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One unique feature of the shrimp white spot syndrome virus (WSSV) genome is the presence of a giant open reading frame (ORF) of 18,234 nucleotides that encodes a long polypeptide of 6,077 amino acids with a hitherto unknown function. In the present study, by applying proteomic methodology to analyze the sodium dodecyl sulfate-polyacrylamide gel electrophoresis profile of purified WSSV virions by liquid chromatography-mass spectrometry (LC-MS/MS), we found that this giant polypeptide, designated VP664, is one of the viral structural proteins. The existence of the corresponding 18-kb transcript was confirmed by sequencing analysis of reverse transcription-PCR products, which also showed that vp664 was intron-less. A time course analysis showed that this transcript was actively transcribed at the late stage, suggesting that this gene product should contribute primarily to the assembly and morphogenesis of the virion. Several polyclonal antisera against this giant protein were prepared, and one of them was successfully used for immunoelectron microscopy analysis to localize the protein in the virion. Immunoelectron microscopy with a gold-labeled secondary antibody showed that the gold particles were regularly distributed around the periphery of the nucleocapsid with a periodicity that matched the characteristic stacked ring subunits that appear as striations. From this and other evidence, we argue that this giant ORF in fact encodes the major WSSV nucleocapsid protein.White spot syndrome virus (WSSV) is one of the most devastating shrimp pathogens, and it has caused serious damage to the worldwide shrimp culture industry. Although this virus can infect several crustacean species, including shrimp, crab, and crayfish (9,12,19,24,42), its virulence is particularly high in infected penaeid shrimp, and mortality can reach 90 to 100% within 3 to 7 days of infection (9, 46). The WSSV virion is a nonoccluded, enveloped particle of approximately 275 by 120 nm with an olive-to-bacilliform shape, and it has a nucleocapsid (300 by 70 nm) with periodic striations perpendicular to the long axis (42,43). The most prominent feature of WSSV is the presence of a tail-like extension at one end of the virion (10, 43), which gives this virus the family name Nimaviridae (27) ("nima" is Latin for "thread").The complete genome sequences of three WSSV isolates have been determined, and 532 open reading frames (ORFs) that contain Ͼ60 amino acids have been identified for the Taiwan isolate (GenBank accession number AF440570). Homology searches against the NCBInr database suggest possible roles or functions for only a few of these ORFs, and most of the ORFs posted for the Taiwan isolate show no significant similarity to other known proteins. Similar results have been reported for the other two isolates (40,44). Until recently, Ͻ5% of the ORFs had been shown to encode either nonstructural proteins (ribonucleotide reductase, protein kinase, the chimeric thymidine kinase/thymidylate kinase, and DNA polymerase [8,22,35,36]) or structural proteins...

PCR and in situ hybridization analysis were used for detection of white spot syndrome virus (WSSV) in an infected, cultured shrimp population over a long period in the absence of disease outbreaks. The shrimp were derived from a single WSSV-carrier brooder and cultured first in a tank and then in outdoor ponds. Prior to harvest at 13 mo, no l-step PCR-positive specimens were found, even though most tested specimens were found to be 2-step PCR-positive. At 7 mo, 2-step PCR-positive tissues were found in 5 sampled shrimp. Heart, gill, integument, muscle and stomach tissues best supported viral replication At 13 mo several shrimp died, and l-step PCR-positive individuals were found for the first time Although superficially healthy, 10% of the surviving adults had tiny white spots on their carapace, and In sjtu hybridization analysis revealed WSSV-positive cells in 40% of the specimens examined. As before, most were found in the stomach, integument and gills, and only very few in the lymphoid organ and other organs. These observations contrasted to those for experimentally infected shrimp with gross signs of terminal WSSV infection, where strong positive signals were also observed in the lymphoid organ and in other organs of ectodermal or mesodermal origin. Our results showed clearly that whatever the source, WSSV was carried in the shrimp population at a low intensity (i.e. nested PCR was required for detection) for a very long time in the absence of massive mortality. We hypothesize that disease outbreaks do not occur if shrimp defense mechanisms manage to contain lowintensity viral infections under low-stress culture conditions. Conversely, outbreaks may occur under stressful conditions.

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By sequencing the entire ribosomal RNA (rRNA) gene of Nosema spodopterae, we show here that its gene organization follows a pattern similar to the Nosema type species, Nosema bombycis, i.e. 5'-large subunit rRNA (2,497 bp)-internal transcribed spacer (185 bp)-small subunit rRNA (1,232 bp)-intergenic spacer (277 bp)-5S rRNA (114 bp)-3'. Gene sequences and the secondary structures of large subunit rRNA, small subunit rRNA, and 5S rRNA are compared with the known corresponding sequences and structures of closely related microsporidia. The results suggest that the Nosema genus may be heterogeneous and that the rRNA gene organization may be a useful characteristic for determining which species are closely related to the type species.

An open reading frame (ORF) that encodes a 715-amino-acid polypeptide was found in an 8421-bp EcoRI fragment of the shrimp white spot syndrome virus (WSSV) genome. The polypeptide shows significant homology to eukaryotic serine/threonine protein kinase (PK) and contains the major conserved subdomains for eukaryotic protein kinases. Coupled in vitro transcription and translation generated a protein having an apparent molecular mass of about 87 kDa according to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. For transcriptional analysis of the pk gene, total RNA was isolated from WSSV-infected shrimp at different times after infection. Northern blot analysis with pk-specific riboprobe found a major and a minor transcript of 2.7 and 5.7 kb, respectively. Rapid amplification of the 5' cDNA ends of the major 2.7-kb pk transcript showed that there were two transcriptional initiation sites located at nucleotide residues -38(G) and -39(G) relative to the ATG translational start codon. Temporal expression analysis by RT-PCR indicated that the transcription of the pk gene started 2 h after infection and continued for at least 60 h. Phylogenetic analysis showed that WSSV protein kinase does not have any close relatives and does not fall into any of the major protein kinase groups.

Invasive events by Bemisia tabaci (Gennadius) biotypes in various parts of the world are of continuing interest. The most famous is biotype B that has caused great economic losses globally. In addition, biotype Q has also recently been reported to be a new invasive pest. These two biotypes have been monitored for some time in the Western North Pacific region, but the invasive events and population genetic structures of these two biotypes are still not clear in this region. In this study, the mitochondrial cytochrome oxidase I (COI) gene was used to reconstruct a phylogenetic tree for identifying biotypes B and Q and to study the relationships between invasive events and ornamental plants. Population genetic analyses of mtCOI sequences were also used to study the genetic relationships within and between populations. A combination of a phylogenetic tree and haplotype analysis suggested the recent invasion of biotype Q in this region is related to the international ornamental trade from the Mediterranean region. Low levels of haplotype diversity and nucleotide diversity indicate that the presence of biotypes B and Q in the Western North Pacific region are caused by multiple invasions. Hierarchical analysis of molecular variance supports the hypothesis of multiple invasions. In addition, high sequence identities and low genetic distances within and between populations of the two biotypes revealed that these invasive events occurred recently. The low levels of genetic differentiation revealed by pairwise F (ST) values between populations also suggests the invasions were recent. Therefore, results of this study suggested that biotypes B and Q entered this region through multiple recent invasions. A quarantine of agricultural crops may be necessary to prevent further invasions.