Isolation, Characterization And Detection Of Macrobrachium Rosenbergii Nodavirus

The giant freshwater prawn, Macrobrachium rosenbergii, is the main cultivatedfreshwater prawn in China since introduced in 1970s. The total production of M.rosenbergii reached to 100,000 tons, with total cultivation area of 33,000 hectares, whichmaking China the biggest M. rosenbergii cultivation in the world. The whitish musclediseases, also called as white prawn disease or white tail disease, was a new epidemicsoccurred in the main area of freshwater prawn throughout China. The disease, first recordedin 1994 in Guandong and Guanxi provinces, spread rapidly to other M.rosenbergiiprovinces including Zhejiang, Jiangsu and Shanghai, mainly infected post larvae three totwenty days after desalting. The typical symptom of the disease is whitish muscle inabdomen or opacity muscles, causing serious loss of M.rosenbergii larvae or post larvae inhatcheries and prawn farms.The typical diseased post larvae (PL) were collected in prawn hatcheries in Huzhou,north of Zhejiang province, and used for bacteria isolation, as well as for viruspathogenicity. The bacteria isolated from diseased PL did not showed virulence to health PLat the concentration of 5×10~8 CFU/ml when immersion challenging was used. Thebacteria-free tissue homogenates can infected healthy PL to developed typical symptom ofwhitish muscle, similar with the nature diseased PL, 7 days after immersionchallenging atconcentration of 1:50 and 1:250. The infectivity was resistant to 10% chloroform treatment.With the ultrathin section, a spherical virion was found with the size of 20nm in the muscleconnection. The virus partly purification was carried out with diseased larvae homogenizedin PBS, treated with chloroform followed by PEG precipitation and 2% PTA negativestaining. It could be found plenty of spherical virions with the size of 23-25nm.The nucleic acid of virus was extracted by using the Trizol reagents and analyzed with1% agarose electrophorosis. The results showed two ssRNA fragment sized 3.1, 1.3 kbrespectively, indicating the virus as the member of Nodaviridae. The two RNA bands wereconfirmed as nodavirus RNA1 and RNA2 by southern blot with the probe of MrNVspecific for virus isolated from Gadeloupe, in Carribean gulf. These results indicated thatnodavirus was the main pathogen of whitish muscle diseases of M. rosenbergii larvae in China in recent years. The virus was named as Maerobrachium rosenbergii Nodavirus(MrNV).The diseased PL was used ofr Macrobrachium rosenbergii nodavirus (MrNV)purification with PEG sedimentation followed by ultra-speed centrifugation. The purifiedMrNV was used for immunization of BABL/c mice. Mouse myeloma cells (SP2/0) wereused for fusion with spleen cells from BALB/c immunized with purified MrNV. Positivecolonies were selected by indirect ELISA with MrNV precoated ELISA-plates. Twelvehybridoma cell lines secreting monocolonal antibodies(Mabs) against MrNV were obtainedafter 2-3 times limited dilution with the positive cloning, The titres of twelve Mabs ascitesranged from 1: 105 to 1: 106 by indirest ELISA. Isotypes of the tweleve Mabs weretested by SBA clonotyping system, the result showed that six Mabs were characterized asIgG1, four Mabs were IgG2a and two Mabs were IgG2b. The Mabs was characterizedagainst 43kDa protein, the main capsid of MrNV.The rabbit antiserum was prepared with New Zealand rabbit injected with purifiedMrNV emulsified with Freund's adjuvant The antiserum and monoclonal antibody 2B5against MrNV were used for establishment of TAS-ELISA. The optical condition of TAS—ELISA: rabbit antibody used for plate precoating was 1μg/ml, work concentration of Mab2B5 was 1: 50, 000. The sensitivity of TAS-ELISA to purified MrNV was 0.98ng, withoutcross reaction to WSSV, TSV and other dead prawn postlarvae caused by bacterial infection.The positive detection rate as well as coincident rate were compared with different methodsand showed that TAS-ELISA was better than indirect ELISA and RNA characterizationwith electrophoresis, when the diseased and suspicious prawn samples were used foranalysis. Besides, the easy TAS-ELISA was developed with lower temperature and shorterincubation time, which making TAS-ELISA more practical in field detection.To more detailed information of MrNV for M.rosenbergii the post larvae of the giantfreshwater prawn, Macrobrachium rosenbergii post-larvae was used for artificialchallenging with MrNV, homogenates prepared with diseased post larvae with typicalsymptom of whitish muscle disease. The results showed the high pathogenicity of MrNV topost larvae of the giant freshwater prawn. The disease peak was observed after 9-12 days ofviral challenging, with the typical symptom of whitish muscle similar with the natural outbreak diseases. The homogenates showed pathogenicity to prawn post larvae with thedilution of 1:62500, which is about 0.6ng MrNV. It is showed the pathogenicity differedwhen the prawn kept at different environments. The prawn kept under light showed thehighest survival rate compared with other groups. Meanwhile, the MrNV pathogenicity toBurmese breeding group and local breeding group was carried out. The Burmese breedinggroup showed the slightly resistance to MrNV, with lower mortality and delayed infection.The MrNV distribution was analyzed with immunological enzyme method, which showedtropism of MrNV to muscle and connected tissues.