Manual of Diagnostic Tests for Aquatic Animals (2003)
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VIRAL ENCEPHALOPATHY AND RETINOPATHY
SUMMARYViral encephalopathy and retinopathy (VER), or viral nervous necrosis (VNN) has been reported as a serious disease of larval and juvenile and sometimes older marine fish that occurs almost world-wide except for Africa (23). To date, the disease has been reported in at least 30 fish species, with the greatest impact being in sea bass (Lates calcarifer  and Dicentrarchus labrax ), groupers (Epinephelus akaara , E. fuscogutatus , E. malabaricus , E. moara , E. septemfasciatus , E. tauvina , E. coioides  and Cromileptes altivelis ), jack (Pseudocaranx dentex ), parrotfish (Oplegnathus fasciatus ), puffer (Takifugu rubripes ), and flatfish (Verasper moseri , Hippoglossus hippoglossus , Paralichthys olivaceus , Scophthalmus maximus ).
Virus particles of about 25-30 nm in diameter have been visualised in affected fish, and the agents in striped jack, barramundi, and European sea bass have been characterised and placed in the genus Betanodavirus, family Nodaviridae (5, 21). Immunological studies have shown relationships between striped jack nervous necrosis virus (SJNNV, the type species of the genus Betanodavirus) and the other betanodaviruses. Genomic classification of betanodaviruses has shown close relationships, with major groupings being SJNNV-type, tiger puffer nervous necrosis virus (TPNNV)-type, barfin flounder nervous necrosis virus (BFNNV)-type and red-spotted grouper nervous necrosis virus (RGNNV)-type (27). Complete nucleotide sequences of RNA1 and RNA2 of SJNNV and GGNNV (a grouper betanodavirus) have been reported (15, 29).
All diseases are characterised by a variety of neurological abnormalities, such as erratic swimming behaviour (spiral, whirling or belly-up at rest) and vacuolation of the central nervous tissues. Usually there is also vacuolation of the nuclear layers of the retina. In general, younger fish have more severe lesions; older fish have less extensive lesions and these may show a predilection for the retina (23). Intracytoplasmic inclusions have been described in brain cells of European sea bass, barramundi, Japanese parrotfish, and brownspotted grouper. Neuronal necrosis has been described in most species.
Interesting differences with regard to the occurrence and severity of the diseases are shown in Table 1. There are considerable variations in the age at which disease is first noted and the period over which mortality occurs. In general, the earlier the signs of disease occur, the greater is the rate of mortality. Although disease occurrence at the juvenile stages in some species is very rare, mass mortalities often occur at juvenile to young stages in the other fish species, but usually do not reach 100%, indicating the age-dependence of susceptibility (23). Mortalities have been reported in production-size European sea bass (18) and grouper (9), but even in these cases mortalities were greatest in younger fish.
It has been demonstrated that vertical transmission of the causative agent occurs in Pseudocaranx dentex and this fact is reflected by the early occurrence of clinical disease. This finding led to the successful control of VNN of larval striped jack, where elimination of virus-carrying broodstock by reverse-transcription polymerase chain reaction and disinfection of fertilised eggs by ozone were applied (20, 24). Paradoxically, ovarian infection has also been reported in Dicentrarchus labrax in which disease is usually not seen until about 30 days post-hatch. The mode of transmission/introduction of the viruses, other than in gametes and by cohabitation, has not been demonstrated, but the possibilities include influent water, juvenile fish held on the same site, and carriage on utensils, vehicles, etc. It is possible that these small viruses are quite resistant to environmental conditions (8) and therefore readily translocated by commercial activities. Vaccination using a recombinant capsid protein is at the experimental stage (14, 30).
Table 1. Important features of VER/VNN of larval and juvenile fish
Earliest occurrence of disease
Latest occurrence of new outbreaks
Usual mortality rate
Highest mortality rate
(24 days post-hatch
<40 mm total length
Up to 100%
14 days post-hatch (7-8 mm total length)
<40 mm total length
Up to 100%
<20 days post-hatch (8 mm total length)
Up to 100%
DIAGNOSTIC PROCEDURESPresumptive diagnosis of viral encephalopathy and retinopathy (VER) or viral nervous necrosis (VNN) can be made on the basis of the appearance of vacuoles in the brain, spinal cord and/or retina as seen by light microscopy. However, individual fish with the presence of only a few vacuoles in the nervous tissues pose a difficult diagnostic problem.
Virus particles can be visualised in affected brain and retina by both positive and negative staining. In positively stained material, the virus is mainly associated with vacuolated cells and, especially, any inclusions. The reported particles vary in size from 22 to 34 nm arranged intracytoplasmically in crystalline arrays, or as aggregates and single virions both intra- and extracellularly. The virus is nonenveloped and icosahedral in shape.
All betanodaviruses can be detected by the indirect fluorescent antibody test (IFAT) or immunohistochemistry with a rabbit anti-SJNNV serum (12, 16). Most can be detected by reverse-transcription polymerase chain reaction (RT-PCR) with a single primer set designed to amplify the T4 region (427 bases) of SJNNV coat protein gene (27, 28) (this primer set can be used for detection of all the genotypic variants of betanodaviruses with only one exception ). Although other immunological methods, such as the enzyme-linked immunosorbent assay (ELISA) or neutralisation test, are available for virus identification, they can only be used for some of betanodaviruses because of limited serological information.
The betanodaviruses can be cultured in a fish cell line, SSN-1, which is derived from striped snakehead (7, 16). A clonal cell line, E-11, derived from the SSN-1 cell line is useful for qualitative and quantitative analyses of all the betanodaviruses (17). It is notable that both SSN-1 and E-11 cells are infected by a spontaneously productive C-type retrovirus designated SnRV (13, 17).
Due to insufficient knowledge of the serological responses of fish to virus infections, the detection of fish antibodies to viruses has not thus far been accepted as a routine screening method for assessing the viral status of fish populations.
Fish material suitable for virological examination is:
Sampling procedures: see Chapter I.1. Section B.
. Asymptomatic fish (apparently healthy fish): whole larvae or small juveniles; brain, spinal cord, and eyes for larger size fish and/or ovarian fluid from broodfish at spawning time.
. Clinically affected fish: whole larvae or small juveniles; brain, spinal cord, and eyes for larger size fish.
1. Standard Screening Method for VER/VNN
1.1. Isolation of betanodaviruses in cell culture
Cell line to be used: SSN-1 or E-11 (a cell clone of SSN-1)
a) Inoculation of cell monolayers
i) Fish samples are homogenised with nine volumes of Hanks' balanced salt solution (HBSS), centrifuged and filtered (0.2 µm membrane filter).
ii) Tenfold dilutions of the virus filtrate are inoculated in monolayers of cells cultured using Leibovitz L-15 medium or other medium supplemented with 5% fetal bovine serum (FBS). Inoculate at least 2 cm2 of drained cell monolayer with 100 µl of each dilution.
iii) Allow to adsorb for 1 hour at room temperature, add the medium supplemented with 5% FBS and incubated at 20-25°C.
Note: Optimal growth temperatures are different among the four genotypic variants: 25-30°C for RGNNV-type, 20-25°C for SJNNV-type, 20°C for TPNNV-type, and 15-20°C for BFNNV-type (16, 17).
b) Monitoring incubation
i) Follow the course of infection in positive controls and other inoculated cell cultures by daily microscopic examination for 10 days.
ii) If cytopathic effect (CPE) appears in those cell cultures inoculated with the dilutions of the tested homogenate supernatants, betanodavirus identification procedures must be undertaken (see Section 1.2. below).
Note: CPE in SSN-1 or E-11 cells is characterised by thin or rounded, refractile, granular cells with vacuoles, the monolayer then partially or completely disintegrates (7, 16).
iii) If no CPE occurs after 10 days of incubation, subcultivation of the inoculated cell cultures must be performed.
c) Subcultivation procedures
i) Collect aliquots of cell culture medium from all monolayers inoculated with organ homogenates.
ii) Inoculate cell monolayers as described in Section1.1.a.
iii) Incubate and monitor as described in Section 1.1.b.
1.2. Identification of betanodavirus isolated in cell culture
a) Indirect fluorescent antibody test
i) Prepare monolayers of cells in 2 cm2 wells of cell culture plastic plates or on cover-slips (or chamber slides) in order to reach around 70% confluency, which is usually achieved within 24 hours of incubation at 25°C.
ii) Inoculate the virus suspensions to be identified by making tenfold dilution steps directly in the cell culture wells or flasks.
iii) Incubate at 20°C or 25°C for 48-72 hours (See Section 1.1.a. Note).
iv) Remove the culture medium, rinse once with PBS, then fix with methanol for 10 minutes.
v) Allow the cell monolayers to air-dry.
vi) Treat the cell monolayers with a rabbit anti-betanodavirus serum for 30 minutes at 37°C in a humid chamber, and rinse four times with PBS-Tween 80 (PBST).
vii) Treat the cell monolayers for 30 minutes at 37°C with commercially available fluorescein isothiocyanate-conjugated anti-rabbit Ig antibody, and rinse with PBST.
viii) Examine the treated cell monolayers on plates immediately, or mount the cover-slips using glycerol saline, pH 8.5, prior to microscopic observation.
b) Reverse-transcription polymerase chain reaction
i) Total RNA is extracted from virus-inoculated cells by a commercially available RNA extraction kit according to the manufacturer's instructions.
ii) There are several published primers for RT-PCR amplification of betanodaviruses. Primers, R3 (5'-CGA-GTC-AAC-ACG-GGT-GAA-GA-3') and F2 (5'-CGT-GTC-AGT-CAT-GTG-TCG-CT-3'), designed to amplify the T4 region (427 bases) of SJNNV coat protein gene, are available for all genotypic variants (27).
2. Diagnostic Methods for Clinically Diseased Fish
2.1. Direct detection in fish tissues
a) Indirect fluorescent antibody test
i) Fish samples fixed in 10% buffered formalin are dehydrated and embedded in paraffin wax. Deparaffinise sections and rehydrate to PBS.
ii) The sections are treated with 0.1% trypsin in PBS at 37°C for 30 minutes.
iii) After washing with cold PBS, proceed as described in Section 1.2.a. steps vi to viii.
iv) Specific fluorescence is observed in the cytoplasm of the affected cells in brain, spinal cord, or retina.
b) Immunohistochemistry (avidin-biotin-alkaline phosphatase technique) (12)
i) Prepare paraffin sections as described above in Section 2.1.a.
ii) Add a blocking solution, for example 5% bovine serum albumin (BSA) in Tris-buffered saline (TBS) for 20 minutes.
iii) Incubate with the anti-betanodavirus rabbit serum for 30 minutes, and wash in TBS.
iv) Add biotinylated goat anti-rabbit Ig for 30 minutes, and wash in TBS.
v) Add streptavidin alkaline phasphatase complex, incubate for 30 minnutes, and wash in TBS.
vi) Add fuchsin chromogen reagent for 5 minutes, and wash in tap water
vii) Counterstain with haematoxylin.
viii) A positive result is indicated by red colour.
c) Reverse-transcription polymerase chain reaction
i) The fish sample is homogenised with distilled water treated with 0.1% diethyl pyrocarbonate.
ii) Centrifuge at 10,000 g for 10 minutes.
iii) Using the supernatant, continue as described in Section 1.2.b.
2.2. Virus isolation in cell culture
See Section 1.1.
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