betanodaviruses ( family nodaviridae ) are the causative agents of viral nervous necrosis ( vnn ) or viral encephalopathy and retinopathy ( ver ) in a variety of cultured marine fishes worldwide .
necrosis and the vacuolation of central nervous tissues ( brain and spinal cord ) and eye retina are the most characteristic lesions of vnn , and affected fish shows abnormal swimming behavior .
the spread of vnn among populations of cultured marine fish may be attributable to either vertical   or horizontal   transmission . the first description of vnn in korea was reported apparently in cultured groupers ( epinephelus septemfasciatus ) ,
recently , oh et al .   reported mass mortality in hatchery - reared red drum ( sciaenops ocellatus ) , which was associated with a betanodavirus .
vnn in the guppy ( poicelia reticulata ) , a freshwater ornamental fish , was described previously , though the viral etiology was not fulfilled .
korea imports a variety of aquarium ( marine or freshwater ) fishes from southeast asian countries ; the number of such imported species increases annually .
however , appropriate quarantine practices are often neglected or overlooked in the importation of aquarium fishes and invertebrates in many countries . as a result ,
imported fishes and invertebrates sometimes die of infections soon after arrival , or during transport . in order to know ,
we evaluated apparently healthy fish and invertebrate samples collected from a commercial aquarium in korea for the presence of betanodavirus , by polymerase chain reaction ( pcr)-based techniques , to determine whether these imported aquarium fishes and invertebrates might be one of the sources of vnn .
from november 2005 to february 2006 , 237 samples of marine ( 65 species ) and freshwater ( 12 species ) fishes and marine invertebrates ( 4 species ) were collected from a commercial aquarium in seoul , korea . from these aquarium fish samples , 20 samples ( 8 species )
other aquarium fishes were imported from different sources , such as japan with 58 samples ( 25 species ) , singapore with 58 samples ( 17 species ) , the amazon river with 45 samples ( 11 species ) , indonesia with 22 samples ( 5 species ) , the philippines with 13 samples ( 5 species ) , australia with 3 samples ( 2 species ) , the united states of america with 9 samples ( 1 species ) , canada with 1 sample ( 1 species ) , the caribbean sea with 1 sample ( 1 species ) and the pacific ocean with 1 sample ( 1 species ) . with regard to the invertebrate samples , 2 samples ( 2 species ) were from japan , 2 samples ( 1 species ) were from hawaii , and 2 samples ( 1 species ) were from the pacific ocean .
the brains or other pooled organs were collected aseptically from the samples , then maintained at a temperature of -80 until use .
total rna was extracted from the brains or pooled organ tissues using an rna extraction kit with trizol reagent ( invitrogen , usa ) , in accordance with the manufacturer 's instructions . in brief , the tissues were homogenized with trizol reagent and shaken with chloroform , then centrifuged for 15 min at 12,000  g. rna in the aqueous phase was precipitated with isopropanol , then dissolved in diethylpyrocarbonate - treated water ( biosesang , korea ) .
pcr amplification was conducted using primers ( bnv - rt , bnv - ur1 , bnv - uf1 ) for rt - pcr and ( bnv - ur2 , bnv - uf2 ) for nested pcr designed for the target regions ( 570 bp and 420 bp ) of the sgnnv rna2 , as previously described .
after reverse transcription using reverse transcriptase superscript ii ( invitrogen , usa ) at 45 for 60 min , pcr was conducted using ex taq polymerase ( takara , japan ) with 30 cycles of denaturation at 94 for 30 s , annealing at 57 for 20 s , and a final extension step at 72 for 60 s. nested pcr was conducted using the protocol described above .
the rnas from the uninfected redspotted grouper ( e. akaara ) larvae were used as negative controls for both rt - pcr and nested pcr .
from november 2005 to february 2006 , 237 samples of marine ( 65 species ) and freshwater ( 12 species ) fishes and marine invertebrates ( 4 species ) were collected from a commercial aquarium in seoul , korea . from these aquarium fish samples , 20 samples ( 8 species )
other aquarium fishes were imported from different sources , such as japan with 58 samples ( 25 species ) , singapore with 58 samples ( 17 species ) , the amazon river with 45 samples ( 11 species ) , indonesia with 22 samples ( 5 species ) , the philippines with 13 samples ( 5 species ) , australia with 3 samples ( 2 species ) , the united states of america with 9 samples ( 1 species ) , canada with 1 sample ( 1 species ) , the caribbean sea with 1 sample ( 1 species ) and the pacific ocean with 1 sample ( 1 species ) . with regard to the invertebrate samples , 2 samples ( 2 species ) were from japan , 2 samples ( 1 species ) were from hawaii , and 2 samples ( 1 species ) were from the pacific ocean .
the brains or other pooled organs were collected aseptically from the samples , then maintained at a temperature of -80 until use .
total rna was extracted from the brains or pooled organ tissues using an rna extraction kit with trizol reagent ( invitrogen , usa ) , in accordance with the manufacturer 's instructions . in brief , the tissues were homogenized with trizol reagent and shaken with chloroform , then centrifuged for 15 min at 12,000  g. rna in the aqueous phase was precipitated with isopropanol , then dissolved in diethylpyrocarbonate - treated water ( biosesang , korea ) .
pcr amplification was conducted using primers ( bnv - rt , bnv - ur1 , bnv - uf1 ) for rt - pcr and ( bnv - ur2 , bnv - uf2 ) for nested pcr designed for the target regions ( 570 bp and 420 bp ) of the sgnnv rna2 , as previously described .
after reverse transcription using reverse transcriptase superscript ii ( invitrogen , usa ) at 45 for 60 min , pcr was conducted using ex taq polymerase ( takara , japan ) with 30 cycles of denaturation at 94 for 30 s , annealing at 57 for 20 s , and a final extension step at 72 for 60 s. nested pcr was conducted using the protocol described above .
the rnas from the uninfected redspotted grouper ( e. akaara ) larvae were used as negative controls for both rt - pcr and nested pcr .
the results of betanodavirus detection by rt - pcr and nested pcr are summarized in table 1 , and the detection rate in nested pcr was 11/237 ( 4.64% ) .
marine aquarium fish with no clinical signs positive for nodavirus by nested pcr were as follows : 3 of the 58 samples from japan , 3 of 58 from singapore , 1 of 13 from indonesia and 1 of 9 from the united states of america .
2 of 45 freshwater aquarium fish from the amazon river also tested positive for nodavirus by nested pcr .
1 of 2 marine invertebrates obtained from japan also tested positive for nodavirus by nested pcr .
other marine aquarium fish from korea ( 20 ) , the philippines ( 13 ) , australia ( 3 ) , canada ( 1 ) , the pacific ocean ( 1 ) , the caribbean sea ( 1 ) as well as freshwater aquarium fish from indonesia ; and marine invertebrates from hawaii ( 2 ) and the pacific ocean ( 2 ) tested negative for nodavirus by both rt - pcr and nested pcr tests .
positive nested pcr results were obtained from the brain samples of 8 apparently healthy marine aquarium fish species : shrimp fish ( aeoliscus strigatus ) , milkfish ( chanos chanos ) , three spot damsel ( dascyllus trimaculatus ) , japanese anchovy ( engraulis japonicus ) , pinecone fish ( monocentris japonica ) , blue ribbon eel ( rhinomuraena quaesita ) , look down fish ( selene vomer ) and the yellow tang ( zebrasoma flavesenes ) .
other positive nested pcr results were obtained from the brains of 2 apparently healthy freshwater fish species , south american leaf fish ( monocirrhus polyacanthus ) , red piranha ( pygocentrus nattereri ) and the marine invertebrate , the spiny lobster ( pamulirus versicolor ) .
representative amplicons of the fish and invertebrate samples tested positive on nested pcr are shown in fig . 1 .
the positive data from the nested pcr results show that betanodavirus is present in both marine and freshwater fish , as well as marine invertebrates , displayed in one commercial aquarium in korea .
korea imports a variety of aquarium fish from different countries , and the majority of these fish or invertebrates are imported without appropriate quarantine protocol . in the present system
, there were no specific rules regarding quarantine practices on the importation of aquarium fishes and invertebrates in korea , and there was also an absence of proper documentation from the exporting countries with regard to whether the fishes or invertebrates were pathogen - free or not prior to export .
positive samples were obtained from the brains of 8 different marine fish species imported from 4 different countries : indonesia , japan , singapore   and the usa , in which vnn has previously occurred , and have also been reported in different marine fish species .
nodavirus positive samples were also obtained from the brains of 2 freshwater fish species imported from the amazon river .
it is interesting to note that , until now , there have been no cases of vnn reported on the latin american continent .
other nodavirus - positive sample was obtained from the brain of the spiny lobster , a marine invertebrate , imported from japan . in taiwan , chi et al .  
reported the detection of vnn in other live food organisms , such as crustaceans including the brine shrimp ( artemia sp . )
nauplii , the copepod ( tigriopus japonicus ) , and the shrimp ( acetesinte medius ) .
it has also been reported in taiwan , china , and the french west indies , that freshwater shrimp ( macrobrachium rosenbergii ) with white tail disease ( wtd ) have also been infected with a virus , and the causative pathogen has been identified as the m. rosenbergii nodavirus ( mrnv ) . however , thus far , there have been no reports of betanodavirus infections in aquarium fish and invertebrates in korea . to our knowledge , this study is the first report of betanodavirus in subclinically infected aquarium fish and invertebrates in a korean commercial aquarium .
so , what is the significance of the detection of betanodaviruses from apparently healthy marine or freshwater aquarium fish or marine invertebrates ?
it is possible that samples from exporting countries in which vnn infections have occurred were already infected vertically from broodstock to larvae , or horizontally from fish via water or other by biological organisms , thus becoming carriers of the nodavirus prior to transport to the importing countries .
the results of the present study indicated that approximately 4.64% of the fish or invertebrate samples imported from different countries were tested positive for nodavirus in the korean commercial aquarium . in the cases in this study ,
the brains were usually negative on rt - pcr , but were confirmed positive on nested pcr , which suggests that the samples had been latently infected with betanodaviruses .
another possibility is that the imported samples might also be infected soon after transport via horizontal transmission , as the fishes are usually maintained together in one aquarium with other subclinically infected wild fishes collected from the korean sea .
sohn et al .   previously reported the first incidence of vnn in a cage - cultured grouper ( e. septemfasciatus ) on the southern coast of korea .
recently , incidences of nodavirus disease outbreaks have been recorded in cultured marine fish such as the red drum ( s. ocellatus ) .
these nodavirus - contaminated fish or invertebrates may cause problems , as they may die soon after arrival or during transport , due to the combination of stress and nodavirus infection which could entail economic losses .
we are also unable to predict what the consequences are likely to be when betanodaviruses are introduced to different countries , due primarily to a lack of proper documentation and limited information regarding the factors relevant to betanodavirus infections in the exporting countries .
the office international des epizooties ( oie ) has detailed provisions regarding the importation and exportation of aquatic animals , as well as aquaculture products targeted at avoiding the risk of spreading aquatic animal diseases .
furthermore , these subclinically infected samples may constitute a persistent potential source of nodavirus from exporting countries for susceptible fish species cultured in korea .
it can not yet be established as to what the source of the virus may be , as well as the most likely route of infection . due to the paucity of reports regarding the detection of betanodaviruses in healthy aquarium fish and invertebrates ,
the results of this study indicate the possibility of a new route of the infection into cultured fish populations .
the fact that this virus was detected in produce imported from foreign countries implies an urgent necessity for the establishment of a quarantine system in korea . until such laws have been implemented ,
imported aquarium fishes or invertebrates should be randomly assessed by pcr - based techniques , either on a routine basis , to verify that they are pathogen - free ; in cases in which fish or invertebrate are infected , a particular batch of samples should not be accepted , or appropriate treatments should be administered prior to clearance and domestic distribution throughout korea .
 betanodaviruses are the causative agents of viral nervous necrosis ( vnn ) in cultured marine fish . 
 a total of 237 apparently healthy aquarium fish , marine ( 65 species ) and freshwater ( 12 species ) fishes and marine invertebrates ( 4 species ) , which were stocked in a commercial aquarium in seoul , south korea , were collected from november 2005 to february 2006 . 
 the brains of the fish and other tissues of the invertebrates were examined by reverse transcriptase - polymerase chain reaction ( rt - pcr ) and nested pcr to detect betanodavirus . 
 positive nested pcr results were obtained from the brains of 8 marine fish species ( shrimp fish aeoliscus strigatus , milkfish chanos chanos , three spot damsel dascyllus trimaculatus , japanese anchovy engraulis japonicus , pinecone fish monocentris japonica , blue ribbon eel rhinomuraena quaesita , look down fish selene vomer , yellow tang zebrasoma flavesenes ) , 1 marine invertebrate species ( spiny lobster pamulirus versicolor ) , and 2 freshwater fish species ( south american leaf fish monocirrhus polyacanthus and red piranha pygocentrus nattereri ) . 
 the detection rate in nested pcr was 11/237 ( 4.64% ) . 
 these subclinically infected aquarium fish and invertebrates may constitute an inoculum source of betanodaviruses for cultured fishes in the korean peninsula . 