Research On Viral Nervous Necrosis Of Half Smooth Tongue Sole (Cynoglossus Semilaevis)

Half smooth tongue sole (Cynoglossus semilaevis) is an emerging mariculturevariety and the industry of half smooth tongue sole increase rapid in the coastal areas ofnorthern China. Due to the industry grow too fast and lack the measures for diseasecontrol, the disease of half smooth tongue sole is serious and cause a large number ofeconomic loss. In August2012, the larvae of half smooth tongue sole, in a hatchery innorthern of China, started to appear explosive death. According to the symptoms of fishdisease and molecular biological detection, we suspected the half smooth tongue solewas infected by nervous necrosis virus. Based on the field investigation of the disease,artificial challenge experiment, histopathology and taxonomic identification ofmolecular biology, we confirmed the pathogen infected half smooth tongue sole was redgrouper nervous necrosis virus (RGNNV) and half smooth tongue sole was a newsusceptible host for nervous necrosis virus.The field survey of virus in hatchery showed the nervous necrosis virus mainlyaffect the13~30dph (days post hatching) of half smooth tongue sole and the cumulativemortality rate could reach90%~100%. The fish above50dph could also affected, butcould not show symptoms. The time of incidence of disease is7~8months, andtemperature of water is22~24oC. The infected fish show a variety of erratic swimmingbehaviour patterns, such as spiralling, lying down at the tank bottom, swim rapidly incircles and loss of appetite, some fish also show body deformity.The challenge experiment of half smooth tongue sole showed that the fish at25dph has a high sensitivity for nervous necrosis virus and has typical symptoms of viralnervous necrosis disease. The fish of25dph began to die at2th day infected by nervousnecrosis virus, and all died at4th. The mortality of fish at50dph was not sensitive tothe virus and did not show the diversity of cumulative mortality rate with the control.The histopathology of natural infected half smooth tongue sole fish (20dph) and thefish (25dph) of challenge experiment showed typically pathological phenomena of viralnervous necrosis. The cells of brain and retina were basophilic increase and vacuolation, and the result of RT-PCR of25dph is positive. But the fish (50dph) of challengeExperiment did not show the vacuolation at the brain and retina. The result of RT-PCRof50dph is positive, but weaker than the25dph. So it demonstrate that the half smoothtongue sole of50dph is not cause disease, but with nervous necrosis virus by the resultof RT-PCR and histopathology. The phenomena was consistent to the low mortality rate.In order to determine the evolution of half smooth tongue sole nervous necrosisvirus, this study measured the complete sequence of viral nervous necrosis of halfsmooth tongue sole (Cynoglossus semilaevis) by5’ RACE and cRACE. The genomesequencing results show that the virus is composed of two positive single-stranded RNA(RNA1and RNA2). The RNA1is3104nt and contains a982-amino-acid open readingframe (ORF) from79nt to3027nt, encoding RNA-dependent RNA polymerase. RNA2is1433nt and has a single ORF from27nt to1043nt, encoding coat protein of338-amino-acid. RNA3started at downstream of the RNA1sequence at2753nt to2980nt, encoding the B2protein of76-amino-acid. The sequence data derived from theRNA1and RNA2gene were submitted to the GenBank database under accessionnumber KJ541747and KJ541748. According to the sequence alignment results ofnervous necrosis virus of half smooth tongue sole. The similarity of RNA1sequence tothe reported RGNNV is96%~99%, and the similarity to SJNNV, BFNNV and TPNNVis approximately82%. The similarity of RNA2sequence to the reported RGNNV is96%~98%, and the similarity to SJNNV, BFNNV and TPNNV is approximately77%~86%. Therefore, we can be sure that the virus found in half smooth tongue sole is a newisolate belong in the RGNNV genotype and named as CsCN128. The phylogenetic treesresults show that the CsCN128shared high sequence similarity to other RGNNVisolates of Japan and China reported, but separation to the SJNNV, BFNNV andTPNNV.Because of lack an effective vaccine, the treatment of ozone is a common measureto inactivate single stranded RNA virus. But it has not effective methods to evaluate thedamaged effects of virus by ozone up to now. In this report, according to the Ozone caninactive viral RNA by fracturing, a special reverse transcription primer and a set of PCRprimers were designed based on the3’ end and5’ end of RGNNV RNA2, respectively.And a special LR RT-PCR (Left reverse transcript Right amplify RT-PCR) wasdeveloped to detect the relatively overall chain of RGNNV RNA2. The concentraion ofprimers, Mg2+, dNTPs as well as annealing temperature of LR RT-PCR were optimized in this study. The sensitivity of LR RT-PCR was1pg to RGNNV RNA2and there wereno cross reactions with genomic RNA from healthy fish and DNA from commonbacteria and viruses. The LR RT-PCR was then used to evaluate damaged effects ofRGNNV RNA2by ozone. As concentration of ozone increased from0.3mg/L to0.5mg/L,1mg/L and2mg/L, the amplified products of LR RT-PCR decreased and wasfinally undetectable. Former reported routine RT-PCR method for RGNNV detectiondid not reflect above tendency. This report demonstrated that the LR RT-PCR canquickly and accurately evaluate disinfection effects of RGNNV by ozone and can bewidely used in hatcheries of groupers.