Yellow-head virus (YHV) causes acute infections in Penaeus monodon that result in very high mortahty First reports of the vlrus suggested that the viral core consisted of DNA and that the virus should b e classified as a granulosls-type baculovirus However, 3 attempts at DNA extraction with high concentratlons of punfied virus (verified by transmission electron micioscopy, TEM) gave only traces of DNA, which could not be visuahzed by ethidium bromide staining of agarose electrophoresls gels Although selected recombinant clones denved from these pooled DNA traces did not hybridize with host s h n m p DNA, they also tailed to react with YHV-~nfected tissue by the In s~t u DNA h y b n d~z ation technique Furthermore, negatively stained virions of YHV viewed by TEM were atypical for baculov~ruses and viral assembly IS cytoplasmic Therefore, renewed attempts to extract n u c l e~c acid fiom punfied YHV preparations focused on RNA rather than DNA Hemolymph was collected aseptlcally by syringe from 200 artificially YHV-infected, live s h n m p in terminal stages of the disease Purlfled vlrions were prepared by a program of centrifugation culminating In 22 % to 45 , Urografin gradient ultracentrifugation A band at the 30-37% Interval of the gradient gave the cleanest preparation with the highest quantity of vinons By TEM these were enveloped measured 150-170 X 40-50 nm and were surrounded by a f r~n g e of knob-hke projections approximately 11 nm in length Nucleic acld was extracted using g u a n i d~u m thiocyanate and punfied by CsCl gradient ultracentnfugation High-molecular-weight nucleic acid was obtained which was degraded by RNase-A but not by DNase I Based on morphology of negatively stained virions by TEM and on RNA content YHV resembles rhabdoviruses or coronaviruses, rather than baculoviruses This 1s an important discovery since i t necessitates cDNA preparation in the process to develop a n u c l e~c -a c~d probe for YHV detection by the In sjtu or dot blot hybndization techniques
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