M. Sarathi

Oral delivery of DNA construct using chitosan nanoparticles to protect the shrimp from white spot syndrome virus (WSSV)

The protective efficacy of oral delivery of a DNA construct containing the VP28 gene of WSSV encapsulated in chitosan nanoparticles was investigated in black tiger shrimp (Penaeus monodon). The results showed that significant survival was obtained in WSSV-challenged shrimp at 7, 15 and 30 days post-treatment (relative survival, 85%, 65% and 50%, respectively) whereas 100% mortality was observed in the control shrimp fed with feed containing chitosan/pcDNA 3.1 or chitosan/PBS complex. The ability of the chitosan to form a complex with the pVP28 and to stabilize it from endonuclease degradation was studied by agarose gel electrophoresis. Cytotoxicity of chitosan-encapsulated pVP28 was also evaluated by the MTT assay, which showed 90% viability of SISK cells incubated with the pVP28/chitosan complexes. Transcription analysis of the chitosan-encapsulated pVP28 gene in different tissues of DNA-treated shrimp and SISK cell line was confirmed by an RT-PCR reaction. The present study also measured the changes in the level of important immunological parameters such as prophenoloxidase, superoxide dismutase and superoxide anion in hemolymph of chitosan-encapsulated VP28 DNA-treated and controls shrimp. The study also correlated the changes in the level of immunological parameters with the survival percentage and protective efficacy of oral route of DNA construct against WSSV in shrimp.

Immunological responses of Penaeus monodon to DNA vaccine and its efficacy to protect shrimp against white spot syndrome virus (WSSV).

White spot disease is an important viral disease caused by white spot syndrome virus (WSSV) and is responsible for huge economic losses in the shrimp culture industry worldwide. The VP28 gene encoding the most dominant envelope protein of WSSV was used to construct a DNA vaccine. The VP28 gene was cloned in the eukaryotic expression vector pcDNA3.1 and the construct was named as pVP28. The protective efficiency of pVP28 against WSSV was evaluated in Penaeus monodon by intramuscular challenge. In vitro expression of VP28 gene was confirmed in sea bass kidney cell line (SISK) by fluorescence microscopy before administering to shrimp. The distribution of injected pVP28 in different tissues of shrimp was studied and the results revealed the presence of pVP28 in gill, head soft tissue, abdominal muscle, hemolymph, pleopods, hepatopancreas and gut. RT-PCR and fluorescence microscopy analyses showed the expression of pVP28 in all these tissues examined. The results of vaccination trials showed a significantly higher survival rate in shrimp vaccinated with pVP28 (56.6-90%) when compared to control groups (100% mortality). The immunological parameters analyzed in the vaccinated and control groups revealed that the vaccinated shrimp showed significantly high level of prophenoloxidase and superoxide dismutase (SOD) when compared to the control groups. The high levels of prophenoloxidase and superoxide dismutase (SOD) might be responsible for developing resistance against WSSV in DNA vaccinated shrimp.

Studies on the immunomodulatory effect of extract of Cyanodon dactylon in shrimp, Penaeus monodon, and its efficacy to protect the shrimp from white spot syndrome virus (WSSV).

The present study investigates the protection of shrimp Penaeus monodon against white spot syndrome virus (WSSV) using antiviral plant extract derived from Cyanodon dactylon and the modulation of the shrimp non-specific immunity. To determine the antiviral activity, the shrimp were treated by both in vitro (intramuscular injection) and in vivo (orally with feed) methods at the concentration of 2mg per animal and 2% of the plant extract incorporated with commercially available artificial pellet feed, respectively. The antiviral activity of C. dactylon plant extract was confirmed by PCR, bioassay and Western blot analysis. In the present study, anti-WSSV activity of C. dactylon plant extract by in vivo and in vitro methods showed strong antiviral activity and the immunological parameters such as proPO, O(2)(-), NO, THC and clotting time were all significantly (P<0.05) higher in the WSSV-infected shrimp treated with plant extract when compared to control groups. These results strongly indicate that in vivo and in vitro administration of C. dactylon plant extract enhances immunity of the shrimp. Based on the present data and the advantages of plant extract available at low price, we believe that oral administration of C. dactylon plant extract along with the pellet feed is a potential prophylactic agent against WSSV infection of shrimp.

Silencing VP28 Gene of White Spot Syndrome Virus of Shrimp by Bacterially Expressed dsRNA

An in vivo expression system to produce large amounts of virus-derived dsRNAs in bacteria to provide a practical control of white spot syndrome virus (WSSV) in shrimp was developed. The bacterially synthesized dsRNA specific to VP28 gene of WSSV promoted gene-specific interference with the WSSV infection in shrimp. Virus infectivity was significantly reduced in WSSV-challenged shrimp injected with VP28-dsRNA and 100% survival was recorded. The inhibition of the expression of WSSV VP28 gene in experimentally challenged animals by VP28-dsRNA was confirmed by RT-PCR and Western blot analyses. Furthermore, we have demonstrated the efficacy of bacterially expressed VP28-dsRNA to silence VP28 gene expression in SISK cell line transfected with eukaryotic expression vector (pcDNA3.1) inserted with VP28 gene of WSSV. The expression level of VP28 gene in SISK cells was determined by fluorescent microscopy and ELISA. The results showed that the expression was significantly reduced in cells transfected with VP28dsRNA, whereas the cells transected with pcDNA-VP28 alone showed higher expression. The in vivo production of dsRNA using prokaryotic expression system could be an alternative to in vitro method for large-scale production of dsRNA corresponding to VP28 gene of WSSV for practical application to control the WSSV in shrimp farming.

Clearance of white spot syndrome virus (WSSV) and immunological changes in experimentally WSSV-injected Macrobrachium rosenbergii.

A time course experimental challenge of WSSV was carried out to examine the clearance of WSSV in Macrobrachium rosenbergii and the consequent immunological changes. The experimental animals were injected with WSSV and the samples of gills, pleopods, head soft tissue and hemolymph were collected at different intervals of 1, 3, 5, 10, 25, 50, 75 and 100 days post infection (p.i.). WSSV infection and clearing were confirmed by single step PCR, nested PCR and bioassay. At 3 days p.i., M. rosenbergii became lethargic and stopped feeding in contrast to the control prawns that behaved and fed normally. However, the WSSV-injected prawns suffered no mortality during the experimental period and recovered without any further gross signs of disease or any mortality over a period of 100 days p.i. The single step PCR analysis showed positive at 1, 3 and 5 days p.i. in gills, head soft tissue, pleopods and hemolymph, and all the organs showed negative at 10 days p.i. onwards. The nested PCR results showed that all organs were positive for WSSV from 3 days p.i. and extended up to 25 days p.i. At 50 days p.i, head soft tissue sample alone showed WSSV-positive while all other organs were negative by nested PCR. All the organs at 75 and 100 days p.i. showed nested PCR negative for WSSV as observed in the control prawn. The hemolymph collected from experimentally infected M. rosenbergii at 1, 3 and 5 days p.i. caused 100% mortality at 40 h p.i., 55 h p.i. and 72 h p.i, respectively in Penaeus monodon whereas hemolymph collected at 10, 25, 50, 75 and 100 days p.i. failed to cause mortality in shrimp. The moribund shrimp showed WSSV-positive and surviving shrimp showed negative by PCR. Immunological parameters such as proPO, O(2)(-) and clotting time in WSSV-injected M. rosenbergii were found to be significantly higher than those of the control groups, whereas THC and superoxide dismutase were significantly lower when compared to control groups.

Development and characterization of cell lines derived from rohu, Labeo rohita (Hamilton), and catla, Catla catla (Hamilton)

Two new cell lines, designated RE and CB, were derived from the eye of rohu, Labeo rohita, and the brain of catla, Catla catla, respectively. The cell lines were maintained in Leibovitzs L-15 supplemented with 20% foetal bovine serum. The RE cell line was sub-cultured for more than 70 passages and the CB cell line for more than 35 passages. The RE cells are rounded and consist predominantly of epithelial cells. The CB cell line consists of predominantly fibroblastic-like cells. Both cell lines are able to grow at temperatures between 25 and 32 degrees C with an optimum of 28 degrees C. The growth rate of the cells increased as the foetal bovine serum concentration increased from 2% to 20% at 28 degrees C, with optimum growth at concentrations of 15% or 20% foetal bovine serum. The cells were successfully cryopreserved and revived at different passage levels. The cell lines were not susceptible to four marine fish viruses. Extracellular products from Aeromonas sp. were toxic to the cell lines. When the cells were transfected with plasmid eukaryotic green fluorescent protein (pEGFP [Clontech, Carlsbad, CA, USA]) vector DNA, a significant fluorescent signal was observed suggesting that these cell lines could be a useful tool for transgenic and genetic manipulation studies. Polymerase chain reaction amplification of mitochondrial 12S rRNA from rohu and catla confirmed that the cell lines originated from these fish species. The cell lines were further characterized by immunocytochemistry using confocal laser scanning microscopy.

Natural aquatic insect carriers of Macrobrachium rosenbergii nodavirus (MrNV) and extra small virus (XSV)

Five different species of aquatic insects were collected from nursery ponds containing the freshwater prawn Macrobrachium rosenbergii infected with Macrobrachium rosenbergii nodavirus (MrNV) and extra small virus (XSV). The insects were screened as potential natural carriers of MrNV and XSV. RT-PCR (reverse transcription polymerase chain reaction) analysis gave positive results for MrNV and XSV in Belostoma sp., Aesohna sp., Cybister sp. and Notonecta sp., and negative results for Nepa sp. An Aedes albopictus mosquito cell line (C6/36) was used for infectivity assays, with viral inoculum prepared from the aquatic insects, since C6/36 cells have recently been shown to be susceptible to infection with MrNV and XSV. The C6/36 cells were harvested 4 d post-challenge for examination by electron microscopy. This revealed aggregation of viral particles throughout the cytoplasm for cells challenged with inocula from all the insect species except Nepa sp. Our results indicate that several aquatic insect species may present a risk for MrNV and XSV transmission to M. rosenbergii.

Efficacy of bacterially expressed dsRNA specific to different structural genes of white spot syndrome virus (WSSV) in protection of shrimp from WSSV infection

White spot syndrome virus (WSSV) is responsible for severe economic losses in shrimp culture worldwide. Any practical method to eradicate or inactivate WSSV in culture systems would have enormous practical benefits for shrimp farmers and hatchery operators. Various studies have been carried out on control of WSSV, including neutralization using antiserum (Van Hulten, Witteveldt, Snippe & Vlak 2001), recombinant subunit vaccines (Namikoshi, Wu, Yamashita, Nishizawa, Nishioka, Arimoto & Muroga 2004; Jha, Xu, Shen, Bai, Sun & Wei 2006), DNA vaccines (Rajesh Kumar, Ishaq Ahmed, Sarathi, Nazeer Basha & Sahul Hameed 2008) and antiviral plant extract (Balasubramanian, Sarathi, Rajesh Kumar & Sahul Hameed 2007). However, there is no efficient strategy to control the spread of this very serious disease. RNA interference (RNAi) has recently emerged as a powerful tool for specific gene silencing in antiviral therapy. The efficiency of RNAi in shrimp was demonstrated by in vivo injection of dsRNA corresponding to the gonad inhibiting hormone (GIH) in the sand shrimp, Metapenaeus ensis, which lowered the expression of GIH (Guan, Mak & Chan 2004). Several recent articles have reported that siRNAs or dsRNA synthesized in vitro are potential therapeutic agents for treating white spot syndrome (Robalino, Bartlett, Shepard, Prior, Jaramillo, Scura, Chapman, Gross, Browdy & Warr 2005; Westenberg, Heinhuis, Zuidema & Vlak 2005; Kim, Kosuke, Nam, Kim & Kim 2007; Xu, Han & Zhang 2007). The in vitro production of dsRNA may not be useful for field applications; however, the present study describes the production of bacterially expressed virus specific dsRNAs, which opens the possibility of developing dsRNA based antiviral therapeutics for commercial use. This study reports the use of an RNase III-deficient strain of E. coli HT115(DE3) (Timmons, Court & Fire 2001) to produce bacterially derived dsRNAs specific to WSSV structural genes and their interference with WSSV infection in shrimp. Our previous studies have shown that intramuscular administration of bacterially expressed VP28dsRNA results in the protection of Penaeus monodon against WSSV (Sarathi, Simon, Ishaq Ahmed, Rajesh Kumar & Sahul Hameed 2008a). The present study determined the protective effect of bacterially expressed dsRNA specific to different structural genes of WSSV other than VP28. We targeted the four Journal of Fish Diseases 2010, 33, 603–607 doi:10.1111/j.1365-2761.2010.01157.x

Experimental exposure of Artemia to Hepatopancreatic parvo-like Virus and Subsequent transmission to post-larvae of Penaeus monodon

The different life stages of Artemia franciscana were experimentally exposed to Hepatopancreatic parvo-like virus (HPV), in order to evaluate the possibility of Artemia acting as reservoir or carrier for HPV. All the five developmental stages of Artemia were challenged with HPV both by immersion and oral infection routes. The viral infectivity to Artemia was studied by PCR but not much difference in mortality between control and challenge groups were observed. To confirm the vector status of Artemia for HPV, the HPV exposed Artemia were fed to postlarval forms of Penaeus monodon. Post-larvae of P. monodon were fed with HPV exposed Artemia and could get infected upon feeding on them. Mortality was observed in the post-larvae, which were fed with HPV exposed Artemia, and whereas no mortality was observed in post-larvae fed with Artemia not exposed to HPV and these post-larvae were PCR negative for HPV, as well. Results of this experiment suggest that Artemia might be a possible horizontal transmission pathway for HPV. Further research however is required with histology, immunohistochemistry and transmission electron microcopy to determine whether the Artemia are actually infected with this virus or whether they are simply mechanical carriers. This will enable us to understand better whether Artemia is a carrier of this virus and if so the mechanism involved.

Artemia is not a vector for monodon baculovirus (MBV) transmission to Penaeus monodon.

Monodon baculovirus (MBV) of penaeid shrimp has been associated with high mortalities in hatchery-reared larval, post-larval and early juvenile stages of Penaeus monodon and is thought to have originated from Taiwan (Lightner, Redman & Bell 1983). MBV is pathogenic to several species of shrimp such as P. monodon Fabricius, P. penicillatus Alcock and Metapenaeus ensis De Haan (Chen, Chang, Kou & Lightner 1989). The principal clinical sign of MBV is the presence of single or multiple, generally spherical occlusion bodies in the hepatopancreas and midgut epithelial cells. In India, the prevalence of MBV and mortalities in hatcheries and farms associated with the virus has been reported (Ramasamy, Brennan & Jayakumar 1995; Karunasagar, Otta & Karunasagar 1998). The virus can be transmitted via horizontal transmission directly through the water column (Paynter, Vickers & Lester 1992), or through cannibalism and it is believed, but not proven, that transmission can also be vertical from brood stock to offspring. Infection can result in substantial economic loss due to poor growth and reduced survival of post-larvae of up to 90% at high densities. Furthermore, stress and overcrowding are predisposing factors that may increase the severity of MBV infection (Lightner et al. 1983). The MBV particles are rod shaped and replicate in the nucleus. They appear either free or within proteinaceous polyhedral occlusion bodies, and contain DNA. However, little is known about the mechanism of MBV production in the host cell (Lu, Tang, Kou & Chen 1995). Diagnosis of MBV depends upon the demonstration of MBV occlusion bodies in hypertrophied nuclei of the anterior midgut epithelium and hepatopancreatic cells by direct light microscopy or standard HE Muroga, Higashi & Keitiku 1989; Nicolas, Robic & Ansquer 1989) and viruses, such as infectious pancreatic necrosis virus (IPNV), Macrobrachium rosenbergii nodavirus and fish nodavirus (Mortensen, Evensen, Rodseth & Hjeltnes 1993; Skliris & Richards 1998; Sudhakaran, YogJournal of Fish Diseases 2008, 31, 631–636 doi:10.1111/j.1365-2761.2008.00926.x