Chainarong Wongteerasupaya

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 Escherischia 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 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 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 farming industry.

Multiplex RT-nested PCR differentiation of gill-associated virus (Australia) from yellow head virus (Thailand) of Penaeus monodon.

Abstract A multiplex RT-nested PCR has been developed to detect and differentiate the closely related prawn viruses, gill-associated virus (GAV) from Australia and yellow head virus (YHV) from Thailand. RT-PCR using primers to conserved sequences in the ORF1b gene amplified a 794bp region of either GAV or YHV. Nested PCR using a conserved sense primer and either a GAV- or YHV-specific antisense primer to a divergent sequence differentially amplified a 277bp region of the primary PCR amplicon. Multiplexing the YHV antisense primer with a GAV antisense primer to another divergent sequence allowed the viruses to be distinguished in a single nested PCR. Nested PCR enhanced detection sensitivity between 100- and 1000-fold and GAV or YHV RNA was detectable in ∼10fg lymphoid organ total RNA. The multiplex RT-nested PCR was also able to co-detect GAV and YHV RNA mixed over a wide range of concentrations to simulate potential dual-infection states. The robustness of the test was examined using RNA samples from Penaeus monodon prawns infected either chronically or acutely with GAV or YHV and collected at different locations in Eastern Australia and Thailand between 1994 and 1998. GAV- (406bp) or YHV-specific (277bp) amplicons were differentially generated in all cases, including five YHV RNA samples in which no primary RT-PCR amplicon was detected. Sequence analysis of GAV and YHV PCR amplicons identified minor variations in the regions targeted by the virus-specific antisense primers. However, none occurred at positions that critically affected the PCR.