Yellow-head virus (YHV), a highly virulent virus of cultured penaeid shrimp, was originally isolated from the black tiger shrimp Penaeus monodon in Thailand. It was initially described as a baculovirus, but was recently reported to be a n RNA-containing virus. The present study reaffirms the genome of highly purified YHV to be an unsegmented single-stranded RNA with negative polarity and of approximately 22 kb size. When analysed by SDS-PAGE, the purified virus yielded at least 4 viral structural proteins of 170, 135,67 and 22 kDa. The 135 kDa protein was determined to be glycosylated. YHV, as with VSV, a rhabdovirus, was found to agglutinate chicken red blood cells. The highly flexible enveloped bacilliform YHV particles measured 50-60 X 190-200 nm. Since the virus had a number of properties in common with rhabdoviruses, particularly plant rhabdoviruses, it was provisionally classified as a rhabdovirus.

Yellow head baculo-like virus infection and disease were demonstrated experimentally in the two main species of penaeid shrimp cultured in Hawaii and the Western hemisphere. Viral infection was induced by intramuscular inoculation of a 10% suspension of cephalothorax tissue filtrate prepared from two tiger shrimp, Penaeus monodon Fabricius, infected with yellow head disease, into sub-adult (3-lOg) P. stylirostris (Stimpson) and P. vannamei (Boone). Signs of disease appeared as early as 2 days post infection (p-i.), and in most cases mortahty reached 100% within 5-7 days p.i. Histopathological examination of the infected animals revealed extensive cellular necrosis in ectodermal and some mesenchymal tissues. Electron microscopical examination of thin sections of the gill and hepatopancreas from the infected shrimp revealed non-occluded rod-shaped baculo-like virus particles measuring 130-197 x 45-58 nm which were primarily localized within the cytoplasm of infected cells. The virus particles were contained within cytoplasmic vacuoles, and occurred singly or in small groups of two or more particles.

A non-occluded baculovirus-like agent recently isolated by this laboratory from moribund Penaeus japonicus shrimps obtained from China and named Chinese baculovirus (CBV) was purified and some of its properties characterized. Under the electron microscope, negatively stained virus particles were rod-shaped, enveloped, and measured 322 to 378 nm in length and 130 to 159 nm in diameter. The nucleoprotein core exhibited a unique striated structure and measured 316 to 350 nm in length and 65 to 66 nm in diameter. The striations appear to be the result of the stacking of ring-like structures. These rings consisted of 2 rows of 12 to 14 globular subunits. Each globular subunit measured approximately 10 nm in diameter. SDS-PAGE gels of purified virus preparations showed, among several, 4 prominent protein bands with approximate molecular weights of 19, 23.5, 27.5 and 75 kDa. The structural viral proteins were identified by western blot analysis using polyclonal hyperimmune serum made against purified CBV. The 19, 27.5, and 75 kDa structural proteins were determined to be non-glycosylated components associated with the viral envelope. The 23.5 kDa protein, also non-glycosylated, was identified with the capsid structure. Viral genomic DNA digested with Hind III restriction endonuclease revealed at least 29 different fragments with a conservatively estimated total size of at least 183 kb.

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Primary cultures of lymphoid (Oka) organ from Penaeus stylirostris were transformed with naked or Lipofectin-mediated pSV-3 neo, a shuttle vector containing the tumor (T) antigen gene from Simian virus-40. The transformed cells, OKTr-1 and OKTr-23, exhibited the following characteristics: rounded morphology forming grapelike aggregates, loosely adhesive, increased growth rate in Medium-199, resistance to G-418 (a neomycin analog marker in the shuttle vector), cloning efficiencies of 68.7% and 36.7% in soft agarose, respectively, and stability in liquid nitrogen storage. Immunofluorescence staining (IFA) of the transformed cells using a monoclonal antibody against SV-40 tumor antigen showed positive results. In contrast, primary cell cultures exhibited fibroblast-like morphology and formed a tight, adhesive monolayer on the surface of the culture vessel. They were sensitive to G-418, and showed negative results with IFA. To date, OKTr-1 and OKTr-23 have undergone 44 and 18 passages, respectively. Primary cultures of the lymphoid organ have not been successfully passaged beyond the primary stage.