Sevenband Grouper Research Articles

Mortality of wild golden grey mullet (Liza auratus) in Iranian waters of the Caspian Sea, associated with viral nervous necrosis-like agent

An acute mortality caused by an uncertain agent occurred in wild golden grey mullet (Liza auratus) population in the Iranian side of the Caspian Sea in February, 2004. Clinical signs of the moribund fish were erratic behavior such as spiral swimming, belly-up at rest and over inflation of swim bladder. Histological examination of brain of dead fish revealed hyperaemia, degeneration and necrosis in external granular layers as well as focal extension in intestine. Severe leucopenia was also observed. No putative causal factors were suggested by bacteriological, parasitological or environmental examinations. In virological examina-tions, however brains of affected golden grey mullet were positive in the reverse transcriptase-polymerase chain reaction test for piscine nodavirus (Nodaviridae, Betanodavirus), the causative agent of Encephalopathy and retinopathy (VER), otherwise known as viral nervous necrosis (VNN) in a variety of cultured marine fishes worldwide. Nucleotide sequence of the PCR amplicons is closely related with the coat protein gene of piscine nodaviruses, particularly redspotted grouper nervous necrosis virus (RGNNV). Although, it was not succeeded to isolate the virus, experimental infection with the brain homogenates of the affected fish in sevenband grouper (Epinephelus septemfasciatus), which is highly susceptible to RGNNV, produced neuro- logical abnormality followed by mortality. These results suggest that the mortality observed in golden grey mullet in the Caspian Sea might have been associated with piscine nodavirus infection.

マハタ仔魚のワムシサイズに対する摂餌選択性

マハタ仔魚のワムシサイズに対する摂餌選択性

Ovarian development and final oocyte maturation in cultured sevenband grouper Epinephelus septemfasciatus

: The histological changes in the ovary of sevenband grouper Epinephelus septemfasciatus were examined during vitellogenesis and final oocyte maturation. Ovaries contained oocytes at the perinucleolus stage at the start of the experiment in January. Oocytes at the primary yolk stage appeared when the gonadosomatic index (GSI) was slightly on the rise in March. After that, vitellogenesis progressed and ovaries contained oocytes at the tertiary yolk stage in May. The GSI increased drastically at the same time. These results suggest that vitellogenesis in sevenband grouper starts or is already in progress in March and is completed by May, In order to clarify the process of final oocyte maturation, luteinizing hormone-releasing hormone (LHRH) analog (des Gly10, [D-Ala6] LHRH ethylamide) was implanted in fish with oocytes at the tertiary yolk stage. The oocytes developed to the migratory nucleus stage in 18 h and reached the maturation stage in 42 h after implantation. This indicates that final oocyte maturation is completed within 42 h after stimulation of gonadotropin-releasing hormone.

Identification of host-specificity determinants in betanodaviruses by using reassortants between striped jack nervous necrosis virus and sevenband grouper nervous necrosis virus.

Betanodaviruses, the causal agents of viral nervous necrosis in marine fish, have bipartite positive-sense RNAs as genomes. The larger genomic segment, RNA1 (3.1 kb), encodes an RNA-dependent RNA polymerase, and the smaller genomic segment, RNA2 (1.4 kb), codes for the coat protein. Betanodaviruses have marked host specificity, although the primary structures of the viral RNAs and encoded proteins are similar among betanodaviruses. However, no mechanism underlying the host specificity has yet been reported. To evaluate viral factors that control host specificity, we first constructed a cDNA-mediated infectious RNA transcription system for sevenband grouper nervous necrosis virus (SGNNV) in addition to that for striped jack nervous necrosis virus (SJNNV), which was previously established by us. We then tested two reassortants between SJNNV and SGNNV for infectivity in the host fish from which they originated. When striped jack and sevenband grouper larvae were bath challenged with the reassortant virus comprising SJNNV RNA1 and SGNNV RNA2, sevenband groupers were killed exclusively, similar to inoculation with SGNNV. Conversely, inoculations with the reassortant virus comprising SGNNV RNA1 and SJNNV RNA2 killed striped jacks but did not affect sevenband groupers. Immunofluorescence microscopic studies using anti-SJNNV polyclonal antibodies revealed that both of the reassortants multiplied in the brains, spinal cords, and retinas of infected fish, similar to infections with parental virus inoculations. These results indicate that viral RNA2 and/or encoded coat protein controls host specificity in SJNNV and SGNNV.

Susceptibility of cultured juveniles of several marine fish to the sevenband grouper nervous necrosis virus.

Piscine nodaviruses (betanodaviruses) have been tentatively divided into four genotypes (SJNNV, RGNNV, TPNNV and BFNNV) and it is suggested that host specificity is different among these genotypes. In the present study, a betanodavirus [sevenband grouper nervous necrosis virus (SGNNV)] belonging to the redspotted grouper nervous necrosis virus (RGNNV) genotype, to which most betanodaviruses from warm water fish are identified, was evaluated for its pathogenicity to hatchery-reared juveniles of several marine fish species. When challenged with the virus by a bath method (10(5.1) TCID50 mL(-1)), sevenband grouper, Epinephelus septemfasciatus, Japanese flounder, Paralichthys olivaceus, and tiger puffer, Takifugu rubripes, displayed behavioural abnormalities and mortalities with distinct histopathological signs of viral nervous necrosis and heavily immunostained cells were observed in the central nervous tissues and retina. Bath-challenged rock fish, Sebastiscus marmoratus, and a hybrid of sevenband grouper and kelp grouper, E. moara, did not display any behavioural abnormality or mortality during the experimental period, although many fish showed slight signs of viral infection in nerve cells. Kelp grouper and red sea bream, Pagrus major, showed no behavioural abnormality, mortality or immunohistopathological changes after the virus challenge. These results are, in part, consistent with the natural host range of RGNNV, indicating the complexity in the host specificity of betanodaviruses.

Molecular cloning of gonadotropin cDNA in sevenband grouper, Epinephelus septemfasciatus

The cDNA for glycoprotein hormone (GPH) α-subunit, β-subunits of the FSH (GtH I) and LH (GtH II) were isolated, cloned, and sequenced from sevenband grouper pituitary using RACE PCR. These results show that sevenband grouper has two different types of gonadotropins FSH (GtH I) and LH (GtH II), as other teleosts.

Betanodavirus as a novel transneuronal tracer for fish

In order to obtain a potential new tool to analyze networks of the central nervous system of teleost fishes, we tested a fish-pathogenic betanodavirus, sevenband grouper nervous necrosis virus (SGNNV), as a transneuronal tracer using the freshwater angelfish Pterophyllum scalare as a test animal. Intravitreous injections of SGNNV into the right eye resulted first in the labelings of neuronal cell bodies in the ganglion cell layers of the retina and then those in the inner and outer nuclear layers in sequence. For the first time, labeled neurons were found also in the stratum periventriculare of the contralateral optic tectum, the ventrolateral and ventromedial thalamic nuclei, and the periventricular nucleus of posterior tuberculum in the brain, then the periventricular pretectal nucleus pars dorsalis and pars ventralis. In contrast, by injections of biotinylated dextran amine into the eye no labeled cell bodies were observed in these brain areas, but axons and terminals were labeled anterogradely. These results suggest that the virus could be transported in both directions in axons of the first order neuron and transfected the second and third order neurons by passing across synaptic clefts, and that this technique is practically applicable to the study of neurobiology in teleost.

Protective immunity of sevenband grouper, Epinephelus septemfasciatus Thunberg, against experimental viral nervous necrosis

This paper describes the protective immune responses of sevenband grouper, Epinephelus septemfasciatus Thunberg, immunized with live piscine nodavirus, the causative agent of viral nervous necrosis (VNN), or the Escherichia coli– expressed recombinant coat protein. Nodavirus‐neutralizing antibodies were detected at titres ranging from 1:158 to 1:1257 in serum of sevenband grouper which survived intramuscular injection with the virus, by a cell culture assay system. The virus‐neutralizing ability of immune serum was also confirmed by injecting virus previously treated with serum into fish. This indicates establishment of acquired immunity in survivors and thus explains why survivors from natural infection are resistant to recurrence of the disease. Young sevenband grouper were immunized twice by intramuscular injections with the recombinant coat protein. Immunized fish produced neutralizing antibodies at high titres for at least 110 days and showed significantly lower mortalities in virus challenge tests. These results suggest the potential for vaccination against VNN in sevenband grouper, which is susceptible to piscine nodavirus at all life‐stages.

Pathogenicity of the Nodavirus Detected from Diseased Sevenband Grouper Epinephelus septemfasciatus.

Viral nervous necrosis (VNN) of sevenband grouper Epinephelus septemfasciatus at grow-out stages, which was characterized by upside down swimming or floating behavior, has been spreading in Japan. The present study describes pathogenicity of a nodavirus found in diseased sevenband grouper. When young sevenband grouper or juvenile redspotted grouper E. akaara were challenged by an intramuscular injection with the filtered homogenate of infected organs (brain and eye), abnormal swimming behavior and mortality were produced, but some affected fish recovered from the disease after exhibiting the characterized behavior. Necrosis and vacuolation of the brain and retinal tissues, characteristic to VNN, were produced in the dead and abnormally-swimming fish and the viral antigens were detected in the degenerated nerve cells by FAT with an anti-SJNNV (striped jack nervous necrosis virus) serum. The infection experiment with redspotted grouper also indicated that rearing water temperature (16-28°C ) influenced development of the disease : higher mortality and earlier appearance of the disease signs were observed at higher water temperatures.

Viral Diseases of Cultured Marine Fish and Shrimp in Korea.

As the marine aquaculture industry has developed, disease problems have increased and caused serious economical losses in the fish production in Korea. Viral diseases are among the major disease problems with mass mortality. Six viruses have been isolated from marine fish and shrimp. This paper reviews the following six viral diseases : viral epidermal hyperplasia (herpesvirus) in flounder, birnaviral disease in flounder and sea breams, viral nervous necrosis (nodavirus) in sevenband grouper, lymphocystis disease (iridovirus) in several marine fishes, snout ulcer disease in tiger puffer and white spot syndrome (baculovirus) in Penaeus chinensis.

Mass Mortality of Cultured Sevenband Grouper, Epinephelus septemfasciatus, Associated with Viral Nervous Necrosis.

Mass mortalities of sevenband grouper Epinephelus septemfasciatus reared in Oita Prefecture were investigated. The outbreaks of the disease occurred in summer seasons (July to October; water temperature 25-28°C) of 1993 and 1994. Affected fish ranging 170 to 1, 850 g in body weight were characterized by upside down swimming and inflation of swimbladder. Histopathologically, necrosis and vacuolation in the central nervous tissues and retina were observed. Although bacteria or parasites associated with the disease were not detected from affected fish and no CPE was observed on RTG-2, BF-2, and EPC cell lines, spherical virus particles, 28 nm in diameter, were found abundantly in the cytoplasm of affected nerve cells of most of the affected fish. The virus was identified as a fish nodavirus (nervous necrosis virus), based on the results of fluorescent antibody technique test with anti-SJNNV rabbit serum and polymerase chain reaction test with primers for amplification of SJNNV coat protein gene. These results suggest that the mortalities of sevenband grouper were at least partly due to viral nervous necrosis (VNN). This is the first report describing VNN of sevenband grouper and the first case of VNN in the adult fish.

Portal Entry and Progression of Betanoda Virus Causing Viral Nervous Necrosis in Sevenband Grouper <i>Epinephelus septemfaciatus</i>

A study was conducted to identify the entry site for the betanodavirus, the causative agent of viral nervous necrosis (VNN) in 375 fish (150 spawn, 150 fry and 75 fingerling) during the period from March to August 2003. Highly susceptible fish, sevenband grouper Epinephelus septemfasciatus to sevenband grouper nervous necrosis virus (SGNNV) was used as experimental fish. Fish were placed into the seawater containing 104 TCID50 mL-1 of the viral isolate for 1 h. Samples for virus isolation were randomly collected at an interval of 3 h, 12 h, 24 h and daily, and continued until 5 to14 days depending on the mortality. The inoculated virus could first recover from the mouth including nostril at 3 h of exposure and then from fin at 12 h, and within 1 d it was detected from all the organs examined at titers ranging from 104 to 107 TCID50 g-1. The highest titer was found at day 5 in the target organs of central nervous system (CNS), i.e., the brain, bone including spinal cord, and eye. The results suggest that nasal route the initial route for the portal entry of betanodavirus into fish. Key words: Betanodavirus; viral nervous necrosis; virus titration; portal entry, nasal route; sevenband grouper doi: 10.3329/bjvm.v2i1.1942 Bangl. J. Vet. Med. (2004). 2 (1) : 83-87