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).

Corresponding genomic regions of isolates of yellow head virus (YHV) from Thailand and gill-associated virus (GAV) from Australia were compared by RT-PCR and sequence analysis. PCR primers designed from sequences in the GAV ORFlb polyprotein gene amplified the corresponding 577 nucleotide region of the YHV genome. Comparison of the amplified region indicated 85.1 % nucleotide and 95.8% amino acid sequence identity. YHV PCR primers designed to amplify a 135 nucleotide product previously described as a YHV &agnostic probe failed to amplify the correspondmg product from GAV RNA. However, the cognate GAV sequence for this and another recently reported YHV sequence were located in an upstream region of the ORFlb gene. A comparison of these sequences indicated identities of 83.0 and 80.9% at the nucleotide level and 86.7 and 86.5% at the amino acid level, respectively. The data indicate that GAV and YHV are closely related but distinct viruses for whlch differential diagnostic probes can be applied.

No abstract

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

White spot syndrome virus (WSSV) of the black tiger prawn Penaeus rnonodon is a recently discovered baculo-like virus disease which is currently the cause of very serious and widespread losses in the shrimp industry in Thailand and elsewhere in Asia. Three suspected crab carriers of this virus commonly found in shrimp-rearing areas were investigated. These were Sesarma sp., Scylla serrata and Uca pugilator. All these crabs could be infected with WSSV by injection and they sustained heavy viral infections for up to 45 d (confirmed by normal histology, specific in situ DNA hybridization and PCR amplification) without visible signs of disease or mortality. All of them also transferred the disease to P. monodon via water while physically separated in aquarium cohabitation tests. Transfer of the virus to the shrimp was monitored using in situ DNA hybridization and PCR assay at 12 h intervals after cohabitation began. With U. pugilator, WSSV could be detected in the shrimp cohabitants after 24 h using PCR amplification and after 60 h using in situ hybridnation. With S. serrata, the shrimp were positive for WSSV after 36 h using PCR and after 60 h using DNA in situ hybridization. With Sesarrna sp. they were positive after 48 h using PCR and 72 h using in situ hybridization. These laboratory studies demonstrated that crab carriers of WSSV may pose a real threat to cultivated shrimp. However, the studies were carried out in containers with a small volume and with relatively clean sea water as compared to shrimp cultivation ponds. Pond-based studies are now needed to determine whether factors such as pond volume, pond water quality and shrimp and crab behavior can influence the rate and success of transfer.