Susceptible Host Survey Of Decapod Iridescent Virus 1(DIV1)

Infection with Decapod iridescent virus 1(IDIV1)is an emerging disease which can cause high rates of mortality in crustaceans(reaching up to 100%).It has been formally accepted by the World Organisation for Animal Health(OIE).The causative agent is Decapod iridescent virus 1(DIV1),which consists of two viral isolates,namely Shrimp hemocyte iridescent virus(SHIV)and C.quadricarinatus iridovirus(CQIV).The genomic similarity of the two strains is 99%.Of them,SHIV was firstly isolated,identified,sequenced and named by our lab.In addition,it was here that a specific detection method was first devised.IDIV1 firstly happened in Fujian,China.In2017,Chinese government officially launched a monitoring program for IDIV1.The monitoring program was implemented in 13 provinces or municipalities.IDIV1 has occurred in 6 provinces.As there are no effective measures to control the spread of IDIV1,this study aims at investigating and assessing the sensibility of various crustaceans against DIV1 infection.This investigation will reveal the potential natural and susceptible hosts thereby providing understructure for both disease prevention and controlling IDIV1,and the healthy culturing of crustaceans.1.Susceptibility of Exopalaemon carinicauda to the infection with decapod iridescent virus 1In this study,ridgetail white prawns Exopalaemon carinicauda were challenged using per os with debris of Penaeus vannamei infected by SHIV strain 20141215 and using intramuscular injection(im)with raw extracts of SHIV from P.vannamei.The infected E.carinicauda showed obvious clinical symptoms,including weakness,empty gut and stomach,pale hepatopancreas,and death.The cumulative mortality in the groups of per os,im,and the control were(50.0±26.5)%,(76.7±18.3)%,and 6.7%,respectively.Results of TaqMan probe-based real-time quantitative PCR showed that the moribund and surviving individuals with clinical signs of E.carinicauda were SHIV-positive.Histological examination showed that there were darkly eosinophilic and cytoplasmic inclusions,of which some were surrounded with or contained tiny basophilic staining,and pyknosis in the hemocytes in hepatopancreatic sinus,hematopoietic cells,and the cuticular epithelium.According to the result of in situ DIG-labeling-loop-mediated DNA amplification(ISDL),positive hybridization signals were observed in the stomach,hematopoietic tissue,the cuticular epithelium,and the hepatopancreatic sinus of infected E.carinicauda.Transmission electron microscopy(TEM)of ultrathin sections showed the presence of icosahedral SHIV particles existed in the hepatopancreatic sinus,the gills,pereiopods,muscles,and uropods of the infected E.carinicauda.The assembled virions,which were mostly distributed along the edge of the cytoplasmic virogenic stromata near cellular membrane of infected cells,were enveloped and about 150 nm in diameter.Taken together,the results of molecular biological tests,histopathological examination,ISDL,and TEM confirmed that E.carinicauda was one of the susceptible hosts of DIV1.2.Investigation on susceptibility to decapod iridescent viruses 1 in several crustaceansIn this study,the challenge tests were carried out on Portunus trituberculatus,Panulirus ornatus,Helice tridens tientsinensis,Paguridae using the methods of per os or injection as previously described,and Artemia fed by SHIV-chlorella adsorption.P.trituberculatus was successfully infected by SHIV,whereas P.ornatus,Helice tridens tientsinensis and Paguridae were not.The diseased P.trituberculatus showed that weakness,empty stomach and gut,and even death.The results of real-time PCR showed strong SHIV-positive detection for samples of epitheliums,gonads,gills,hearts,hepatopancreas,stomachs,muscles and guts from P.trituberculatus.Positive signals in blue-violet were also observed in these tissues,based on the results of ISDL.TEM photography showed that large amounts of icosahedral viruses existed in the before mentioned tissues of P.trituberculatus.The viral particles were(136.4±16.1)nm(n=90)in diameter.In the challenge test of Artemia,the load of SHIV decreased with the increase of time after infection.The load of SHIV in Artemia reached 10~6copies/ng-DNA after one day post challenge(1 dpc).However,no SHIV was detected in Artemia at 7 dpc.Similarly,positive signals were only observed in the surrounding of appendages in Artemia at 3 dpc,but not in Artemia at 7 dpc.Together these results show that,P.trituberculatus is a susceptible host of DIV1,while P.ornatus,Helice tridens tientsinensis,Paguridae and Artemia are not.3.Description of a natural infection with decapod iridescent virus 1 in farmed giant freshwater prawn,Macrobrachium rosenbergiiMacrobrachium rosenbergii is a valuable freshwater prawn species in Asian aquaculture.In recent years,a new symptom,generally called‘white head'disease,has caused severe economic losses in China.However,to date,the causative agent has not been determined.In this study,samples of M.rosenbergii,M.nipponense,Procambarus clarkii,M.superbum,Penaeus vannamei,and Cladocera were collected from a farm suffering from‘white head'disease in Jiangsu Province and analyzed in etiology.Pathogen diagnosis based on qPCR tests showed that all these samples were DIV1positive.Histopathological examination revealed dark eosinophilic inclusions and pyknosis in hematopoietic tissue,hepatopancreas and gills of M.rosenbergii and M.nipponense.Positive signals of ISDL were also observed in hematopoietic tissue,hemocytes,the hepatopancreatic sinus,and the antennal gland in M.rosenbergii and M.nipponense.In addition,TEM microscopy showed a large number of SHIV particles with a mean diameter of approximately 157.9 nm in the hematopoietic tissue of M.rosenbergii,and in the virogenic stromata and budding virions in hematopoietic cells of M.rosenbergii.Quantification of DIV1 using TaqMan probe based real-time PCR in different tissues of naturally infected M.rosenbergii showed that the highest DIV1 load was detected in the hematopoietic tissue with a relative abundance of(25.4±16.9)%,whereas the lowest SHIV load was detected in the hepatopancreas and musclewith a relative abundance of(2.44±1.24)%and(2.44±2.16)%,respectively.These preliminary results indicated that DIV1 was the pathogen causing‘white head'disease,and M.nipponense and Pr.clarkii are also susceptible hosts of SHIV.The results of the study concluded that(1)Macrobrachium rosenbergii,Macrobrachium nipponense,Procambarus clarkii,Exopalaemon carinicauda and Portunus trituberculatus are susceptible hosts of DIV1,while Helice tridens tientsinensis,Paguridae,Artemia and Panulirus ornatus are not;(2)crustaceans infected by DIV1 displayed clinical signs of weakness,empty gut and stomach,and E.carinicauda and M.rosenbergii have an obvious symptom that hematopoietic tissue under the base of rostrum showed a slight whiteness;(3)histopathological characteristics of infected tissues such as hepatopancreas,gill and etc.included darkly eosinophilic and cytoplasmic inclusions,of which some were surrounded with or contained tiny basophilic staining,and pyknosis.All these findings will facilitate the understanding the pathogenesis of DIV1 and further research in prevention and controlling IDIV1.Moreover,it will provide technological supports for making strategies to control the spread of IDIV1.