DNA fragment of Penaeus monodon baculovirus PmNOBII gives positive in situ hybridization with white-spot viral infections in six penaeid shrimp species

Abstract

PmNOBII was first described from experimentally infected shrimp, but contemporary reports showed that white-spot virus infections in several penaeid shrimp species exhibited similar gross signs and histopathology. Using laboratory infected specimens of Penaeus monodon, DNA of the non- occluded baculovirus PmNOBII was extracted and digested with BamHI and EcoRI. Resulting DNA fragments were ligated with Bluescribe vector using T4 ligase and competent cells of Escherichia coli JM 107 were 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 EcoR1. 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 infected nuclei identified by H and E staining in parallel samples, the 4.2 kbp fragment gave a stronger DNA hybridization signal than did the 0.9 kbp fragment. The 4.2 kbp fragment was then used for in situ DNA hybridization tests with commercially or experimentally cultivated shrimp specimens showing gross signs and histopathology characteristic of white-spot virus infection. Field signs of the disease included general reddish coloration, white granules of 1-2 mm under the cuticle and rapid mortality. Normal histology (H and E) revealed Cowdry-A type nuclear inclusions that developed to produce basophilic hypertrophied nuclei typical of PmNOBII, and transmission electron microscopy revealed characteristic rod shaped virions. All these specimens gave positive hybridization results, and included cultivated shrimp specimens of Penaeus chinensis, P. indicus, P. japonicus. P. merguiensis, P. monodon and P. vannamei obtained from various countries in Asia between August 1993 and January 1995. The data indicate that PmNOBII or closely related variants, are currently responsible for a widespread epizootic in the Asian shrimp fanning industry.