C. Wongteerasupaya et al., DNA FRAGMENT OF PENAEUS-MONODON BACULOVIRUS PMNOBII GIVES POSITIVE IN-SITU HYBRIDIZATION WITH WHITE-SPOT VIRAL-INFECTIONS IN 6 PENAEID SHRIMP SPECIES, Aquaculture, 143(1), 1996, pp. 23-32
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.