Farmed Flounder (paralichthys Olivaceus) Viral Nervous Necrosis Disease Investigation And Diagnostic Technology Research

Recently,viral nervous necrosis(VNN)or viral encephalopathy and retinopathy (VER)had emerged as a serious threat to cultured marine and freshwater fish worldwide.Currently,more than 40 species of fish including Anguilliformes, Gadiformes,Perciformes,Pleuronectiformes,Tetraodontiformes have been reported to be affected,The disease can transmitted both horizontally via the water pathway and vertically via the egg.It usually occurs in larvae or juveniles resulting in high mortality to over 90%often up to 100%.Hence,this infection had been listed as the severe fishing disease by Office International Desépizooties(OIE).Viral nervous necrosis(VNN)was first discovered in cultured Paralichthys olivaceu in China.At present,antibiotics and chemic drugs have little function,there's also no efficent therapeutic method.So,the detecting methods must be developed for the NNV in order to prevent VNN as soon as possible.On the basis of the epidemic investigation of the disease,we developed the nested RT-PCR method to dectect VNN of Paralichthys olivaceu.The results as follows:1.Viral nervous necrosis(VNN)of Paralichthys olivaceu was investigated in Tianjin.The investigation confirmed that it usually occurs in juveniles, 3.50±0.48cm in average length and 1.2±0.43g in average weight.Clinical signs of VER.include lack of appetite,changes in pigmentation,neurological signs such as abnormal swimming,and mortality rates of up to 70%.Aggregates of virus particles and the vacuolation of the cytoplasm of infected brain cells have been observed by transmission electron microscopy.The particles were found in paracrystalline arrays,approximately 30～35nm in diameter,icosahedron and non-enveloped.2.Through the aetiological investigation,we used the OIE's primer designed in conformity to highly conserved region of the virus gene,which amplified a fragment of 421bp and been confirmed that PONNV belongs to RGNNV genotype through the nucleotide sequence analysis and phylogenetic analysis.This virus has the highest similarity with the ECNNV and MNNV in China,are 98%and 97%respectively.The similarities with RGNNV,DLEV and DNNV are also more than 90%respectively.As for other genotypes,the similarities are less than 80%.For alphanodaviruses like BBV,BOV and FHV,the similarities are less than 10%.Consequently,this virus belongs to RGNNV genotype.3.The author used Primer Premier5.0 to designe three suits of nested reverse transcription-polymerase chain reaction(nested RT-PCR)primers by genebank,they referred to three kinds of marine fish's genome sequences(DNNV,JFNNV and DLEV)which were infected by nervous necrosis virus(NNV).Through the optimize the reaction system,the methords for detecting Paralichthys olivaceu which were the pathogen associated with VNN were developed.Three suits of primers(M2,L2,M5,L5;M2,L4,M5,L5; M3,L3,M3,L5)amplified 866bp,398bp,605bp,297bp,400bp,299bp respectively.No specific fragments were amplified when other principal oceanic fish pathogens were used as negative controls.The sensitivity of the three kinds of RT-PCR methods for detecting PONNV were 6.7pg,67 pg and 67pg respectively.