Whispovirus.

During the last two decades, a combination of poor management practices and intensive culturing of penaeid shrimp has led to the outbreak of several viral diseases. White spot disease (WSD) is one of the most devastating and it can cause massive death in cultured shrimp. Following its first appearance in 1992-1993 in Asia, this disease spread globally and caused serious economic losses. The causative agent of WSD is white spot syndrome virus (WSSV), which is a large, nonoccluded, enveloped, rod- or elliptical-shaped, dsDNA virus of approximately 300 kbp. WSSV has a very broad host range among crustaceans. It infects many tissues and multiplies in the nucleus of the target cell. WSSV is a lytic virus, and in the late stage of infection, the infected cells disintegrate, causing the destruction of affected tissues. The WSSV genome contains at least 181 ORFs. Most of these encode proteins that show no homology to known proteins, although a few ORFs encode proteins with identifiable features, and these are mainly involved in nucleotide metabolism and DNA replication. Nine homologous regions with highly repetitive sequences occur in the genome. More than 40 structural protein genes have been identified, and other WSSV genes with known functions include immediate early genes, latency-related genes, ubiquitination-related genes, and anti-apoptosis genes. Based on temporal expression profiles, WSSV genes can be classified as early or late genes, and they are regulated as coordinated cascades under the control of different promoters. Both genetic analyses and morphological features reveal the uniqueness of WSSV, and therefore it was recently classified as the sole species of a new monotypic family called Nimaviridae (genus Whispovirus).

Cloning, characterization, and phylogenetic analysis of a shrimp white spot syndrome virus gene that encodes a protein kinase.

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.

Hepatopancreas is the extraovarian site of vitellogenin synthesis in black tiger shrimp, Penaeus monodon.

The site of yolk protein synthesis in crustaceans has long been a subject of controversy. The vitellogenin gene structure was partially reported only very recently in Macrobrachium rosenbergii, after which the hepatopancreas was confirmed as the extraovarian site of vitellogenin synthesis in that species. Ovaries are the most frequently reported as the site of yolk protein synthesis in penaeid shrimp. Using cDNA reversed-transcribed from mRNA isolated from the hepatopancreas of vitellogenic female shrimp, Penaeus monodon, we found that its deduced amino acid sequence had high identity of 48% with that from M. rosenbergii vitellogenin. A similar location of the intron in the sequenced region of genomic DNA was also found between these two species. We therefore concluded that the hepatopancreas the extraovarian site of vitellogenin synthesis in P. monodon in vivo. The partial structure of vitellogenin gene is presented in this study.

Sequencing and amplified restriction fragment length polymorphism analysis of ribonucleotide reductase large subunit gene of the white spot syndrome virus in blue crab (Callinectes sapidus) from American Coastal Waters.

In the present study, the existence of white spot syndrome virus (WSSV) in blue crab (Callinectes sapidus) collected from 3 different American coastal waters (New York, New Jersey, and Texas) was confirmed by 2-step diagnostic polymerase chain reaction and in situ hybridization analysis. When geographic isolates were also compared using a gene that encodes the WSSV ribonucleotide reductase large subunit RR1 (WSSV rr1), a C(1661)-to-T point mutation was found in the New Jersey WSSV isolated. This point mutation, which resulted in the creation of an additional RsaI endonuclease recognition site, was not found in the WSSV from the New York and Texas blue crab samples, or in the WSSV Taiwan isolate, or in any of the other WSSV geographical isolates for which data are available. WSSV rr1-specific RsaI amplified restriction fragment length polymorphism of an amplified 1156-bp fragment thus distinguished the New Jersey blue crab samples from the other WSSV isolates.

Transcriptional analysis of the ribonucleotide reductase genes of shrimp white spot syndrome virus.

The causative agent of white spot syndrome (WSS) is a large double-stranded DNA virus, WSSV, which is probably a representative of a new genus, provisionally called Whispovirus. From previously constructed WSSV genomic libraries of a Taiwan WSSV isolate, clones with open reading frames (ORFs) that encode proteins with significant homology to the class I ribonucleotide reductase large (RR1) and small (RR2) subunits were identified. WSSV rr1 and rr2 potentially encode 848 and 413 amino acids, respectively. RNA was isolated from WSSV-infected shrimp at different times after infection and Northern blot analysis with rr1- and rr2-specific riboprobes found major transcripts of 2.8 and 1.4 kb, respectively. 5' RACE showed that the major rr1 transcript started at a position of -84 (C) relative to the ATG translational start, while transcription of the rr2 gene started at nucleotide residue -68 (T). A consensus motif containing the transcriptional start sites for rr1 and rr2 was observed (TCAc/tTC). Northern blotting and RT-PCR showed that the transcription of rr1 and rr2 started 4-6 h after infection and continued for at least 60 h. The rr1 and rr2 genes thus appear to be WSSV "early genes."

Identification and characterization of a shrimp white spot syndrome virus (WSSV) gene that encodes a novel chimeric polypeptide of cellular-type thymidine kinase and thymidylate kinase.

From previously constructed genomic libraries of a Taiwan WSSV isolate, a putative WSSV tk-tmk gene was identified. Uniquely, the open reading frame (ORF) of this gene was predicted to encode a novel chimeric protein of 388 amino acids with significant homology to two proteins: thymidine kinase (TK) and thymidylate kinase (TMK). Northern blot analysis with a WSSV tk-tmk-specific riboprobe detected a major transcript of 1.6 kb. When healthy adult Penaeus monodon shrimp were inoculated with WSSV, the tk-tmk gene transcript was first detected by RT-PCR analysis at 4 h postinfection and transcription levels continued to increase over the first 18 h. The gene's major in vitro transcription and translation product, equivalent to the predicted size (43 kDa), is a single chimeric protein that includes both the TK and TMK functional motifs. Evidence for phylogenetic analysis and sequence alignment suggested that the gene may have resulted from the fusion of a cellular-type TK gene and a cellular-type TMK gene. Its unique arrangement may also provide a valuable gene marker for WSSV.

Ultrastructure of white spot syndrome virus development in primary lymphoid organ cell cultures.

Primary cell cultures from the lymphoid organ of Penaeus monodon were used to investigate in vitro propagation and morphogenesis of white spot syndrome virus (WSSV). Double-strength Leibovitz's L15 supplemented with 20% fetal bovine serum, pH 7.5, with a final osmolarity of 530 +/- 5 mOsm kg-1 was identified as the most suitable culture medium. In this medium, the lymphoid cells remained viable for more than 1 wk. Migrating cells were inoculated with WSSV, and the consequent cytopathic effects documented by light and electron microscopy. WSSV appears capable of following 2 alternative assembly sequences, one similar to the morphogenesis of the Oryctes rhinocerus virus and another which is more typical of baculoviral assembly. Possible relationships between WSSV, Oryctes virus, and baculoviruses are discussed.

Natural and experimental infection of white spot syndrome virus (WSSV) in benthic larvae of mud crab Scylla serrata.

White spot syndrome virus (WSSV), the causative agent of white spot syndrome in shrimp, has a wide host range which extends to crabs, copepods and other arthropods. In this study, benthic larvae of the mud crab Scylla serrata were captured from Taiwan's coastal waters and screened for the presence of WSSV by polymerase chain reaction (PCR) and in situ hybridization. WSSV was detected in around 60% of the larvae, and this prevalence rate remained fairly constant when the captured larvae were subsequently maintained in an aerated system in the laboratory. WSSV-free larvae obtained from a hatchery were challenged by immersion in a WSSV inoculum. Fifteen days after challenge, cumulative mortality in the experimental group reached 43% compared to 20% in the control group. PCR detection of WSSV in both moribund and surviving specimens clearly implicated the virus as the cause of death in most cases. Histological and in situ hybridization data confirmed that WSSV tissue tropism in Scylla serrata crab larvae is similar to that found in shrimp.

Analysis of a genomic segment of white spot syndrome virus of shrimp containing ribonucleotide reductase genes and repeat regions.

White spot syndrome is a worldwide disease of penaeid shrimp. The disease agent is a bacilliform, enveloped virus, white spot syndrome virus (WSSV), with a double-stranded DNA genome that probably contains well over 200 kb. Analysis of a 12.3 kb segment of WSSV DNA revealed eight open reading frames (ORFs), including the genes for the large (RR1) and small (RR2) subunits of ribonucleotide reductase. The rr1 and rr2 genes were separated by 5760 bp, containing several putative ORFs and two domains with multiple sequence repeats. The first domain contained six direct repeats of 54 bp and is part of a coding region. The second domain had one partial and two complete direct repeats of 253 bp at an intergenic location. This repeat, located immediately upstream of rr1, has homologues at several other locations on the WSSV genome. Phylogenetic analysis of RR1 and RR2 indicated that WSSV belongs to the eukaryotic branch of an unrooted parsimonious tree and, further, seems to suggest that WSSV and baculoviruses probably do not share an immediate common ancestor. The present analysis of WSSV favours the view that this virus is either a member of a new genus (Whispovirus) within the Baculoviridae or a member of an entirely new virus family.

Nested polymerase chain reaction and in situ hybridization for detection of nucleopolyhedrosis.

A nested polymerase chain reaction (PCR) and in situ hybridization were developed for detection of baculoviruses in insects or other arthropods with nucleopolyhedrosis. The nested PCR was based on the sequences of polyhedrin genes from baculoviruses. Two sets of primers were designed, primers set, 35/36, was for the first step of amplification and yielded a product of around 680 bp, the second primer, 35-1/36-1, was designed to yield a product of around 335bp from the fragment amplified by the first primer set. The sensitivity of this two-step amplification was 100 to 1000 times higher than that of the one-step amplification by primer set (35/36). Samples which contained baculovirus DNA yielded an amplification product showing the expected DNA fragment mobility, whereas nucleic acid extracted from tissue samples of clinically healthy insects or uninfected cells showed no such DNA fragment, thereby confirming the specificity of the primers. Using the 35/36 amplicon as a probe, the PenuNPV-infected cells show positive reaction by in situ hybridization. Two-step DNA amplification and in situ hybridization with the DNA probe developed in the present paper provide effective detection and diagnostic tools for screening insects or other arthropods, especially crustacean species, crabs and shrimps, for baculovirus infections, and may be important in preventing (and/or controlling/enhancing) the infection of baculoviruses.

Ultrastructural justification for the transfer of Pleistophora anguillarum Hoshina, 1959 to the genus Heterosporis Schubert, 1969.

This study presents the ultrastructure of the microsporidian infecting the trunk musculature of Anguilla japonica and originally described as Pleistophora anguillarum Hoshina, 1959. All stages develop within a special structure, the sporophorocyst (SPC), which is equipped with a thick dense wall. This wall grows along with the growth of the parasites within it. Meronts are uni- to binucleate, which divide and steadily give rise to sporonts. During transition to sporonts the cell coat of the meronts increases its thickness, temporarily featuring thick irregular projections. Eventually a uniformly thick sporont wall is formed, then the sporont cells detach themselves from the wall (= future wall of the sporophorous vesicle, SPV) and start a series of divisions to produce sporoblasts. The SPV wall is compact, has no pores and consists of 2 layers. The presence of the SPC justifies the transfer of the species into the genus Heterosporis. Spores from disrupted SPCs are ingested by macrophages and within them are spread into various body tissues including the outermost layers of the epidermis. From here, they can easily be released to the outside and can contaminate the environment while the host is still alive.

Studies on effective PCR screening strategies for white spot syndrome virus (WSSV) detection in Penaeus monodon brooders.

We re-tested stored (frozen) DNA samples in 5 independent polymerase chain reaction (PCR) replicates and confirmed that equivocal test results from a previous study on white spot syndrome virus (WSSV) in brooders and their offspring arose because amounts of WSSV DNA in the test samples were near the sensitivity limits of the detection method. Since spawning stress may trigger WSSV replication, we also captured a fresh batch of 45 brooders for WSSV PCR testing before and after spawning. Replicates of their spawned egg batches were also WSSV PCR tested. For these 45 brooders, WSSV prevalence before spawning was 67% (15/45 1-step PCR positive, 15/45 2-step PCR positive and 15/45 2-step PCR negative). Only 27 (60%) spawned successfully. Of the successful spawners, 56% were WSSV PCR positive before spawning and 74% after. Brooders (15) that were heavily infected (i.e. 1-step PCR positive) when captured mostly died within 1 to 4 d, but 3 (20%) did manage to spawn. All their egg batch sub-samples were 1-step PCR positive and many failed to hatch. The remaining 30 shrimp were divided into a lightly infected group (21) and a 2-step PCR negative group (9) based on replicate PCR tests. The spawning rates for these 2 groups were high (81 and 78%, respectively). None of the negative spawners (7) became WSSV positive after spawning and none gave egg samples positive for WSSV. In the lightly infected group (21), 6 brooders were 2-step WSSV PCR negative and 15 were 2-step WSSV PCR positive upon capture. However, all of them were WSSV PCR positive in replicate tests and after spawning or death. Four died without spawning. The remaining 17 spawned but only 2 gave egg samples PCR negative for WSSV. The other 15 gave PCR positive egg samples, but they could be divided into 2 spawner groups: those (7) that became heavily infected (i.e. 1-step PCR positive) after spawning and those (8) that remained lightly infected (i.e. became or remained 2-step PCR positive only). Of the brooders that became heavily infected after spawning, almost all egg sample replicates (91 %) tested 2-step PCR positive. One brooder even gave heavily infected (i.e. 1-step PCR positive) egg samples. For the brooders that remained lightly infected after spawning, only 27% of the egg sample replicates were 2-step PCR positive. Based on these results, we recommend that to avoid false negatives in WSSV PCR brooder tests screening tests should be delayed until after spawning. We also recommend, with our PCR detection system, discarding all egg batches from brooders that are 1-step PCR positive after spawning. On the other hand, it may be possible with appropriate monitoring to use eggs from 2-step PCR positive brooders for production of WSSV-free or lightly infected postlarvae. These may be used to stock shrimp ponds under low-stress rearing conditions.

The small subunit ribosomal RNA gene sequence of Pleistophora anguillarum and the use of PCR primers for diagnostic detection of the parasite.

Using the polymerase chain reaction (PCR) and two primers for conserved regions of the small subunit ribosomal RNA (SSU-rRNA) of Microsporidia, a DNA segment about 1,195 base pairs long was amplified from a DNA template prepared from purified spores of the microsporidian species Pleistophora anguillarum. These spores had been isolated from adult eels (Anguilla japonica) with "Beko Disease." A comparison of sequence data from other microsporidian species showed P. anguillarum SSU-rRNA to be most similar to Vavraia oncoperae. When juvenile eels were artificially infected with P. anguillarum, enzyme-linked immunosorbent assay could detect a positive infection only 12 days post-infection. However, when suitable PCR primers were used, a DNA fragment of about 0.8 kb was detected from these juvenile eels after only 3 days post infection. No PCR product was obtained with templates prepared from clinically healthy control animals.

Identification and characterization of a hyperglycemic hormone from freshwater giant prawn, Macrobrachium rosenbergii.

Crustacean hyperglycemic hormone (CHH), a physiologically important neurohormone stored in the sinus gland of eyestalks, primarily regulates carbohydrate metabolism and also plays significant roles in reproduction, molting and other physiological processes. In the freshwater giant prawn, Macrobrachium rosenbergii, an injection of X-organ sinus gland (XOSG) extract evoked a hyperglycemic response, peaked in 1 h. The hyperglycemic effect of the eyestalk extract was maximal at the dose of 0.5 eyestalk equivalent. CHH fractionated by RP-HPLC, in M. rosenbergii was identified by its hyperglycemic activity and partial amino acid sequence, and the molecular weight of 8534 was determined by matrix-assisted laser desorption ionization mass spectrometry--time of flight analysis (MALDI-TOF). The amino acid sequence of the first 25 residues of CHH showed 72% homology with the first 25 residues of CHH A and CHH B of the American lobster Homarus americanus.

Negative regulation of a heterologous promoter by human cytomegalovirus immediate-early protein IE2.

The HCMV IE2 protein promiscuously activates transcription of many viral and cellular genes. IE2 also negatively autoregulates its own expression by binding to a strategically positioned IE2 binding site, called CRS, located immediately downstream of the TATA box of the HCMV major IE promoter. Here we show that IE2 is able to repress transcription driven by a heterologous promoter, RSV LTR. Repression of RSV LTR by IE2 is completely dependent of DNA sequences downstream of the TATA box of RSV LTR. A DNA sequence, 5'-CGATACAATAAACG-3', evidently matching the proposed CRS consensus sequence, is located between nucleotides -13 and +1 (relative to the transcription start site) of RSV LTR. Three lines of evidence support the notion that this RSV CRS element is involved in the IE2-mediated repression of RSV LTR. First, introduction of mutation to the RSV CRS element renders to the mutant RSV LTR resistance to IE2-mediated repression. Second, a mutant IE2 defective in DNA binding cannot downregulate transcription from RSV LTR. Third, IE2 specifically binds to the wild-type, but not the mutant, RSV CRS element in vitro. These data, in conjunction with previous works, demonstrate that IE2 can passively repress transcription of homologous and heterologous promoters that contain a CRS element.

Passive immunization of the tiger prawn, Penaeus monodon, using rabbit antisera to Vibrio harveyi.

Passive immunization, toxicity neutralization and the persistence of passive protection in the tiger prawn (Penaeus monodon) were investigated using rabbit antisera to the formalinized extracellular products (ECP) (R alpha ECP) and/or formalinized bacterial cells (R alpha BC) of luminescent Vibrio harveyi strain 820514 originally isolated from diseased tiger prawns. Rabbit antiserum to bovine serum albumin (R alpha BSA) or phosphate-buffered saline (PBS, pH 7.2) both served as controls. The toxicity of ECP to prawns was neutralized by pre-incubation with R alpha ECP. Passive immunization by pre-injection of R alpha BC or R alpha ECP into prawns 3 d in advance protected against a lethal dose challenge of bacteria. To determine the persistence of passive protection by rabbit antiserum in tiger prawns, the R alpha BC, R alpha ECP, R alpha BSA or PBS were injected into prawns. At 10, 17 or 24 d post-immunization, groups of prawns were given a lethal dose challenge of bacteria. The prawns in the two control groups were all killed within the first 2 d following challenge at all three challenge dates. Pre-injection with R alpha BC and R alpha ECP provided total protection for 10 and 17 d, respectively, with all treated prawns surviving for at least 2 weeks post-challenge. This is the first study using mammalian antisera to investigate toxicity neutralization, passive immunization and persistence of passive protection by rabbit antisera in prawns. The results could be useful in future studies on virulence mechanisms and disease control of vibriosis in cultured prawns.

Isolation of Vibrio harveyi from diseased kuruma prawns Penaeus japonicus.

Outbreaks of high mortality among the cultured kuruma prawn Penaeus japonicus without overt gross signs occurred during August and December of 1994 in I-Lan, Taiwan. Eleven luminous bacterial strains were isolated from the hepatopancreas of moribund prawns from five different farms by use of tryptic soy agar (TSA, supplemented with 2% NaCl) and/or thiosulfate citrate bile salt sucrose (TCBS) agar. These strains, together with our two previously unpublished isolates, were characterized and identified to be Vibrio harveyi in comparison with two ATCC Type strains and one strain previously isolated from the tiger prawn, P. monodon.

Characterization of Perina nuda nucleopolyhedrovirus (PenuNPV) polyhedrin gene.

The Perina nuda nucleopolyhedrovirus (PenuNPV) polyhedrin gene was located in EcoRI-G (6.3 kilobase pairs; kbp) and PstI-G (4.3 kbp) fragments of its genomic DNA. A portion of 1333 nucleotides (nt) containing this gene was sequenced. An open reading frame of 735 nt encoded a 245-amino-acid-long polyhedrin. A conserved TAAG motif which is associated with transcriptional start sites was identified 51 nt upstream of the translation initiation codon of PenuNPV polyhedrin gene. A putative polyadenylation signal, AATAAA, was found 116 nt downstream of the termination codon (TAA). Comparison of the amino acid sequences of PenuNPV polyhedrin with those of other NPVs showed that PenuNPV polyhedrin was most closely related to Orgyia pseudotsugata multiple NPV (OpMNPV) polyhedrin.

Human cytomegalovirus immediate-early protein IE2 tethers a transcriptional repression domain to p53.

The IE2 gene of human cytomegalovirus has been implicated in the development of coronary restenosis, and the gene product appears to inhibit p53-dependent transactivation. Here we describe an analysis of the IE2-p53 interaction. Repression of p53 function by IE2 requires two separable domains of IE2. The N terminus of IE2 interacts with p53. IE2 has little effect on the ability of p53 to bind specific DNA sequences. Reduction of the transactivation activity of p53 is caused by a transcriptional repression function contributed by the C-terminal domain of IE2. These findings suggest that IE2 may function as a transcriptional repressor, which is recruited to p53's target genes by interacting with p53.

[Survey of disease of cultural shrimp in Taiwan].

The large number of hemocytes infiltrated several abnormal tissues of kuruma shrimp (Penaeus japonicus), including musculature, hepatopancreas, lymphoid organ, gill filament and sponge tissue. In addition, there were many denatured hemocytes existing inside acidophilic particles and forming granules. Futhermore, in hepatopancreas of kuruma shrimp, a white spot baculovirus (WSBV; 40-50 x 50-300 nm) was discovered in UH (undifferential haemocyte). The epithelium cells, which including stomach cuticle and underlying epidermis of exoskeletal cuticle, could also be infected by WSBV in another main cultural species--grass shrimp (P. monodon). During a period of high water temperature, with pond shrimp in normal condition, the CFU/ml of water bacteria rose from 10(5) to 10(7), but this number had decreased to 10(5) CFU/ml by the time moribund shrimp began to appear. Coincidentally, the total bacterial number isolated from hepatopancreas and musculature of moribound shrimp was over 10(5) (CFU/g) and 10(3)-10(5), respectively. The fauna of bacteria was taken over by the active metabolitic species which were represented by Vibrio species causing the pond shrimp to undergo either behavioral changes, such as swimming on the water surface, or histological changes, such as having whitish muscle color, hemocyte infiltration and granuloma formation etc. Pathogenetic species of Vibrio including V. parahaemolyticus, V. alginoly ticus V. anguillarum, V. fischery and V. damsela were isolated from those tissues of moribund shrimp. The main pathogens, isolated from musculature and hepatopancreas, were V. parahaemolyticus and V. alginolyticus. On the other hand there, was no bacterium could be isolated from the musculature of healthy shrimp and only a single species of Gram (+) coccus--Micrococcus--was isolated from the tissue of hepatopancreas.