Tissue distribution of hepatopancreatic parvo-like virus of shrimp in freshwater rice-field crab, Paratelphusa hydrodomous (Herbst).

An attempt was made to determine the replication efficiency of hepatopancreatic parvo-like virus (HPV) of shrimp in different organs of freshwater rice-field crab Paratelphusa hydrodomous (Herbst) using bioassay, PCR, RT-PCR, ELISA, Western blot and q-PCR analyses. Another attempt was made to use this crab as an alternative to penaeid shrimp for the large-scale production of HPV. This crab was found to be highly susceptible to HPV by intramuscular injection. The systemic HPV infection was confirmed by PCR and Western blot analyses in freshwater crab. The expression of capsid protein gene in different organs of infected crab was revealed by RT-PCR analysis. Indirect ELISA was used to quantify the capsid protein in different organs of the crab. The copy number of HPV in different organs of the infected crab was quantified by q-PCR. The results revealed a steady decrease in CT values in different organs of the infected crab during the course of infection. The viral inoculum that was prepared from different organs of the infected crab caused significant mortality in post-larvae of tiger prawn, Penaeus monodon (Fabricius). The results revealed that this rice-field crab could be used as an alternative host for HPV replication and also for large-scale production of HPV.

Neutralization of cobra venom by cocktail antiserum against venom proteins of cobra (Naja naja naja).

Naja naja venom was characterized by its immunochemical properties and electrophoretic pattern which revealed eight protein bands (14 kDa, 24 kDa, 29 kDa, 45 kDa, 48 kDa, 65 kDa, 72 kDa and 99 kDa) by SDS-PAGE in reducing condition after staining with Coomassie Brilliant Blue. The results showed that Naja venom presented high lethal activity. Whole venom antiserum or individual venom protein antiserum (14 kDa, 29 kDa, 65 kDa, 72 kDa and 99 kDa) of venom could recognize N. naja venom by Western blotting and ELISA, and N. naja venom presented antibody titer when assayed by ELISA. The neutralization tests showed that the polyvalent antiserum neutralized lethal activities by both in vivo and in vitro studies using mice and Vero cells. The antiserum could neutralize the lethal activities in in-vivo and antivenom administered after injection of cobra venom through intraperitoneal route in mice. The cocktail antiserum also could neutralize the cytotoxic activities in Vero cell line by MTT and Neutral red assays. The results of the present study suggest that cocktail antiserum neutralizes the lethal activities in both in vitro and in vivo models using the antiserum against cobra venom and its individual venom proteins serum produced in rabbits.

Chitosan tripolyphosphate (CS/TPP) nanoparticles: preparation, characterization and application for gene delivery in shrimp.

The present study examines the use of CS/TPP nanoparticles for gene delivery in different tissues of shrimp through oral route. The viral gene of WSSV was used to construct DNA vaccines using pcDNA 3.1, a eukaryotic expression vector and the constructs were named as pVP28. The CS/TPP nanoparticles were synthesized by ionic gelation process and these particles were characterized. The structure and morphology of the nanoparticles were studied by field emission scanning electron microscopy (FE-SEM) and FTIR (Fourier Transform Infrared Spectra). The cytotoxicity of CS/TPP nanoparticles was evaluated by MTT assay using fish cell line. The expression of gene was confirmed by Immuno-dot blot, ELISA and RT-PCR analyses. The results indicate that DNA can be easily delivered into shrimp by feeding with CS/TPP nanoparticles.

Comparison of betanodavirus replication efficiency in ten Indian fish cell lines.

Ten cell lines established from Indian marine, brackishwater and freshwater fish were tested for their susceptibility to fish nodavirus. In addition, the efficiency of betanodavirus replication was tested in these cell lines. Multiple vacuolation, a typical cytopathic effect for virus infection, was observed in infected SISK, SISS, SIGE and ICF cells. Infection of the different fish cell lines was confirmed by RT-PCR, immunodot blot assay and indirect ELISA. The virus concentration in culture supernatant collected from infected sea bass and grouper cell lines increased progressively from 10(3) at day 1 postinfection to 10(8) TCID50 ml(-1) at day 9. The amount of virus in different cell lines was also quantified by real-time PCR. These results indicate the suitability of the SISK, SISS, and SIGE cell lines for fish nodavirus propagation for developing viral diagnostics and vaccines.

In vitro propagation of hepatopancreatic parvo-like virus (HPV) of shrimp in C6/36 (Aedes albopictus) cell line.

Hepatopancreatic parvovirus (HPV) which causes infection in many species of penaeid shrimp is a serious viral pathogen in the young life stages of shrimp. An attempt was made to develop an in vitro system using C6/36 subclone of Aedes albopictus cell line for propagation of HPV. The results revealed that C6/36 cells were susceptible to this virus and the infected cells showed CPE in the form of vacuole formation. The results of PCR, immunocytochemistry and Western blot revealed the HPV-infection in C6/36 cell line. The RT-PCR analysis confirmed the replication of HPV in C6/36 cell line. The HPV load was quantified at different time intervals by ELISA and real time PCR, and the results showed the increase of viral load in C6/36 cell line in time course of infection. HPV propagated in C6/36 cell line was used to infect post-larvae of shrimp and the results showed that the twentieth passage of HPV propagated in C6/36 cell line caused 100% mortality in post-larvae after 6 weeks post infection (d.p.i.). The infected post-larvae showed clinical signs of reduced growth, reduced preening, muscle opacity and atrophy of hepatopancreas. The HPV-infection was confirmed by PCR. The results of the present study showed that C6/36 cell line can be used as an in vitro model for HPV replication instead of whole animal.

Establishment and characterization of permanent cell line from gill tissue of Labeo rohita (Hamilton) and its application in gene expression and toxicology.

Rohu gill cell line (LRG) was established from gill tissue of Indian major carp (Labeo rohita), a freshwater fish cultivated in India. The cell line was maintained in Leibovitz's L-15 supplemented with 10 % foetal bovine serum (FBS). This cell line has been sub-cultured more than 85 passages over a period of 2 years. The LRG cell line consists of both epithelial and fibroblastic-like cells. The cells were able to grow at a wide range of temperatures from 22 to 32 °C, the optimum temperature being 28 °C. The growth rate of gill cells increased as the FBS proportion increased from 2 to 20 % at 28 °C. The plating efficiency was also high (34.37 %). The viability of the LRG cell line was 70-80 % after 6 months of storage in liquid nitrogen. The karyotype analysis revealed a diploid count of 50 chromosomes. The gill cells of rohu were successfully transfected with pEGFP-N1. Amplification of mitochondrial Cox1 gene using primers specific to L. rohita confirmed the origin of this cell line from L. rohita. The cytotoxicity of malathion was assessed in LRG cell line using multiple endpoints such as 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Neutral Red assay, Alamar Blue assay and Coomassie Blue protein assay. Acute toxicity assay on fish was conducted by exposing L. rohita for 96 h to malathion under static conditions. Statistical analysis revealed good correlation with r (2) = 0.946-0.990 for all combinations between endpoints employed. Linear correlations between each in vitro effective concentration 50 and the in vivo lethal concentration 50 data were highly significant.

High efficacy of white spot syndrome virus replication in tissues of freshwater rice-field crab, Paratelphusa hydrodomous (Herbst).

An attempt was made to determine the replication efficiency of white spot syndrome virus (WSSV) of shrimp in different organs of freshwater rice-field crab, Paratelphusa hydrodomous (Herbst), using bioassay, PCR, RT-PCR, ELISA, Western blot and real-time PCR analyses, and also to use this crab instead of penaeid shrimp for the large-scale production of WSSV. This crab was found to be highly susceptible to WSSV by intramuscular injection. PCR and Western blot analyses confirmed the systemic WSSV infection in freshwater crab. The RT-PCR analysis revealed the expression of VP28 gene in different organs of infected crab. The indirect ELISA was used to quantify the VP28 protein in different organs of crab. It was found that there was a high concentration of VP28 protein in gill tissue, muscle, haemolymph and heart tissue. The copy number of WSSV in different organs of infected crab was quantified by real-time PCR, and the results revealed a steady increase in copy number in different organs of infected crab during the course of infection. The viral inoculum prepared from different organs of infected crab caused significant mortality in tiger prawn, Penaeus monodon (Fabricius). The results revealed that this crab can be used as an alternate host for WSSV replication and production.

In vitro white spot syndrome virus (WSSV) replication in explants of the heart of freshwater crab, Paratelphusa hydrodomous.

Explants from different organs of freshwater crab, Paratelphusa hydrodomous were prepared to establish an in vitro system for replication of white spot syndrome virus (WSSV) of shrimp. Heart explants were maintained for 53 days without any morphological changes in EX-CELL™ 405 medium with and without serum whereas the explants of eye muscle, gill, shell membrane and appendage muscle died within 15 days of culture period. The heart explants on different days of culture were exposed to WSSV for 10 days to study the viral replication. The infection of WSSV in explants of the heart was confirmed by PCR, RT-PCR, Western blot, histology, immunohistochemistry, bioassay and transmission electron microscopy. The WSSV was quantified by real-time PCR and indirect ELISA. The WSSV inoculum prepared from the heart explants of crab caused significant mortality in Penaeus monodon in challenge experiments and the results indicate that the WSSV which replicated in the heart explants of freshwater crab maintains its infectivity as in marine shrimp. The results indicate that the heart explants of P. hydrodomous would be a good alternative to whole animals for production of WSSV.