Susceptibility of a number of Australian freshwater fishes to dwarf gourami iridovirus (Infectious spleen and kidney necrosis virus)

Megalocytiviruses cause high mortality diseases that have seriously impacted aquaculture, with the most frequent outbreaks occurring in East and South‐East Asia. The international trade of juvenile fish for food and ornamental aquaculture has aided the spread of these viruses, which have spread to Europe and Australia and other regions. Australian freshwater fishes were examined for susceptibility to infection with the exotic megalocytivirus, dwarf gourami iridovirus (DGIV), which belongs to a group with the type species, Infectious spleen and kidney necrosis virus (ISKNV). Fish were held at 23 ± 1 °C and challenged by intraperitoneal (IP) injection or by cohabitation with Murray cod, Maccullochella peelii (Mitchell) infected with DGIV. A species was deemed to be susceptible to DGIV based on evidence of viral replication, as determined by qPCR, and megalocytic inclusion bodies observed histologically. Horizontal transmission occurred between infected Murray cod and golden perch, Macquaria ambigua (Richardson), Macquarie perch, Macquaria australasica (Cuvier) and Murray cod. This indicated that DGIV shed from infected fish held at 23 °C can survive in fresh water and subsequently infect these naïve fish. Further, DGIV administered IP was highly pathogenic to golden perch, Macquarie perch and Murray cod. Compared to these species, the susceptibility of southern pygmy perch, Nannoperca australis (Gunther) was lower. Freshwater catfish (dewfish), Tandanus tandanus (Mitchell), were not susceptible under the experimental conditions based on the absence of clinical disease, mortality and virus replication. This study showed the potential risks associated with naïve and DGIV‐infected fish sharing a common water source.     

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.     

Determination of the muscle residue levels and withdrawal time of florfenicol in healthy and experimentally Lactococcus garvieae infected rainbow trout (Oncorhynchus mykiss,Walbaum 1792) grown in the Black Sea water

The aim of this study was to determine, muscle residue level (MRL) and withdrawal time (WT) of florfenicol (FF), in healthy and infected rainbow trout (Oncorhynchus mykiss) with Lactococcus garvieae. Fish were divided into the four groups, Group I (Infected fish fed with FF free diet, n = 10), Group II (Healthy fish fed with FF free diet, n = 10), Group III (Infected fish fed with FF containing diet, n = 80) and Group IV (Healthy fish fed with FF containing diet, n = 80). Fish in Group I and Group III were infected with 1.8×10⁶ cfu/ml Lactococcus garvieae by immersion. FF containing diet was applied fish (10 mg kg^‐1 day^‐1) in Group III and IV for 10 days. FF residues in fish muscles were analysed by validated HPLC method. The limit of detection (LOD) and limit of quantification (LOQ) values of the method were determined to be 0.31 μg/g and 0.64 μg/g, respectively, and the recovery was 92.61%. As a result of the experiments, there was no significant difference between the infected and healthy group in term of MRLs. However, in both groups, it was determined that third‐day values were below MRLs (1,000 μg/kg). Also, the WT values were lower than the 500°C‐day values reported in the EC commission decision in both groups and were determined as 14.5 and 14.12 (water temperature mean 18°C ± 1) per day, respectively.     

Ultrastructural morphogenesis of salmonid alphavirus 1

Studies on the ultrastructural morphogenesis of viruses give an insight into how the host cell mechanisms are utilized for new virion synthesis. A time course examining salmonid alphavirus 1 (SAV 1) assembly was performed by culturing the virus on Chinook salmon embryo cells (CHSE‐214). Different stages of viral replication were observed under electron microscopy. Virus‐like particles were observed inside membrane‐bound vesicles as early as 1 h following contact of the virus with the cells. Membrane‐dependent replication complexes were observed in the cytoplasm of the cells, with spherules found at the periphery of late endosome‐like vacuoles. The use of intracellular membranes for RNA replication is similar to other positive‐sense single‐stranded RNA (+ssRNA) viruses. The number of Golgi apparatus and associated vacuoles characterized by ‘fuzzy’‐coated membranes was greater in virus‐infected cells. The mature enveloped virions started to bud out from the cells at approximately 24 h post‐infection. These observations suggest that the pathway used by SAV 1 for the generation of new virus particles in vitro is comparable to viral replication observed with mammalian alphaviruses but with some interesting differences.     

Physiological characteristics and stress resistance of great sturgeon (<Emphasis Type="Italic">Huso huso</Emphasis>) juveniles fed with vitamins C,E, and HUFA-enriched <Emphasis Type="Italic">Artemia urmiana</Emphasis> nauplii

This study was carried out to examine the effect of Artemia urmiana nauplii enriched with HUFA, and vitamins C and E on stress tolerance, hematocrit, and biochemical parameters of great sturgeon, Huso huso juveniles. Cod liver oil (EPA 18% and DHA 12%), ascorbyl-6-palmitate and α-tocopherol acetate were used as lipid, and vitamin C and E sources, respectively. Beluga juveniles at the stage of first feeding (69.7 ± 5.9 mg body weight) were randomly divided into five treatments and three tanks were assigned to each diet. All fish groups were fed non-enriched Artemia for the initial 5 days and then fed enriched Artemia for 7 days. Juveniles were fed with Artemia enriched with HUFA + 20% vitamin C (C group); HUFA + 20% vitamin E-enriched Artemia nauplii (E group); HUFA + 20% vitamin C + 20% vitamin E (C and E group); HUFA without vitamins (HUFA) and non-enriched Artemia (control). After the period of enrichment, Juveniles were fed with Daphnia sp. from the 13th to the 40th day. At day 40, the fish were transferred directly from fresh water (0.5 ppt) to brackish water (6 ppt for 4 days and 12 ppt for 2 days) and warm water (from 27 to 33°C) to evaluate juvenile resistance to salinity and thermal shocks. Moreover, all treatments were separately exposed to freshwater in tanks with the same capacity as used for osmotic and thermal tests (as fresh water control). The addition of vitamins C, E, and C + E to HUFA significantly increased fish resistance to 12 ppt salinity and temperature stress tests, whereas survival was not significantly different among challenges at 6 ppt. There was no significant difference in the hematocrit index under stress conditions. Enrichment had significant influence on plasma Na⁺ level in the C group on the 4th day at 6 ppt. Na⁺ and Ca²⁺ concentrations in C, E, and C and E groups on the 1st day at 12 ppt, and Ca²⁺ level in E group on the 2nd day at 12 ppt were lower than the other groups. The glucose level in the C and C and E groups was lower than the other treatments on the 1st day at 12 ppt and the 2nd day at 33°C. Regardless of Artemia enrichment, plasma ions (Na⁺, K⁺, Ca²⁺, and Mg²⁺) and glucose concentrations in fish exposed to salinity stress tests were higher than fish in fresh water. Glucose concentration in plasma also increased after 2 days at 33°C. Although most of our results were not significantly different, the use of vitamins C, E, and HUFA in Artemia enrichment can improve Juveniles tolerance under stress conditions, and regardless of enrichment, these data show that beluga juveniles are partly sensitive to high salinity and temperature.     

The response of intestinal mucous cells to the presence of enteric helminths: their distribution,histochemistry and fine structure

Histochemical and ultrastructural investigations were conducted on the mucous cells of the intestine of brown trout, Salmo trutta L., naturally infected with the cestode Cyathocephalus truncatus (Pallas, 1781) and the acanthocephalan Echinorhynchus truttae Shrank, 1788. A subpopulation of 45 S. trutta were examined of which 15 specimens harboured E. truttae, 15 of which were infected with C. truncatus and 15 fish, the control group, were uninfected. In histological sections, hyperplasia and hypertrophy of the mucous cells were evident at the site of parasite infection. Enhanced mucus secretion was also recorded in infected fish. The number of mucous cells close to the site of parasite attachment within the intestine was significantly higher than the number detected in uninfected individuals and in infected individuals at sites 1 cm or greater from the point of parasite attachment. There were no significant differences between the number of mucous cells found at the latter two sites. Alcian blue and periodic acid‐Schiff’s staining of representative histological sections revealed a significant increase in the number of mucous cells staining positively for acid glycoconjugates compared to the number of cells found in the intestines of uninfected S. trutta. In transmission electron microscopy sections, each mucous cell typically possessed an elongated, basally positioned nucleus. The cytoplasm was observed to possess numerous electron dense and lucent vesicles, in addition to well‐developed rough endoplasmic reticulum, Golgi apparatus and a few round mitochondria.     

Comparative genome analysis reveals a distinct influence of nucleotide composition on virus–host species‐specific interaction of prawn‐infecting nodavirus

Discovery of species‐specific interaction between the host and virus has drawn the interest of many researchers to study the evolution of the newly emerged virus. Comparative genome analysis provides insights of the virus functional genome evolution and the underlying mechanisms of virus–host interactions. The analysis of nucleotide composition signified the evolution of nodavirus towards host specialization in a host‐specific mutation manner. GC‐rich genome of betanodavirus was significantly deficient in UpA and UpU dinucleotides composition, whilst the AU‐rich genome of gammanodavirus was deficient in CpG dinucleotide. The capsid of MrNV and PvNV of gammanodavirus retains the highest abundance of adenine and uracil at the second codon position, respectively, which were found to be very distinctive from the other genera. ENC‐GC3 plot inferred the influence of natural selection and mutational pressure in shaping the evolution of MrNV RdRp and capsid, respectively. Furthermore, CAI/eCAI analysis predicts a comparable adaptability of MrNV in squid, Sepia officinalis than its natural host, Macrobrachium rosenbergii. Thus, further study is warranted to investigate the capacity of MrNV replication in S. officinalis owing to its high codon adaptation index.     

Enhancing rotifer Brachionus calyciflorus population growth with commercial probiotics

The effect of a commercial probiotic (NanoCrusta, Altacrusta, Mexico City, Mexico) on the growth performance of Brachionus calyciflorus Pallas, 1766, was evaluated. In a first approach, probiotics were supplied in four densities (2.0 × 10³, 1.1 × 10⁵, 2.1 × 10⁵ and 2.1 × 10⁶ cells/ml), alone and in combination with Chlorella vulgaris (1 × 10⁶ cells/ml). The test rotifer did not grow on the probiotic alone. However, when probiotics + C. vulgaris were added, the maximum densities (D_max; ind/ml) and population growth rates (r) observed were higher. In the second experiment, probiotics were supplied at five higher densities (2.1 × 10⁶, 4.2 × 10⁶, 8.5 × 10⁶, 1.7 × 10⁷ and 3.4 × 10⁷ cells/ml) with C. vulgaris and a control treatment with only C. vulgaris (probiotic‐free). Treatments supplied with probiotics between 2.1 × 10⁶ and 1.7 × 10⁷ cells/ml showed significantly higher D_max and r than the control treatment. The results showed a positive effect of probiotic bacteria when supplied with C. vulgaris. The best outcome showed a D_max 2.16 times and an r 1.63 times higher than the density of the control treatment. Growth rates were higher in the treatments with probiotics compared to the control. We conclude that application of NanoCrusta is feasible to improve B. calyciflorus production, but the effects need to be tested in larger scales.     

Identification and characterization of a late gene encoded by grouper iridovirus 2L (GIV‐2L)

Grouper iridovirus (GIV) belongs to the Ranavirus genus and is one of the most important viral pathogens in grouper, particularly at the fry and fingerling stages. In this study, we identified and characterized the GIV‐2L gene, which encodes a protein of unknown function. GIV‐2L is 1242 bp in length, with a predicted protein mass of 46.2 kDa. It displayed significant identity only with members of the Ranavirus and Iridovirus genera. We produced mouse monoclonal antibodies against the GIV‐2L protein by immunizing mice with GIV‐2L‐His‐tag recombinant protein. By inhibiting de novo protein and DNA synthesis in GIV‐infected cells, we showed that GIV‐2L was a late gene during the viral replication. Finally, immunofluorescence microscopy revealed that GIV‐2L protein accumulated in both the nucleus and cytoplasm of infected cells. These results offer important insights into the pathogenesis of GIV.     

Resistance of pearlspot larvae,Etroplus suratensis,to redspotted grouper nervous necrosis virus by immersion challenge

Viral nervous necrosis (VNN) affects more than 120 species mostly belonging to the order Perciformes. However, none of the brackishwater species belonging to the family Cichlidae under the order Perciformes are reported to be susceptible. Hence, the present experiment was undertaken to study the susceptibility of the brackishwater cichlid, pearlspot, Etroplus suratensis to NNV. Thirty‐day‐old pearlspot larvae were infected with NNV by immersion. Mortality was recorded till 14 days post‐infection, and the infected larvae were subjected to nested RT‐PCR and histology. The virus was isolated from infected larvae using SSN‐1 cells. To study the replication of the virus in vitro, primary cultured brain cells of E. suratensis and IEK cells were infected with NNV. No mortality was observed in any of the control or experimentally infected larvae. However, the experimentally infected larvae were positive for NNV by nested RT‐PCR and the virus was isolated using SSN‐1 cells. Further, the infected pearlspot brain cells and IEK cells showed cytopathic effect at second and third passage of the virus and they were positive for NNV by nested RT‐PCR. Pearlspot is relatively resistant to VNN although the virus could replicate in the larvae and in cell culture.     

Low pathogenicity of flounder iridovirus (FLIV) and the absence of cross‐protection between FLIV and rock bream iridovirus

The genus Megalocytivirus is known to infect a wide range of cultured marine fish. In this study, we examined the pathogenicity of FLIV (Megalocytivirus from olive flounder, genotype III) and RBIV (Megalocytivirus from rock bream, genotype I) to their homologous and heterologous host species. Olive flounder (7.5 ± 1.3 cm) injected with FLIV [major capsid protein (MCP) gene copies, 6.8 × 10³–6.5 × 10⁶/fish] at 24 °C did not die until 90 days post‐infection (dpi). The average virus replication in the spleen peaked (1.27 × 10⁶/fish) at 20 dpi. Rock bream (6.5 ± 1.5 cm) injected with FLIV (8.8 × 10⁵ and 6.5 × 10⁶/fish of MCP copies) showed no mortality until 50 dpi. The rock bream that survived after FLIV infection were rechallenged with RBIV at 50 dpi had 100% mortality, showing that there is no cross‐protection between FLIV and RBIV. Temperature shifting (26 °C and 20 °C at 12 h intervals) did not cause FLIV‐specific mortality into olive flounder, but higher virus copies were observed in the fish exposed to higher stocking density. This study demonstrates that FLIV and RBIV have different antigenic and pathogenic characteristics and that FLIV has low pathogenicity to olive flounder.     

Testing the yield of a pilot‐scale bubble column photobioreactor for cultivation of the microalga Rhodomonas salina as feed for intensive calanoid copepod cultures

A dual column photobioreactor (PBR) (2 × 47 L) with mixed CO₂/air bubbling was tested for cultivation of the microalga Rhodomonas salina as food for live feed copepods. In the continuous growth phase, the cell density was relatively stable at 2.40 ± 0.13 × 10⁶ cells/ml at an average dilution rate of 0.46 ± 0.02 per day throughout the 30‐day experiment. The produced algae had a high content of both total fatty acids (TFA) and free amino acids (FAA). Especially, the harvested algae contained a high proportion of poly‐unsaturated fatty acids that made up 80% of the TFA and of essential amino acids (35% of all FAA), implicating desirable components as feed for copepods. The current PBR was sufficient to feed a culture of the calanoid copepod Acartia tonsa at a density of 2,500 adult/L in ca. 500 L culture with a daily yield of approximately 17 × 10⁶ eggs. To be able to sustain the integrated copepods production, the suggested volume of the algae cultures should be ca. 20% of the copepod culture volume.     

The level of 1C diets fed prior to cell isolation affects lipid metabolism in primary liver cells isolated from Atlantic salmon (Salmo salar)

Liver cells were isolated from 6 fish fed a diet containing 12.1 g methionine/kg, 11.02 mg vitamin B6/kg, 0.20 mg vitamin B12/kg and 7.80 mg folate/kg (named high‐1C diet). These cells were compared to liver cells isolated from 6 fish fed a diet containing 6.7 g methionine/kg, 7.01 g vitamin B6/kg, 0.15 mg vitamin B12/kg and 2.60 mg folate/kg (named low‐1C diet). Isolated cells were plated on 6‐well plates in Leibovitz medium and treated with 10 mM metformin, 10 mM metformin for 24 hr followed by 0.4 mM oleic acid (OA) for 24 hr or only 0.4 mM OA for 24 hr. The cells were compared to untreated controls added only the medium. All cells were harvested 48 hr after being plated. Cells isolated from Atlantic salmon fed low‐1C diets showed higher gene expression of MGAT‐2 (p < .0001), CPT‐1 (p = .028), FAS (p = .0006), LXR (p = .020), ACC (p = .032) and MnSOD (p < .0001). The low‐ or high‐1C diets fed prior to cell isolation had no effect on gene expression of ApoB100, PPARa, CD36, SREBP‐2 or Bcl‐2. Metformin treatment increased the expression of the anti‐apoptotic protein Bcl‐2 (p = .0001) indicating an anti‐apoptotic effect. Metformin generally increased the expression of genes associated with lipid oxidation and transport, but decreased the expression of genes associated with cholesterol metabolism confirming our earlier results using this model.     

Changes in Na+,K+-ATPase activity and gill chloride cell morphology in the grouper Epinephelus coioides larvae and juveniles in response to salinity and temperature

The activity of the enzyme Na⁺,K⁺-ATPase and morphological changes of gill chloride cells in grouper, Epinephelus coioides larvae and juveniles were determined 6–48 h after abrupt transfer from ambient rearing conditions (30–32 ppt, 26.5–30 °C) to different salinity (8, 18, 32, 40 ppt) and temperature (25, 30 °C) combinations. Na⁺,K⁺-ATPase activity in day 20 larvae did not change at salinities 8–32 ppt. Activity decreased significantly (P <0.01) after exposure to 40 ppt at 25–30 °C, which was accompanied by an increase (P <0.05) in density and fractional area of chloride cells. Enzyme activity in 40 ppt did not reach a stable level and larvae failed to recover from an osmotic imbalance that produced a low survival at 25 °C and death of all larvae at 30 °C. Enzyme activity and chloride cell morphology in day 40 groupers did not change in 8–40 ppt at 25 °C and 8–32 ppt at 30 °C. A significant decrease and a subsequent increase in Na⁺,K⁺-ATPase activity in 40 ppt at 30 °C was associated with the increase in chloride cell density resulting in an increased fractional area but a decreased cell size. Enzyme activity and chloride cells of day 60 grouper were unaffected by abrupt transfer to test salinities and temperatures. These results demonstrate that grouper larvae and juveniles are efficient osmoregulators over a wide range of salinities. Salinity adaptation showed an ontogenetic shift as the larvae grew and reached the juvenile stage. This development of tolerance limits may reflect their response to actual conditions existing in the natural environment.     

Ultrastructural analysis of sequential cyprinid herpesvirus 3 morphogenesis in vitro

Cyprinid herpesvirus 3 (CyHV‐3) is an alloherpesvirus, and it is the aetiological agent of koi herpesvirus disease. Although the complex morphogenic stages of the replication cycle of CyHV‐3 were shown to resemble that of other members of the Herpesvirales, detailed analysis of the sequence and timing of these events was not definitively determined. This study describes these features through a time course using cyprinid cell cultures (KF‐1 and CCB) infected with CyHV‐3 (KHV isolate, H361) and analysed by transmission electron microscopy. Rapid viral entry was noted, with high levels of intracellular virus within 1–4 h post‐infection (hpi). Intranuclear capsid assembly, paracrystalline array formation and primary envelopment of capsids occurred within 4 hpi. Between 1 and 3 days post‐infection (dpi), intracytoplasmic secondary envelopment occurred, as well as budding of infectious virions at the plasma membrane. At 5–7 dpi, the cytoplasm contained cytopathic vacuoles, enveloped virions within vesicles, and abundant non‐enveloped capsids; also there was frequent nuclear deformation. Several morphological features are suggestive of inefficient viral assembly, with production of non‐infectious particles, particularly in KF‐1 cells. The timing of this alloherpesvirus morphogenesis is similar to other members of the Herpesvirales, but there may be possible implications of using different cell lines for CyHV‐3 propagation.     

An experimental means of transmitting pancreas disease in Atlantic salmon Salmo salar L. fry in freshwater

A challenge model for pancreas disease in Atlantic salmon, Salmo salar L. fry, was developed comparing two salmonid alphavirus (SAV) subtypes: SAV1 and SAV5. Viral doses of 3 × 10⁵ TCID₅₀ mL⁻¹ for SAV1 and 3 × 10⁴ for SAV5 were tested in triplicate tanks, each containing 450 salmon fry. Cumulative mortalities of 1.2% were recorded. Titres of virus recovered from the mortalities ranged from 10² to 10⁷ TCID₅₀ mL⁻¹. Fry were sampled at 3, 5 and 7.5 weeks post-challenge. Sampling after 3 weeks revealed a high prevalence of infection in the absence of clinical signs, and infectious virus was recovered from 80% and 43% of sampled fry infected with SAV1 and SAV5, respectively. After 5 weeks pancreas, heart and red skeletal muscle lesions were generally observed, whilst degeneration in white skeletal muscle was observed only in fish infected with SAV1. In situ hybridisation confirmed the presence of viral genome in infected pancreas, heart and muscle. After 7.5 weeks, infectious virus (both isolates) was recovered from 13.3% of the fish sampled, with a viral titre of 10² TCID₅₀ mL⁻¹. Clearly, salmon fry are susceptible to SAV infection and pancreas disease.     

Establishment of a new cell line susceptible to Cyprinid herpesvirus 3 (CyHV‐3) and possible latency of CyHV‐3 by temperature shift in the cells

A new cell line named CCF‐K104 predominantly consisting of fibroblastic cells showed optimal growth at temperatures from 25 °C to 30 °C. Serial morphological changes in the cells induced by Cyprinid herpesvirus 3 (CyHV‐3) included cytoplasmic vacuolar formation, cell rounding and detachment. Mature virions were purified from CyHV‐3‐infected CCF‐K104 cells by sucrose gradient ultracentrifugation and had a typical herpesvirus structure on electron microscopy. Infectious CyHV‐3 was produced stably in CCF‐K104 cells over 30 viral passages. Our findings showed that CCF‐K104 is a useful cell line for isolation and productive replication of CyHV‐3. A temperature shift from 25 °C to 15 °C or 35 °C did not allow serial morphological changes as observed at 25 °C for 14 days. Under the same conditions, real‐time PCR showed that CyHV‐3 was present with low viral DNA loads, suggesting that CyHV‐3 may establish latent infection in CCF‐K104 cells. Amplification of the left and right terminal repeat sequences of the CyHV‐3 genome arranged in a head‐to‐tail manner was detected by nested PCR following an upshift in temperature from 25 °C to 35 °C. The PCR results suggested that the circular genome may represent a latent form of CyHV‐3.     

Establishment of a new cell line from the heart of giant grouper,Epinephelus lanceolatus (Bloch), and its application in toxicology and virus susceptibility

A new marine fish cell line, derived from the heart of giant grouper, Epinephelus lanceolatus (Bloch), was established and characterized. The cell line was designated as ELGH and subcultured with more than 60 passages. The ELGH cells were mainly composed of fibroblast-like cells and multiplied well in Leibovitz's L-15 medium supplemented with 10% foetal bovine serum (FBS) at 28 °C. Chromosome analysis indicated that the modal chromosome number was 48. The fluorescent signals were detected in ELGH when transfected with green fluorescent protein reporter plasmids. The 50% cytotoxic concentration (CC₅₀) of the extracellular products (ECPs) from Streptococcus iniae and Vibrio alginolyticus E333 on ELGH cells was 60.02 and 12.49 μg mL⁻¹, respectively. Moreover, the ELGH cells showed susceptibility to Singapore grouper iridovirus (SGIV), but not to soft-shelled turtle iridovirus (STIV), red-spotted grouper nervous necrosis virus (RGNNV) and spring viremia of carp virus (SVCV), which was demonstrated by the presence of a severe cytopathic effect (CPE) and increased viral titres. In addition, electron microscopy observation showed that abundant virus particles were present in the infected cells. Taken together, our data above provided the potential utility of ELGH cells for transgenic and genetic manipulation, as well as cytotoxicity testing and virus pathogenesis.     

Preparation of monoclonal antibody against Macrobrachium rosenbergii Nodavirus and application of TAS-ELISA for virus diagnosis in post-larvae hatcheries in east China during 2000-2004

“Whitish muscle disease” of Macrobrachium rosenbergii, also called “whitish disease” or “white tail disease”, is a new serious epizootic disease that has occurred in recent years in giant freshwater prawn culture regions, mainly in southern China. This disease occurred in post-larvae 3-5 days to 3 weeks after desalting. Clinical signs include the development of white spot in muscles or milky muscles throughout the body, causing serious loss in few days, with a mortality rate of 40-90%. A 26-27 nm icosahedral non-enveloped virus, identified as M. rosenbergii Nodavirus (MrNV), was confirmed as the aetiological agent. Twelve hybridomas strongly secreting monoclonal antibodies (Mabs) against MrNV were shown to be specific for MrNV and reacted with MrNV 42 kDa coat protein by Western blot. A triple antibody enzyme-linked immunosorbent assay (TAS-ELISA) was developed and shown to be a useful diagnostic tool for MrNV infection.