Development and characterization of brain cell line from Trachinotus blochii and its application in virological and gene expression studies.

A permanent cell line, SPB (Snubnose pompano brain) was established from Trachinotus blochii by the explant culture method. It has been sub-cultured more than 75 passages and showed optimal growth at 28°C using L-15 medium supplemented with 15% to 20% FBS. The SPB cells were cryopreserved at different passage levels for various applications. SPB cells were composed of fibroblastic and epithelial-like cells. The SPB cells were tested for mycoplasma contamination which was found to be negative. The origin of the SPB cell line from T. blochii was confirmed by amplification of the mitochondrial cytochrome oxidase I (COI) gene. The transfection efficiency of SPB cell line is 15% assessed by expression of green fluorescent protein using pEGFP-N1 plasmid. In addition, two CMV promotor plasmids pFNCPE42-DNA and pcDNAVP28 were transfected to SPB cells and it shows high expression levels of FNCP of fish nodavirus and VP28 protein of white spot syndrome virus by immunostaining. The SPB cells showed susceptibility to SJNNV and the infection was confirmed by RT-PCR, Western blot, ELISA, TCID50 and RT-qPCR. Experimental infection was carried out in T. blochii using SJNNV propagated in SPB cell line and found 100% mortality with clinical signs. The infection was confirmed by RT-PCR. The SPB cell line can be used for propagation of fish viral pathogens and production of the recombinant proteins.

Probing Biothiols Using a Red-Emitting Pyridoxal Derivative by Adopting Copper(II) Displacement Approach and Cell Imaging.

An aggregation-induced emission (AIE) active Schiff base L was obtained by reacting pyridoxal and 2-hydroxy-1-naphthaldehyde with p-phenylenediamine in two simple steps. The colorimetric, UV/VIS and fluorescence studies of L revealed that the yellow emissive L (λem =540 nm, λex =450 nm) in pure DMSO turned to a red-emissive L, when the poor solvent fraction (HEPES buffer, 10 mM, pH 7.4) was increased above 50 % in DMSO. The SEM and DLS results indicated the formation of self-aggregates of L that restricted the intramolecular motion and promoted the excited state intramolecular proton transfer (ESIPT) process. The cations sensing ability of the AIEgen L was explored in HEPES buffer (5 % DMSO, 10 mM, pH 7.4), where Cu2+ selectively quenched the fluorescence at 608 nm due to the chelation-enhanced fluorescence quenching (CHEQ) effect with an estimated sensitivity limit of 0.9 µM. Subsequently, the in situ formed AIEgen L-Cu2+ complex was applied for the cascade detection of glutathione (GSH), cysteine (Cys) and homocysteine (Hcy). The decomplexation of Cu2+ from the AIEgen L-Cu2+ by GSH, Cys and Hcy restored the quenched fluorescence emission of AIEgen L at 608 nm. With this Cu2+ displacement approach, the concentration of Cys, Hcy and GSH can be detected down to 2.8 µM, 3.12 µM and 2.0 µM, respectively. The practical utility of AIEgen L and AIEgen L-Cu2+ was examined by monitoring the selective analytes in real environmental and biological samples, and also applied successfully for the cell imaging applications.

In vitro propagation of infectious myonecrosis virus in C6/36 mosquito cell line.

Infectious myonecrosis (IMN) is an important shrimp viral disease caused by infectious myonecrosis virus (IMNV). Based on previous reports, an attempt was made to propagate IMNV in apparently healthy C6/36 subclone of Aedes albopictus cell line. The confirmatory assays such as RT-PCR, real-time PCR and bioassay revealed that C6/36 cells were found to be susceptible to IMNV and these cells could be used easily for isolation and propagation of IMNV. The results of real-time PCR assay showed that a lower CT value of 22.25 in IMNV-infected cells was obtained on 10 day post-infection (d p.i.), whereas the higher CT value of 35.21 was obtained in IMNV-infected cells on 2 d p.i. There is no significant difference between CT values of IMNV production in vitro using C6/36 cell line and in vivo using shrimp. The IMNV propagated in C6/36 cells is capable of infecting shrimp and caused 100% mortality in shrimp. Clinical signs observed in shrimp injected with IMNV propagated in C6/36 cell line were found to be similar to naturally infected shrimp.

Silencing of prophenoloxidase (proPO) gene in freshwater prawn, Macrobrachium rosenbergii, makes them susceptible to white spot syndrome virus (WSSV).

Prophenoloxidase (proPO) is very important to protect the invertebrates from microbial infections. Our previous studies revealed that proPO was up-regulated in WSSV-injected Macrobrachium rosenbergii and is responsible for protecting M. rosenbergii from WSSV. In order to prove this mechanism, an attempt was made in the present study to silence the proPO gene in freshwater prawn by injection of dsRNA-proPO followed by WSSV challenge. Two partial fragments of proPO with the size of 251 and 331 bp were used to synthesize dsRNA using LITMUS38i vector and E. coli. The bacterially synthesized dsRNA-proPO was used to silence proPO gene to determine its involvement in developing resistance in prawn against WSSV. In proPO gene-silenced prawn, 100% mortality was observed after WSSV challenge whereas no mortality was observed in prawn injected with WSSV alone. The WSSV infection in gene-silenced prawn was confirmed by PCR, and its propagation was quantified by ELISA and real-time PCR at different time intervals. Real-time PCR assay revealed a significant reduction in the expression of proPO gene in WSSV-challenged proPO-silenced prawn when compared to normal prawn. Level of proPO was reduced significantly in the haemolymph of proPO-silenced prawn when compared to prawn injected with PBS.

ICTV Virus Taxonomy Profile: Nodaviridae.

The family Nodaviridae includes two genera, Alphanodavirus and Betanodavirus. The family name derives from the Japanese village of Nodamura where Nodamura virus was first isolated from Culex tritaeniorhynchus mosquitoes. Virions are non-enveloped and spherical in shape with icosahedral symmetry (T=3) and diameters ranging from 25 to 33 nm. The genome consists of two molecules of single-stranded positive-sense RNA: RNA1 and RNA2. The virion capsid consists of 180 protein subunits arranged on a T=3 surface lattice. Alphanodaviruses infect insects, whereas betanodaviruses are pathogens of fish. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the taxonomy of the Nodaviridae, which is available at www.ictv.global/report/nodaviridae.

Global distribution of white spot syndrome virus genotypes determined using a novel genotyping assay.

White spot disease, caused by infection with white spot syndrome virus (WSSV), is a serious panzootic affecting prawn aquaculture. The disease has spread rapidly around the prawn-culturing regions of the world through a number of previously identified mechanisms. The ability to distinguish and trace strains of WSSV is of great benefit to identify, and then limit, the translocation routes of the disease. Here, we describe a novel genotyping method using 34 short tandem repeat regions of the viral genome concurrently. This technique is highly sensitive to strain differences when compared to previous methods. The efficacy of the described method is demonstrated by testing WSSV isolates from around the globe, showing regional genotypic differences. The differences in the genotypes were used to create a global minimum spanning network, and in most cases the observed relationships were substantiated with verification of transboundary movement. This novel panel of STR markers will provide a valuable epidemiological tool for white spot disease. We have applied this to an outbreak of the disease in Queensland, Australia, that occurred in 2016. While the results indicate that the source of this outbreak currently remains cryptic, the analyses have provided valuable insights with which to further study the origins of the strains involved.

Multiple infections caused by white spot syndrome virus and Enterocytozoon hepatopenaei in pond-reared Penaeus vannamei in India and multiplex PCR for their simultaneous detection.

White leg shrimp, Penaeus vannamei, were collected on a monthly basis from grow-out ponds located at Tamil Nadu and Andhra Pradesh states along the east coast of India for screening of viral and other pathogens. Totally 240 shrimp samples randomly collected from 92 farms were screened for white spot syndrome virus (WSSV), infectious hypodermal and haematopoietic necrosis virus (IHHNV), infectious myonecrosis virus (IMNV) and Enterocytozoon hepatopenaei (EHP). The number of shrimp collected from shrimp farms ranged from 6 to 20 based on the body weight of the shrimp. All the shrimp collected from one farm were pooled together for screening for pathogens by PCR assay. Among the samples screened, 28 samples were WSSV-positive, one positive for IHHNV and 30 samples positive for EHP. Among the positive samples, four samples were found to be positive for both WSSV and EHP, which indicated that the shrimp had multiple infections with WSSV and EHP. This is the first report on the occurrence of multiple infections caused by WSSV and EHP. Multiplex PCR (m-PCR) protocol was standardized to detect both pathogens simultaneously in single reaction instead of carrying out separate PCR for both pathogens. Using m-PCR assay, naturally infected shrimp samples collected from field showed two prominent bands of 615 and 510 bp for WSSV and EHP, respectively.

ICTV virus taxonomy profile: Sarthroviridae.

The family Sarthroviridae includes a single genus, Macronovirus, which in turn includes a single species, Macrobrachium satellite virus 1. Members of this species, named extra small virus, are satellite viruses of Macrobrachium rosenbergii nodavirus, an unclassified virus related to members of the family Nodaviridae. Both viruses have isometric, spherical virions, infect giant freshwater prawns and together cause white tail disease, which is responsible for mass mortalities and severe economic losses in hatcheries and farms. Infection is caused by both vertical and horizontal transmission of virus. Aquatic insects act as a carrier to transmit the disease in prawns. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the taxonomy of the Sarthroviridae, which is available at www.ictv.global/report/sarthroviridae.

A new strain of white spot syndrome virus affecting Litopenaeus vannamei in Indian shrimp farms.

White spot syndrome virus (WSSV)-infected shrimp samples collected from grow-out ponds located at Nellore, Andhra Pradesh, India, showed WSSV negative and positive by PCR using primer sets specific to ORF119 and VP28 gene of WSSV, respectively. This indicated the deletion of genetic fragments in the genome of WSSV. The WSSV isolate along with lab strain of WSSV was subjected to next-generation sequencing. The sequence analysis revealed a deletion of 13,170 bp at five positions in the genome of WSSV-NS (new strain) relative to WSSV-TH and WSSV-LS (lab strain). The PCR analysis using the ORF's specific primer sets revealed the complete deletion of 10 ORFs in the genome of WSSV-NS strain. The primer set was designed based on sequence covering ORF161/162/163 to amplify a product of 2,748 bp for WSSV-LS and 402 bp for WSSV-NS. Our surveillance programme carried out since 2002 revealed the replacement of WSSV-LS by WSSV-NS in Indian shrimp culture system.

Lateral flow assay for rapid detection of white spot syndrome virus (WSSV) using a phage-displayed peptide as bio-recognition probe.

White spot disease caused by the white spot syndrome virus (WSSV) has a major socio-economic impact on shrimp farming in India. It has been realized that a field-usable diagnostic capable of rapid detection of WSSV can prevent huge economic losses in disease outbreaks. In this work, we explored the possibility of using a peptide as bio-recognition probe in a field-usable device for the detection of WSSV from infected shrimps and prawns. A commercially available random phage-display library was screened against rVP28 (a major structural protein of WSSV, expressed as a recombinant protein in Escherichia coli). A bacteriophage clone VP28-4L was obtained, and its binding to purified rVP28 protein as well as WSSV from infected shrimp Litopaeneus vannamei tissue was confirmed by ELISA and western blot. The apparent equilibrium dissociation constant (Kd,app) was calculated to be 810 nM. VP28-4L did not show cross-reactivity with any other shrimp viruses. A 12-mer peptide (pep28, with the sequence 'TFQAFDLSPFPS') displayed on the VP28-4L was synthesized, and its diagnostic potential was evaluated in a lateral flow assay (LFA). Visual detection of WSSV could be achieved using biotinylated-pep28 and streptavidin-conjugated gold nanoparticles. In LFA, 12.5 µg/mL of the virus could be detected from L. vannamei gill tissue homogenate within 20 min. Pep28 thus becomes an attractive candidate in bio-recognition of WSSV in field-usable diagnostic platforms benefitting the aquaculture sector.

Biochemical changes and tissue distribution of Enterocytozoon hepatopenaei (EHP) in naturally and experimentally EHP-infected whiteleg shrimp, Litopenaeus vannamei (Boone, 1931), in India.

Stunted growth in pond-reared Litopenaeus vannamei was observed in different farms located in Tamil Nadu and Andhra Pradesh, India. No mortality was associated with stunted growth. PCR assay on these samples revealed the presence of Enterocytozoon hepatopenaei (EHP) in stunted shrimp. Tissue distribution of EHP in naturally and experimentally infected shrimp was studied by PCR and histology. Histological examination revealed the presence of EHP in hepatopancreas and gut, but not in other organs. The PCR assay revealed the presence of EHP in all the organs tested in both naturally and experimentally infected shrimp. Healthy shrimp were challenged with E. hepatopenaei by intramuscular injection and oral route, and no mortality was observed in both routes after 30 days post-challenge. Different developmental stages of the microsporidian parasite were observed in the hepatopancreatic epithelial cells. Biochemical parameters such as total protein, albumin, aspartate transaminase (AST), alanine transaminase (ALT) and alkaline phosphatase were measured in the haemolymph of naturally and experimentally EHP-infected shrimp. All biochemical parameters mentioned were found to be significantly higher in EHP-infected shrimp when compared to normal shrimp. This is the first report relating AST and ALT levels to EHP infection in naturally and experimentally infected shrimp.

Studies on the occurrence of infectious myonecrosis virus in pond-reared Litopenaeus vannamei (Boone, 1931) in India.

Whiteleg shrimp, Litopenaeus vannamei, with clinical sign of muscle opaqueness with reddish colour at the distal abdominal segments were observed in farms located in West Bengal State, India. The mortality of shrimp in all disease outbreak ponds ranged from 20% to 50%, and mortality increased gradually. The RT-PCR assay of these samples using primer sets specific to infectious myonecrosis virus (IMNV) revealed its presence in the disease outbreak ponds. The IMNV infection was reproduced in healthy shrimp by intramuscular injection to satisfy River's postulates. The virus caused mortality in intramuscularly challenged shrimp, but failed to cause mortality by oral route. Tissue distribution of IMNV in infected shrimp by RT-PCR assay revealed the presence of this virus in haemolymph, gill, hepatopancreas and muscle. This study confirms that the disease outbreak which occurred in the shrimp farms located at Purba Medinipur District, West Bengal, India, was due to IMNV.

Immune responses of whiteleg shrimp, Litopenaeus vannamei (Boone, 1931), to bacterially expressed dsRNA specific to VP28 gene of white spot syndrome virus.

In this study, dsRNA specific to VP28 gene of white spot syndrome virus (WSSV) of shrimp was synthesized in Escherichia coli in large scale and studied the immune response of shrimp to dsRNA-VP28. The haematological parameters such as clotting time and total haemocytes counts, and immunological parameters such as prophenoloxidase (proPO), superoxide dismutase (SOD), superoxide anion (SOA) and malondialdehyde content, as well as the mRNA expression of ten immune-related genes were examined to estimate the effect of dsRNA-VP28 on the innate immunity of Litopenaeus vannamei. The activities of proPO, SOA and SOD significantly increased in haemocyte after dsRNA-VP28 treatment, whereas MDA content did not change significantly. Among the ten immune-related genes examined, only the mRNA expression of proPO, cMnSOD, haemocyanin, crustin, BGBP, lipopolysaccharides (LPs), lectin and lysozyme in haemocytes, gill and hepatopancreas of L. vannamei, was significantly upregulated at 12 h after dsRNA-VP28 treatment, while no significant expression changes were observed in Toll receptor and tumour receptor genes. The increase of proPO and SOD activities, and SOA level and mRNA expression level of proPO, cMnSOD, haemocyanin, crustin, BGBP, LPs, lectin and lysozyme after dsRNA-VP28 stimulation indicate that these immune-related genes were involved in dsRNA-VP28-induced innate immunity in shrimp.

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.

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.

In vitro cytotoxic, genotoxic and oxidative stress of cypermethrin on five fish cell lines.

The indiscriminate use of pesticides and herbicides to enhance crop production has aroused great concern, because these products are likely to reach the aquatic environment, thereby posing a health concern for humans and aquatic species. Cypermethrin (CYP), a type II pyrethroid insecticide, is widely used in agriculture and for other purposes. Therefore a study was conducted for the assessment of cytotoxic, genotoxic and oxidative stress of CYP in IEG, CB, ICG, LRG and CSG cell lines at 24h exposure. The cytotoxic effect of CYP in IEG, CB, ICG, LRG and CSG cell lines was assessed using MTT, NR, AB and CB assays. Linear correlations between each EC50 values, of CYP resulting in 50% inhibition of cytotoxicity parameters after 24h exposure to CYP were calculated for IEG, CB, ICG, LRG and CSG cell lines using MTT, NR, AB and CB assays. Statistical analysis revealed good correlation with R(2)=0.90-0.939 for all combinations between endpoints employed. The percentage of DNA damage was assessed by comet assay in IEG, CB, ICG, LRG and CSG cells exposed to CYP. The results of antioxidant parameters obtained show a significant increase in lipid peroxidation (LPO) level and decreased level of GSH, SOD and CAT in IEG, CB, ICG, LRG and CSG cell lines after exposure to increasing CYP in a concentration-dependent manner. This work proves that fish cell lines could be used not only for cytotoxicity and genotoxicity studies but also for studying oxidative stress when exposed to environmental contaminants such as pesticides and other pollutants.

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.

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.

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.

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.