SSN-1 Cell Line Research Articles

Humoral immune response in Asian seabass vaccinated with inactivated and recombinant viral nervous necrosis vaccine

Viral nervous necrosis is a serious viral disease of marine and brackishwater finfish hatcheries. The red-spotted grouper nervous necrosis virus (RGNNV) is the most prevalent genotype among fishes in tropical and temperate regions. RGNNV was propagated in SSN-1 cell line and inactivated using BEI. The capsid protein of RGNNV was cloned and expressed in Escherichia coli and purified. Inactivated and recombinant VNN vaccines were produced by emulsifying the antigen with commercial adjuvant and administered to Asian seabass, Lates calcarifer, fingerlings and broodstock intraperitoneally. The immune response was assessed at regular intervals by indirect ELISA. The maternal antibodies in one-day post-hatch larvae obtained from the vaccinated and control broodstock were assessed by indirect ELISA. The larvae were subjected to challenge with virulent RGNNV and the relative per cent survival (RPS) was estimated. The efficacy of the vaccine was also assessed under field conditions. The recombinant vaccine produced significantly higher specific antibody levels than the inactivated vaccine in fingerlings and broodstock and the immune response was dose-dependent. Recombinant vaccine at 50 and 100 μg/fish produced significantly higher antibody levels and immune response could be observed as early as one-week post vaccination. Vaccinated broodstock produced larvae with maternal antibodies and had higher RPS when challenged at 18 days post-hatch. The vaccine produced a significant immune response even under field conditions. The vaccine can be used to vaccinate broodstock to reduce loss due to VNN in early larval stages.

Characterization of red-spotted grouper nervous necrosis virus isolated from ovarian fluids of asymptomatic wild Asian seabass, Lates calcarifer

Viral nervous necrosis (VNN) caused by nervous necrosis virus is a serious disease affecting a wide range of marine, brackishwater and freshwater fishes. The disease causes acute mortalities in larval and early juvenile stages while adult fishes are asymptomatic and remain as carriers of the virus. In the present study, nervous necrosis virus was isolated from ovarian fluids of asymptomatic wild-collected Asian seabass, Lates calcarifer in SSN-1 cell line. The complete genome of the virus was amplified by PCR from plaque purified virus and sequenced. Bioinformatic analysis of the sequence revealed that the isolate belonged to red-spotted grouper nervous necrosis virus. The virus purified from infected SSN-1 cell culture supernatants by cesium chloride gradient ultracentrifugation resulted in two bands in the gradient. Both the bands on SDS-PAGE analysis and western blot revealed a single 40 kDa capsid protein. The virus replication was optimum at 26–28 °C in vitro and the virus can be completely inactivated using 3 mM binary ethylenimine in 32 h at 25 °C. The isolated virus was infectious and caused 100% mortality in Asian seabass larvae. The inactivated virus when injected to Asian seabass fingerlings produced neutralizing antibody titre ranging from 1:320 to 1: 1280. The isolate is a potential candidate for production of an inactivated vaccine against VNN.

Betanodavirus infection in Kuhlia rupestris and Ambassis marianus and isolation of betanodavirus from infected pond water.

This is the first report of betanodavirus infection in 2 species of finfish, Kuhlia rupestris (jungle perch) and Ambassis marianus (estuary perchlet). This report also describes isolation of betanodavirus from infected pond water using the SSN-1 cell line. Histopathology of K. rupestris larvae revealed vacuolation in the eye and brain, which was confirmed using betanodavirus-specific immunohistochemistry. The eye and brain from A. marianus and betanodavirus isolated from pond water were confirmed using real-time PCR and Sanger sequencing. High throughput sequencing was used to obtain betanodavirus sequences from paraffin blocks containing infected K. rupestris. The phylogenetic analysis of betanodavirus RNA1 and RNA2 sequences from all 3 sources were associated with the red-spotted grouper nervous necrosis virus (RGNNV) genotype. The RNA1 nucleotide sequence from jungle perch showed 100% identity with the betanodavirus water isolate and 99.37% identity with A. marianus. Furthermore, we have demonstrated the usefulness of combining recovery of viable virus from environmental samples through fish cell line culture with PCR testing as a means of validating the efficacy of chlorination to eradicate betanodavirus from the pond environment.

Bicistronic DNA vaccine against Edwardsiella tarda infection in Labeo rohita: Construction and comparative evaluation of its protective efficacy against monocistronic DNA vaccine

DNA-based vaccination or genetic immunization is one of the most promising, effective and prophylactic measures to control aquatic animal diseases. This immunization strategy involves administration of eukaryotic antigen expression vectors (DNA vaccine) into the host that encode for an antigen under the control of a eukaryotic promoter which resulted into elicitation of strong cellular and humoral immune responses. In the present study, a bicistronic DNA vaccine (designated as pGPD+IFN) was constructed which contains an additional immune adjuvant gene (Interferon gamma gene of Labeo rohita) along with a regular antigenic gene (glyceraldehyde-3-phosphate dehydrogenase gene of Edwardsiella tarda) with the purpose to maximize the protective efficacy of the vaccine against E. tarda infection. After construction of the vaccine a pilot study was orchestrated in vitro to ascertain the positive co-expression of the dual genes in vaccine-transfected SSN-1 cell line. Successful co-expression of GAPDH and IFN-γ genes in the transfected cell were confirmed by Western blot and RT-PCR respectively. Further, an in vivo vaccine trial was conducted in which rohu (L. rohita) fingerlings were intramuscularly (I/M) injected (initial and booster immunised) with two DNA constructs one group with pGPD+IFN and the other with pGPD (containing GAPDH gene only) and challenged with E. tarda (1×105CFU/fish) at 35day post-initial vaccination. The protective immune responses were determined in terms of relative percentage survival (RPS), specific antibody production, non-specific immune response and expression kinetics of immune-related iNOS gene. Evaluation of RPS analysis revealed that pGPD+IFN group recorded highest RPS of 63.16% while the pGPD vaccinated group showed 47.37% when compared with 63.33% cumulative mortality of control group. The results regarding respiratory burst activity, myeloperoxidase activity as well as antibody titre also showed pGPD+IFN group with highest activities at all the time points. Furthermore, the current study displayed pGPD+IFN group having significant (p<0.05) upregulation of iNOS gene transcript at 24–48h post-immunization (both initial and booster dose) as well as after challenge. Thus, from this study, we can conclude that the bicistronic vaccine can be an effective immunization strategy in orchestrating a coordinative immune response against E. tarda in L. rohita.

Silencing and augmentation of IAG hormone transcripts in adult Macrobrachiumrosenbergii males affects morphotype transformation.

Morphotypic differentiation is the external manifestation of dominance hierarchy in Macrobrachium rosenbergii The intermediate morphotype orange claw (OC) male exhibits the highest growth rate and is subordinate in hierarchy to blue claw (BC) male while dominant on small male (SM). The present study was undertaken to examine the specific role of insulin-like androgenic gland (iag) hormone in morphotype differentiation of M. rosenbergii To achieve this, RNAi mediated knockdown as well as augmentation of iag transcripts were effected in ∼60 g OC males using plasmid-based constructs pcD-IAG-lh and pcD-IAGorf, respectively. The treatments were administered to animals maintained in isolation as well as in community. The knockdown plasmid construct that expresses iag-specific long hairpin RNA caused 16-fold reduction of iag transcripts in the SSN1 cell line in vitro When injected into OC males living in a community, 2.3-fold iag knockdown was recorded, while in isolated OC males it was 4.2-fold initially, but returned to normal subsequently. Compared with the respective controls, OC to BC transformations in the iag silenced animals were significantly lower in the community-reared group, while no difference was observed in the isolated animals. It is reported here for the first time that iag augmentation in OC males resulted in significantly higher OC to BC transformations, when animals were reared in community. This plasmid-based IAG knockdown approach could be developed into a low stress, feed or immersion treatment for controlling heterogeneous individual growth of M. rosenbergii males in aquaculture.

Autophagy induced by snakehead fish vesiculovirus inhibited its replication in SSN-1 cell line.

Autophagy plays an important role in host protection against pathogen infection through activating innate and adaptive immunity. In the present study, we observed that the infection of snakehead fish vesiculovirus (SHVV) could induce apparent autophagy in striped snakehead fish cell line (SSN-1), including clear double-membrane vesicles, fluorescent punctate pattern of microtubule-associated protein 1 light chain 3B (SSN-LC3B) and the conversion of SSN-LC3B-Ⅰ to SSN-LC3B-Ⅱ. Furthermore, we verified that autophagy inhibited the replication of SHVV by assessing mRNA and protein level of nucleoprotein as well as virus titer in the supernatant. These results will shed a new light on the prevention of the infection of SHVV.

Molecular analysis of RNA1 and RNA2 sequences from a betanodavirus isolated from giant grouper (Epinephelus lanceolatus) in Australia

Betanodavirus infections have a significant impact through direct losses and trade restrictions for aquaculture sectors in Australia. The giant grouper, Epinephelus lanceolatus, is a high-value, fast-growing species with significant aquaculture potential. With subacute to chronic mortalities reported from a commercial aquaculture facility in northern Queensland, the viral nervous necrosis in the affected fish was confirmed using a RT-qPCR followed by virus isolation using the SSN-1 cell line. The RNA1 and RNA2 segments were sequenced and nucleotide sequences were compared with betanodavirus sequences from GenBank. Phylogenetic analysis revealed that both these sequences clustered with sequences representing red spotted grouper nervous necrosis virus genotype and showed high sequence identity to virus sequences affecting other grouper species. This is the first report confirming infection by betanodavirus in E. lanceolatus from Australia with successful isolation of the virus in a cell culture system, and analysis of nearly full length RNA1 and RNA2 sequences.

Isolation and identification of Betanodavirus causing mass mortalities in golden grey mullet (Liza auratus) in the Caspian Sea

Outbreaks causing mass mortalities have been observed in the golden grey mullet (Liza auratus) population in southern coastal area of the Caspian Sea since 2002. The clinical signs include neurological abnormalities, such as erratic swimming behavior, spiral swimming, belly-up at rest and over inflation of the swim bladder. Three hundred and twelve samples of moribund fish with sizes that ranged from 15-26 cm showing abdominal enlargement were collected from 2006-2008. No pathogenic bacteria and parasite were found. The observed histopathological changes in 29 fish were necrosis and vacuolation of the brain, optic nerve and retina. Cytopathological effects of nodavirus characterized by vacuolation were observed in the SSN-1 cell line at 25?C, 5 d after inoculation with the filtered supernatant of the brain and eye of four affected fish. The recovered virus from cell line exhibited 104 TCID50 per mL when titrated. Indirect immunoflorescent antibody test showed nodavirus antigens in the retina and positive-CPE SSN-1 cells. Also, a RT-PCR product of approximately 289 bp was amplified from the brain and retina of the 23 samples, including the SSN-1 positive samples. This is the first report of the isolation and characterization of nodavirus from golden grey mullet from the Caspian Sea and, therefore, possible infections in other valuable species of the Caspian Sea warrant further studies.

Outbreak of betanodavirus infection in tilapia, Oreochromis niloticus (L.), in fresh water

A betanodavirus associated with a massive mortality was isolated from larvae of tilapia, Oreochromis niloticus, maintained in fresh water at 30 degrees C. Histopathology revealed vacuolation of the nervous system, suggesting an infection by a betanodavirus. The virus was identified by indirect fluorescent antibody test in the SSN1 cell line and further characterized by sequencing of a PCR product. Sequencing of the T4 region of the coat protein gene indicated a phylogenetic clustering of this isolate within the red-spotted grouper nervous necrosis virus type. However, the tilapia isolate formed a unique branch distinct from other betanodavirus isolates. The disease was experimentally reproduced by bath infection of young tilapia at 30 degrees C. The reservoir of virus at the origin of the outbreak remains unidentified. To our knowledge, this is the first report of natural nodavirus infection in tilapia reared in fresh water.

Partial susceptibility of the SSN‐1 fish cell line to a crustacean virus: a defective replication study

This study evaluated the possible use of the fish SSN-1 cell line to investigate the development of Macrobrachium rosenbergii nodavirus (MrNV). Cells were incubated with viral particles and cytopathic effects were observed. De novo synthesis of viral capsid proteins was shown by immuno-fluorescence labelling and a sandwich ELISA test. Viral genomic replication was demonstrated by RT-PCR using primers specific to RNA-1 as well as by quantitative RT-PCR (RT-qPCR). Using electron microscopy, only a few empty particles were observed and attempts to isolate complete infectious particles or to re-infect healthy cells (second passage) were unsuccessful. As complete viral particles were rarely observed, it appeared that defaults in MrNV virogenesis might arise resulting in the formation of scarce and non-infectious particles. SSN-1 cells were found to be partially permissive to MrNV infection that induced cell lysis, but key elements for viral infection were lacking such as regulatory factors for gene replication or post-translational modifications.

Temperature-dependency of Betanodavirus infection in SSN-1 cell line

This study examined the in vitro effects of temperature on Betanodavirus infection in the SSN-1 cell line. A Betanodavirus isolated from moribund sea bass fry Dicentrarchus labrax farmed in the Adriatic Sea and characterised as a RGNNV (Redspotted Grouper Nervous Necrosis Virus) genotype was used. Virus-infected SSN-1 cells were incubated at temperatures between 10 and 30 degrees C and observed for cytopathic effects daily for 15 d. Cell-free and cell-associated viral growth were evaluated by 50% tissue culture infectious dose (TCID50) titration at 0, 24, 48, 72, 96, 144, 192, 240, 312 and 360 h post-infection. Virus replication was observed at all temperatures from 15 to 30 degrees C. The optimal temperature for virus growth was 25 degrees C. A temperature of 10 degrees C was detrimental to the growth of the SSN-1 cells and cell death interfered with interpretations of viral growth. The isolate of Betanodavirus from Italian sea bass in this study demonstrates a different temperature range for growth compared to previous reports for related Betanodavirus strains, most likely due to an adaptation to the normal environmental temperatures of the host fish species of origin.

Effect of temperature on Betanodavirus infection in SSN-1 cell line.

Effect of temperature on Betanodavirus infection in SSN-1 cell line.

Cloning of the fish cell line SSN-1 for piscine nodaviruses.

Six cell clones were derived from the SSN-1 cell line, which is composed of a mixed cell population and persistently infected with a C-type retrovirus (SnRV). These clones were susceptible to 4 piscine nodavirus strains belonging to different genotypes (SJNNV, RGNNV, TPNNV and *BFNNV [striped jack, redspotted grouper, tiger puffer and barfin flounder nervous necrosis viruses]). Three clones, designated A-6, E-9, and E-11, were highly permissive to nodavirus infection and production. The virus-induced cytopathic effects appeared as cytoplasmic vacuoles and intensive disintegration at 3 to 5 d post-incubation. These observations were highly reproducible and formed the basis for a successful virus titration system. Quantitative analysis using the cloned E-11 cell line clearly revealed differences in the optimal growth temperatures among the 4 genotypic variants: 25 to 30 degrees C for strain SGWak97 (RGNNV), 20 to 25 degrees C for strain SJNag93 (SJNNV), 20 degrees C for strain TPKag93 (TPNNV), and 15 to 20 degrees C for strain JFIwa98 (BFNNV). Electron microscopy demonstrated SnRV retrovirus particles only in A-6 and E-9 cells, but PCR amplification for the pol gene and LTR region of the proviral DNA indicated the presence of the retrovirus in the other clones, including E-11. The cell clones obtained in the present study will be more useful for qualitative and quantitative analyses of piscine nodaviruses than the SSN-1 cell line.

Isolation in cell culture of nodavirus from farmed Atlantic halibut Hippoglossus hippoglossus in Norway.

Isolation in cell culture of nodavirus from Atlantic halibut Hippoglossus hippoglossus suffering from viral encephalopathy and retinopathy (VER) is described. The cell line SSN-1 was inoculated with tissue material from affected juveniles (60 d after first feeding). Extensive cytopathic effects (CPE) developed approximately 5 d after inoculation, and were also observed after several passages in the same cell line. Cells from infected cultures showed reactivity with an antiserum against sea bass Dicentrarchus labrax nodavirus in an indirect immunofluorescence test. Analysis of infected cells with reverse transcriptase-polymerase chain reaction (RT-PCR) resulted in a product of the predicted size using primers specific for striped jack Pseudocaranx dentex nodavirus. Electron micrographs of infected SSN-1 cells demonstrated virus particles that were approximately less than 30 nm. Challenge of Atlantic halibut larvae (4 d post-hatching) with supernatants from infected SSN-1 cells resulted in development of VER as verified by immunohistochemistry performed on larvae sampled from Day 9 after challenge. The present results show that a nodavirus from Atlantic halibut has been isolated using the SSN-1 cell line and that virus propagated in cell culture retained virulence.

Induction of nodavirus disease in seabass, Dicentrarchus labrax, using different infection models

The aim of this study was to investigate the susceptibility of juvenile and adult seabass, which are generally thought to be refractory to nodavirus. Moreover, preliminary immunological studies were performed to examine the immune response of adult seabass. Successful transmission of the disease was experimentally demonstrated in juvenile and adult seabass as ascertained by the presence of the clinical signs of the disease, re-isolation of the virus in the SSN-1 cell line and subsequent confirmation by histopathology and immunohistochemistry. Bigger seabass not only developed the clinical disease but also suffered mortalities. Serum neutralisation titres were considered low in this study.