YELLOW-HEAD VIRUS OF PENAEUS-MONODON IS AN RNA VIRUS

Yellow-head virus (YHV) causes acute infections in Penaeus monodon that result in very high mortality. First reports of the virus suggested that the viral core consisted of DNA and that the virus should be classified as a granulosis-type baculovirus. However, 3 attempts at DNA extraction with high concentrations of purified virus (verified by transmission electron microscopy, TEM) gave only traces of DNA, which could not be visualized by ethidium bromide staining of agarose electrophoresis gels. Although selected recombinant clones derived from these pooled DNA traces did not hybridize with host shrimp DNA, they also failed to react with YHV-infected tissue by the in situ DNA hybridization technique. Furthermore, negatively stained virions of YHV viewed by TEM were atypical for baculoviruses and viral assembly is cytoplasmic. Therefore, renewed attempts to extract nucleic acid from purified YHV preparations focused on RNA rather than DNA. Hemolymph was collected aseptically by syringe from 200 artificially YHV-infected, Live shrimp in terminal stages of the disease. Purified virions 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 virions. By TEM these were enveloped, measured 150-170 x 40-50 nm and were surrounded by a fringe of knob-like projections approximately 11 nm in length. Nucleic acid was extracted using guanidium thiocyanate and purified by CsCl gradient ultracentrifugation. 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 is an important discovery, since it necessitates cDNA preparation in the process to develop a nucleic-acid probe for YHV detection by the in situ or dot blot hybridization techniques.