for 10 min. The amplified PCR products were electrophoresed in 1.0 % agarose gel stained with ethidium bromide and visualized by ultraviole t 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 ÃŽ ²-actin PCR (Fig. 3B). For histological analysis, a small portion of gill and head-soft tissue was taken from all the three experimental groups and was fixed in Davidson’s 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 significant changes were observed 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. Acknowledgements References Balasubramanian, 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 handbook 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 the 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. Synthesis of novel 6-fluoro-3-(4-piperidinyl)-1, 2-benzisoxazole derivatives as antiproliferative agents: A structure–activity 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. Anti–white 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 resistance against white spot syndrome virus (WSSV) in shrimp. Fish Shellfish immunol. 36, 52-60. Table 1 Primers used for the RT-PCR Primer name Sequence (5’- 3’) Annealing temperature Product size VP28-F ATG GAT CTT TCT TTC AC VP28-R TTA CTC GGT CTC AGT GC 50à ¯Ã¢â‚¬Å¡Ã‚ °C 615 bp ÃŽ ²-actin-F ÃŽ ²-actin-R GTG CCC ATC TAC GAG GGA TA GTG TTG GCG TAC AGG TCC TT 55à ¯Ã¢â‚¬Å¡Ã‚ °C 404 bp Fig. 1. Single crystal ORTEP diagram of the 3-(1-chloropiperidin-4-yl)-6-fluoro benzisoxazole 2 Fig. 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 tissue; 6, heart; 7, muscle tissue. (B) RT-PCR results of the same samples for ÃŽ ²-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: 1000 X.

Judging on Apperance

Justin Tipton What is the first thing we do when meeting a first. We judge! Judging someone based on their appearance is an inherent human instinct. Many times when people judge a person, people end up making wrong decisions. We create mental images in our minds that categories people based upon their appearance. When you see a man in a suit, tie, and a briefcase you figure is a lawyer, professor, or a businessman. In reality the guy that you see could have been out for a special event and could be no more than a car mechanic. We base our opinions on perceptions on people based upon what we can biologically see with our eyes.In her article how the eye works Ker Than explain how we use our eyes to visually see things. â€Å"Vision begin when lights rays are reflected off an object and enter the eyes through the cornea, The transparent outer covering of the eyes. The cornea refracts the rays that pass through a round hole, called the pupil. The Irish is the colored portion of the eye that surrounds the pupil, and then become larger or smaller to regulate the amount of light passing through. The rays then travel through the lenses to the retina at the back of the eye. The retina conducts light into electrical impulses.The optic nerve sends impulses to the brain where a visual image or perception is produced. † We use our eye to visually see people, and to make an array of judgment about that person based on what we see. While it’s true we can come times tell certain thing about people by looking at their external appearance, it is not the best of measurements. I believe people should not judge people by their external appearance, and should at least speak with a person before making a judgment. You can’t judge a person character base on appearance and that why many of us make unfair and bias assumption of others.People sometimes change their appearance to fit different situations. According to a Washington post article, Joshua bell one of the world greatest violinists set out to do an experiment to test people’s perception on appearance. By most, he was nondescript: a youngish white man in jeans, a long sleeved t-shirt and Washington National baseball cap. He positioned himself against a wall beside a trash basket, from a case; he moved his violin and began to play. It was a Friday and the middle of morning rush hour. The violinist played for nearly an hour, and performed six classical pieces.Nearly 1,100 people passed by, many of them on their way to work. No one knew it, but the fiddler standing against a bare wall outside was one of the finest classical musicians in the world, playing some of the most elegant music ever written on one of the most valuable violins ever made. In the hour that Joshua Bell played, seven people stooped what they were doing to hang around and take in the performance, at least for minute. Twenty-seven gave money, for a total of thirty-two dollars and seventeen cents. That means over 1 ,070 people passed on by, with few even turning to look.Three days before conducting this experiment on a street corner in Washington, Joshua bell filled the house at Boston’s stately symphony Hall, where the average seat went for one-hundred dollars. the appearance of a guy in plain clothes playing a violin at the street corner, made many people ignore his sound before ever even taking the time to listen. Many times people judge what they see, and ignore the beauty that right in front of their face. To begin with, someone external appearance is not mirror of one’s inner self. I can relate to from examples in my own life.Being a young African-American male, growing up near Memphis one of Americas most dangerous cities. I came from a poorer family, I been working full time since attending college. In the day time I was a college student, but at work I was a normal sweaty and dirty factory worker. When people saw me at work, they assumed I was uneducated and not very int elligent. In reality, I was a good academic student, has a good reputation among my fellow students and professors. My co-workers couldn’t see the college side of me, and many of my fellow students couldn’t see working in a factory which was known for harsh working conditions.Since leaving the factory, I know have a security job just outside of downtown Nashville. TRICOR is the name of the corporation is profits in the million, by utilizing inmates for production and paying their cheap labor wages. This cooperation is ran by your typical CEO, a short old white man, that dresses in suits and ties and owns many expenses vehicles. For many months while working there, he passed by me every day and never once he even acknowledging my presence. One day I was reading a book about written from a famous entrepreneur, and the boom caught the eye of the CEO.He walked up to me and asked why I reading a book like that, I told I was a college student that was a business major, and t hat it has beneficial information in this nook, and that I read many book from this author. He then brought to my attention that he has read the same book and he too was a fan of the author books. After conversation for a few minutes he then brought to my attention that he too grew up from a poor family, and that he also work at a factory and once at a security job while too attending college. I am a business management major and he too has a degree in business management .Ever since that day of our conversation he speak to me daily, and we hold brief but interesting conversation often. Prior to that conversation we had never spoken to another, once we looked past the external appearance, we realized we two men who shared many things in common. You may not realize it, but perception based on appearance happen all the time. Studies show that fifty-five percent of opinion of a person is based upon physical appearance, and that it takes around fifteen second for a person to realize if he or she like your or not, or want to do business with you.Do honestly we feel like we get accurate of someone inner self with over half of what we perceive come from appearance, and while only judging them for one-fourth of a minute? Many people believe that judging people on the appearance is beneficial and effective. Mangers often use appearance as a asset while evaluating a candidate for a job. You could have two good candidates with impressive resume, but choosing a candidate based on race, or which dressed too me is ineffective. In reality just because one candidate is dressed better, doesn’t make him any smarter or better qualified than the other.When it comes to judging a lover or friend, external appearance shouldn’t be a factor on your judgment about that person. Much time we see that a man or women date someone because she is beautiful and he is handsome. In high school and college you see on many occasions when someone hangs around a certain person because it will increase his/her popularity. In a poem I once read it state â€Å"we see best when we look for the less†. In just a few words, that poem speaks a lot, we see the best when look past the artificial obvious, and see the beauty that deep within a person.Beauty and the Beast just a fairy tale and a kid’s story but moral of this tale has such a deeper meaning. The story about a beautiful women falling in love with an ugly beast, passed artificial obvious and see the beauty that lied deep within him. Making a judgment on some based only external appearance is an unfair and accurate judgment of someone. It is not accurate to judge someone based on we biologically see wit our eyes. A appearance can adjust and change their appearance to fit many different situation where there true selves do not show, but a person cannot change his/her character.For these reason, it is important to get to know someone before making a judgment. â€Å"The best and most beautiful things in the world cannot be seen or touched†. â€Å"But must be felt by the heart†. The best and most beautiful things in people cannot be seen by the eyes. Judging someone on external appearance is an inherent and instinct that happen almost instantaneously. We as people have the ability to control our judgment, we have the ability to use our minds to see more within people than what we see with our eyes.Stero-typing is something we do without even noticing, but placing people into different categories based on external appearance limits our ability to meet new people. Sometimes the people that we expect knot to have anything in common with, could be a totally false assumption. If we allow ourselves to judge and meet people of different culture, many would be surprised to know they share many things in common with people they would have never suspected. As long as people allow ourselves to be blinded by external appearance then we will fully see the inner beauty within so m any people.

Needle Cast Tree Disease Prevention and Control

Needle cast is a broad group of fungal diseases that cause conifers to shed needles. The symptoms of needle cast first appear on needles as light green to yellow spots, which eventually turn red or brown. Growth of the fungal pathogen from the spots on the needle will cause the death of the entire needle. This shedding of needles can be more serious to conifers than losing leaves is to deciduous hardwoods. There are over 40 kinds of needle casts in North America. Recognition Infected needles usually turn red to brown from their tips beginning in winter or early spring. By mid to late spring the death of infected needles is well advanced giving diseased trees a red to brown fire-scorched appearance. Tiny black fruiting bodies (spore producing structures) form on the surface of the needles before or after the infected needles are shed. Prevention Avoid planting trees on sites not suited for a particular species. Needle cast seems to thrive when conifers are under stressful conditions including drought. Young seedlings and saplings are susceptible, as well as pure and crowded stands. Keeping your tree healthy can minimize the harmful effects of this disease.   Control Control is unnecessary in most non-commercial situations. However, Christmas tree growers must take some action against the disease. If control is desired for cosmetic reasons, protection of newly emerging needles through June with regular applications of an appropriate fungicide may be helpful.

Pediatric Cancer Is A Scary Concept - 1874 Words

Pediatric Cancer is a scary concept. We think as a society that children are innocent and should not have to go through such a deadly and scary adult disease. Unfortunately that is not true. Cancer has no limits or prejudices on what the age, sex, or race of its victim will be. With this paper I want to discuss multiple topics about pediatric cancer. First the statistics, then the treatment options, finally the treatment/prognosis for the family as a whole and not just the patient. There are different treatments that should occur when dealing with the family members of a patient that has cancer. A social worker that works in pediatric oncology truly wears many hats. Statistics In 2014 it was estimated that approximately 16000 children were diagnosed with cancer and approximately 2000 will die from the disease.(nih) However in 2010 it was estimated that there are almost 400,000 survivors that were diagnosed before the age of 19. That is amazing news. Almost half a century ago the outlook was not so bright almost 50% of children diagnosed with cancer would die within 5 years now there is an 80% survival rate. There are different rates for different types of cancer. The most common type of pediatric cancer that is diagnosed involves brain tumors and blood cancers. The mortality rates have greatly improved for leukemia going from 10% to 90% survival rates since the early 1970’s. On the flip side there are still some forms of cancer such as diffuse pontine glioma (brainShow MoreRelatedThe Definition Of Nursing And Share My Thoughts On What Makes The Ideal Nurse Essay1505 Words   |  7 Pagesmy way of giving back and helping those in my community. I t ruly enjoy working with children and believe I can make a difference in each patient’s life. I ultimately decided to join the profession of nursing after my experience with cancer. Battling cancer was scary, but my nurses and physicians truly fought to keep me alive. During this experience I felt the nurses saw me as a human being and not just â€Å"another patient.† My treatment focused on the mind, body, and spirit aspect and I do believeRead MoreA Little More About Gender And Sex1014 Words   |  5 PagesHunter-Angel Gregory Mrs. Williams ELA 3/4th Hour 7 April 2017 A LITTLE MORE ABOUT GENDER SEX One thing people have thought until recently is that the concepts of gender and sex are the same thing. This is now something that is changing and proving to be false. That being said, they can be troublesome to learn. Acceptance is another tough thing people now deal with since having these thoughts shared with them. Once upon a time the earth was thought to have beenRead MoreOrganizational Leadership and Interprofessional Team Development10384 Words   |  42 Pagesit comes to your health, you can feel confident knowing that the new MCHS is your one source for patient and family centered care as the hospital system has developed a full service Integrated delivery system, services offered are listed below: ï‚ § Cancer ï‚ § Cardiology ï‚ § Cauterization Lab ï‚ § Chest Pain Center ï‚ § Cardiac Rehab ï‚ § Electrophysiology ï‚ § Cardiac Surgery ï‚ § Community Outreach ï‚ § A Tu Salud (To Your Health) ï‚ § Permian BasinRead MoreToo Far Ahead of the It6117 Words   |  25 Pagespoint. Eventually, he always fell back on his clinical experience. â€Å"You can standardize the testing of ball bearings for manufacturing defects,† he said. â€Å"But as far as I know, you can’t—at least not yet—standardize the protocol for treating colon cancer.† As a physician, Max believed that the last word in all matters of patient care should rest with the doctor and the patient. But as a CEO he believed in best practices. So his compromise position was to favor selective (Max called it â€Å"surgical†)