DNA FRAGMENT OF PENAEUS-MONODON BACULOVIRUS PMNOBII GIVES POSITIVE IN-SITU HYBRIDIZATION WITH WHITE-SPOT VIRAL-INFECTIONS IN 6 PENAEID SHRIMP SPECIES

PmNOBII was first described from experimentally infected shrimp, but c ontemporary reports showed that white-spot virus infections in several penaeid shrimp species exhibited similar gross signs and histopatholo gy. Using laboratory infected specimens of Penaeus monodon, DNA of the non-occluded Baculovirus PmNOBII was extracted and digested with BamH I and EcoRI. Resulting DNA fragments were ligated with Bluescribe vect or using T4 ligase and competent cells of Escherischia coli JM 107 wer e transformed. Two recombinant clones that gave negative hybridization with P. monodon DNA but positive hybridization with PmNOBII DNA were selected. Inserted DNA fragments of 0.9 kbp and 4.2 kbp were obtained from these clones after plasmid digestion with BamHI and EcoRI. These fragments were subsequently labeled with digoxygenin for visualization and tested using the in situ DNA hybridization technique with tissues from PmNOBII infected and non-infected laboratory shrimp. For viral i nfected nuclei identified by H and E staining in parallel samples, the 4.2 kbp fragment gave a stronger DNA hybridization signal than did th e 0.9 kbp fragment. The 4.2 kbp fragment was then used for in situ DNA hybridization tests with commercially or experimentally cultivated sh rimp specimens showing gross signs and histopathology characteristic o f white-spot virus infection. Field signs of the disease included gene ral reddish coloration, white granules of 1-2 mm under the cuticle and rapid mortality. Normal histology (H and E) revealed Cowdry-A type nu clear inclusions that developed to produce basophilic hypertrophied nu clei typical of PmNOBII, and transmission electron microscopy revealed characteristic rad shaped virions. All these specimens gave positive hybridization results, and included cultivated shrimp specimens of Pen aeus chinensis, P. indicus, P. japonicus, P. merguiensis, P. monodon a nd P. vannamei obtained from various countries in Asia between August 1993 and January 1995. The data indicate that PmNOBII, or closely rela ted variants, are currently responsible for a widespread epizootic in the Asian shrimp farming industry.