for virus isolation and purification, 1 batch of prawns yielded hemolymph fractions dominated by a previously undescribed non-occluded baculovirus rather than YHV. Injection of test shrimp with a semipurified preparation of this virus gave rapid mortality, and examination with the transmission electron microscope revealed a dual infection where cells containing the new virus dominated, but some cells containing YHV could also be seen. The tissues infected by the 2 viruses were similar. However, in contrast to YHV, the new virus was assembled completely in the nucleus and in the absence of occluding protein (polyhedrin). By normal histology, the most characteristic feature of infection was eosinophilic Cowdry A-type inclusions in hypertrophied nuclei with marginated chromatin, especially in epithelial cells of the stomach. These intranuclear inclusions became lightly basophilic in late stages of infection. In the epithelial cells of the gills, ultrastructural pathology included nuclear hypertrophy and cytoplasmic disintegration leading to large voids at lysed cell sites. By negative staining, completely assembled, enveloped virions were ellipsoid to obovate with a distinctive multifibrillar appendage and they measured 276 x 121 nm (excluding the appendage). Enveloped and unenveloped nucleocapsids were significantly different In size, indicating posslble shortening and thickening of the viral core and nucleocapsid during viral assembly. Isolation and punficat~on of the nucleic acid from the new virus yielded double-stranded DNA of approximately 168 lulo base pairs. This DNA did not cross-hybridize with DNA fragments isolated from YHV-infected shrimp or from monodon baculovirus (MBV). The features placed t h~s virus in the family Baculoviridae, subfamily Nudibaculovirinae as PmNOBII, but for convenience we have named it informally as Systemic Ectodermal and Mesodermal Baculovirus (SEMBV).
A recently repor1t.d dlsease s y n d~o m e of Penaeus monodon In Thalland IS called 'yellow-head' or hua leung In Thal It is usually charactenzed by llght yellow coloratlon of the dorsal cephalothorax area and generally pale or bleached appearance of a f f~c t e d prawns The yellow color In the cephalothorav reglon iesults from the underlying y e l l o~~ hepatopancreas showlng through the translucent carapace In m o~i b u n d shrimp In h~stological prepdrations of moribund yellow-head specimens for the light mlcloscope. no consistent bacteiial fungal or parasltlc agents could be found The lylnphold organs ot yellow-head specimens sho\ved extensive abnormalities These Included obv~ously necrotlc cells and vacuolated cells with hypertrophied nuciel Also evldent were vely densely basophil~c, globose cytoplasmic ~nclusions located adjacent to some ot the hypertroph~ed nuclei Slrn~lar basophil~c inclusions were found In interst~tl~il hepatopancreat~c tissue In connective tlssue u n d e~ lylng the mld gut, In cardlac tissue, In glll t~s s u e and In hematopoetlc tissue T l a n s m~s s~o n electron mlclographs revealed the presence of pic\iously undescrlbed rod-shaped, enveloped virions in the cytoplasm adjacent to the nuclei of cells f~ om various tissues Free vlnons \yere also present In intercellular spaces The virions were s~r n d a to those of the ~tlsect grahulosls vlruses (Baculov~r~dae) In terms of cytoplasmic locatlon size, morphology and development. However, they were not occluded by granulln
Yellow head virus (YHV) causes a serious disease that can result in high mortality of penaeid shrimp within 2-3 days after the first appearance of gross signs of disease in rearing ponds. Current detection systems for YHV are based on molecular and immunological techniques. Immunological detection methods require continual production of relatively large, complex proteins using living animals or hybridoma cells. An alternative possibility is to use the phage display technique to find short peptides that can bind strongly with YHV particles and replace antibodies in such tests. Once identified, these peptides could also be tested for efficacy in blocking YHV infection. YHV was purified and then immobilized on microtiter plates for 4 rounds of biopanning against a pool of phage displayed peptide variants. From 89 sequentially generated phage pools, 13 variants were selected for strong binding with YHV by ELISA assay. DNA sequencing led to the deduced amino acid sequences LNAKSRN, KSKKSSS, GPQRKRS, KLKRLSS, RTNKKNA, SNISNAS, SNSKKRN, RTKKMRT, NTKRPAR, GPQRKRS, VSNKKRS, RKKSNAS and GPKKNRS. Preliminary tests using two representative phages (SNSKKRN and GPQRKRS) with high YHV binding activity showed that they were unable to block YHV infection in shrimp. However, when immobilized, these clones could bind YHV from probe solutions, indicating that they have potential for use as YHV detection reagents.
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