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).

Plasma and urine pharmacokinetics of niacin and its metabolites from an extended-release niacin formulation.

OBJECTIVE: To characterize plasma and urine pharmacokinetics of niacin and its metabolites after oral administration of 2,000 mg of extended-release (ER) niacin in healthy male volunteers. METHODS: Niacin ER was administered to 12 healthy male subjects following a low-fat snack. Plasma was collected for 12 h post dose and was analyzed for niacin, nicotinuric acid (NUA), nicotinamide (NAM) and nicotinamide-N-oxide (NNO). Urine was collected for 96 h post dose and analyzed for niacin and its metabolites, NUA, NAM, NNO, N-methylnicotinamide (MNA) and N-methyl-2-pyridone-5-carboxamide (2PY). RESULTS: Mean niacin Cmax and AUC(0-t) values were 9.3 microg/ml and 26.2 microg x h/ml and were the highest of all analytes measured. Peak niacin and NUA levels occurred at 4.6 h (median) while tmax for NAM and NNO were 8.6 and 11.1 h, respectively. The mean plasma terminal half-life for niacin (0.9 h) and NUA (1.3 h) was shorter as compared to NAM (4.3 h). Urine recovery of niacin and metabolites accounted for 69.5% of the administered dose; only 3.2% was excreted as niacin. The highest recovery was for 2PY (37.9%), followed by MNA (16.0%) and NUA (11.6%). Mean half-lives for 2PY and MNA calculated in urine were 12.6 and 12.8 h, respectively. CONCLUSIONS: Niacin was extensively metabolized following oral administration, and about 70% of the administered dose is recovered in urine in 96 h as niacin, NUA, MNA, NNO, NAM and 2PY. The plasma levels of the parent niacin were higher than its metabolites though only about 3% of the unchanged drug is recovered in urine.

Genomic organization and the promoter region of the round-spotted pufferfish (Tetraodon fluviatilis) CDC37 gene.

The CDC37 gene was isolated from a round-spotted pufferfish genomic library and characterized. This gene is composed of nine exons spanning 3.5 kb. Exon 1 contains the 5'-untranslated region and exon 2 contains the putative translation initiation site. By 5'-RACE (rapid amplication of cDNA ends) and sequence analysis, we deduced the promoter region for the CDC37 gene and found that it does not contain typical TATA or CCAAT box. The 1.8 kb DNA fragment upstream of the putative transcription initiation site contains numerous potential binding sites for transcription factors including CREB, E2A, Ets-1, GATA, NF-IL6 and PEA3. When this DNA fragment was placed upstream of the chloramphenicol acetyltransferase (CAT) reporter gene and transfected into a carp CF cell line, it could drive the synthesis of CAT enzyme four times more efficiently than the promoterless pCAT-Basic did. In addition, the CDC37 gene is linked to the TYK2 gene in a tail-to-head manner with a small intergenic region of 292 bp.

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.

Complete genomic organization and promoter analysis of the round-spotted pufferfish JAK1, JAK2, JAK3, and TYK2 genes.

We have previously reported the isolation of the JAK1 gene from the round-spotted pufferfish. In the present study, we cloned and characterized genomic sequences encoding pufferfish JAK2, JAK3, and TYK2, which are other members of JAK family. To our knowledge, this is the first report to demonstrate the existence of four JAK genes in fish. All pufferfish JAK genes except JAK1 are composed of 24 exons; JAK1 has an additional exon. A comparison of the exon-intron organization of these genes revealed that the splice sites of JAK genes are nearly identical. In addition, all pufferfish JAK genes have one intron in the 5' untranslated region. Taken together, these data suggest that the pufferfish JAK genes may have evolved from a common ancestor. By 5' rapid amplification of cDNA ends and sequence analysis, we deduced the promoter regions for all JAK genes and found they do not contain typical TATA or CCAAT boxes but rather numerous other potential binding sites for transcription factors. Interestingly, the TYK2 gene is linked to CDC37 in a head-to-tail manner with a small intergenic region of 292 bp. Within this region, there are two potential binding sites for transcriptional factors such as c-Myb and NF-IL6. The putative promoter regions of all JAK genes were tested either in a carp CF cell line or in zebrafish embryos using CAT or lacZ as reporter genes. Both assays confirmed the transcriptional activities of these promoters in vitro and in vivo.

Molecular cloning and characterization of a hemolymph clottable protein from tiger shrimp (Penaeus monodon).

To investigate the coagulation system in crustacean decapoda, a homodimeric glycoprotein of 380 kDa was purified from the hemolymph of tiger shrimp (Penaeus monodon) by sequential DEAE anion exchange chromatography. The purified protein was coagulated by the shrimp hemocyte transglutaminase in the presence of Ca2+. The clottable protein contains 44% alpha helices and 26% beta sheets as determined by circular dichroism spectra. Its conformation is stable in buffer of pH 4-9. To solve its primary structure, partial sequences of the purified polypeptides from cyanogen bromide cleavage and endopeptidase digestion were also determined. A shrimp cDNA expression library was constructed. By combination with antibody screening, reverse transcriptase PCR using degenerate primers from determined amino acid sequences and cDNA library screening with digoxigenin-labeled DNA probes, the entire cDNA of 6124 bp was obtained. This cDNA encodes a protein of 1670 amino acids, including a 14-amino acid signal peptide. With four potential N-glycosylation sites, the clottable protein was found to contain 3.8% high-mannose glycan; and Man8GlcNAc and Man9GlcNAc were released upon endo-beta-N-acetylglucosaminidase hydrolysis. Upon conducting a protein sequence database survey, the shrimp clottable protein shows 36% identities to the crayfish clotting protein and lower similarities to members of insect vitellogenins, apolipoprotein B and mammalian von Willebrand factor. Notably, a region rich in Gln residues, a polyGln motif and five Ser-Lys-Thr-Ser repeats are present in the shrimp protein, suggesting this protein might be a transglutaminase substrate. Northern blot analysis revealed that the clottable protein is expressed in most of the shrimp tissues but not in the mature hemocytes.

Isolation and identification of novel protein kinase genes from the round-spotted pufferfish (Tetraodon fluviatilis) genomic DNA.

The round-spotted pufferfish Tetraodon fluviatilis has a genome size of 380 Mb which is slightly smaller than that of another pufferfish, Fugu rubripes rubripes (Fugu). Due to their compact genome and small introns, both pufferfishes have been proposed as model organisms for genome studies. In this study, we have used genomic DNA as template to perform PCR to screen for protein kinase (pk) genes. Forty-one T. fluviatilis pk genes encoding 7 receptor tyrosine kinases, 14 nonreceptor tyrosine kinases, 16 serine/threonine kinases, 1 dual kinase and 3 novel kinases have been identified. The success of this approach depends on the size and location of the introns. Most of the identified pk gene fragments contain introns, ranging from 71 to 300 bp, with an average of 120 bp. It is noteworthy that the intron/exon boundaries of certain genes which belong to the same family are identical. We also analyzed by specific RT-PCR primers the expression profile of those 3 novel genes as well as some selected pk genes in a variety of tissues. We found that erbB3, pku a, mrk, CaMK I, CaMKIIgamma, and two novel kinase genes (133 and 3-26) are expressed in all tissues examined. However, the novel clone 146 is strongly expressed in the brain and weakly in the intestine, kidney and heart.

Round-spotted pufferfish (Tetraodon fluviatilis) snf5 gene is oriented in a tail-to-tail manner with the set gene which encodes an inhibitor of protein phosphatase 2A.

The round-spotted pufferfish Tetraodon fluviatilis has a genome size of 380 Mb which is slightly smaller than that of another pufferfish Fugu rubripes rubripes (Fugu). Due to its compact genome and small introns, Fugu has been introduced as a model for genome studies. Recently, the round-spotted pufferfish has also been proposed as a new model for genome studies because of the ease in obtaining material and high-sequence homology to that of Fugu. In this study, we have cloned and characterized the snf5 and set genes from the round-spotted pufferfish. The snf5 gene is composed of 9 exons spanning about 2.9 kb whereas the set gene consists of 8 exons spanning about 2.7 kb. They are linked in a tail-to-tail manner with an intergenic region of about 6.5 kb. So far, the genomic structures of human snf5 and set genes are unknown. Based on our data, the pufferfish SNF5 and SET display high amino acid sequence identity (>90%) with the respective human genes. By primer extension and sequence analysis, we found that putative promoter region of the snf5 gene contains a typical TATA box and numerous potential binding sites for transcription factors including AP1, AP2, AP3, c-Myb, HNF-5, and NF-IL6. As for the set gene, its promoter region does not have any TATA or CCAAT motif and contains a few potential binding sites for transcriptional factors such as c-Myb and gamma-IRE. When these promoter regions were placed upstream of the CAT reporter gene and transfected into a carp CF cell line, the 5'-upstream 1.6-kb DNA fragment of the snf5 gene displayed stronger promoter activity, approximately three-fold higher than that of the 5'-upstream 1.3 kb DNA fragment of the set gene. By transient expression and immunofluorescent staining, we also showed that the pufferfish SNF5 and SET are nuclear proteins, consistent with their postulated roles as transcriptional factors.

Genomic organization and characterization of the promoter region of the round-spotted pufferfish (Tetraodon fluviatilis) JAK1 kinase gene.

Seventeen kilobases of genomic DNA containing the promoter and the coding region of the round-spotted pufferfish JAK1 gene was isolated and completely sequenced. This gene consists of 25 exons and 24 introns spanning about 13.5 kb, compared to > 30kb in carp JAK1 gene. Primer extension analysis revealed one transcription initiation site which was 376 bp upstream of the translation initiation site. The sequence of the 2.9 kb region upstream of the transcription initiation site contains numerous potential binding sites for transcription factors including HNF-5, GCF, Sp1, CRE, AP2, GATA, GAGA, E2A, p53, and NF-IL6. When this region was placed upstream of the chloramphenicol acetyltransferase (CAT) reporter gene and transfected into a carp CF cell line, it could drive the synthesis of CAT enzyme three times more efficiently than could the common carp JAK1 promoter.

Expression, characterization, and genomic structure of carp JAK1 kinase gene.

A 3.7-kb cDNA encodes the carp JAK1 kinase of 1,156 amino acid residues. The overall amino acid sequence identity between carp JAK1 and murine JAK1, JAK2, JAK3, and human TYK2 is 57%, 35.5%, 31.3%, and 42.4%, respectively. In addition, carp JAK1 shows higher sequence homology to mammalian JAK1 in both the kinase-like (JH2) and kinase (JH1) domains (approximately 70% identity). Therefore, carp JAK1 is a homolog of mammalian JAK1. To investigate the possible function of JH2 domain, full-length, and various truncated forms of carp JAK1 were produced in the baculovirus system. Our results demonstrate that c-JH1 and c-JH2 associate with each other and c-JH2 can be tyrosine-phosphorylated by c-JAK1 and by c-JH(1 + 2). The JAK1 gene was also isolated from a carp genomic library and characterized. This gene is divided into 24 exons spanning at least 31 kb of genomic DNA. Exon 1 contains the 5'-untranslated region and exon 2 contains the putative translation initiation site. The 2.5-kb DNA region upstream of the transcription initiation site contains numerous potential binding sites for transcription factors including NF-IL6, HNF-5, AP1, GHF-5, and E2A. When this DNA fragment was placed upstream of the chloramphenicol acetyltransferase (CAT) reporter gene and transfected into a carp CF cell line, it could drive the synthesis of CAT enzyme 16 times more efficiently than the promoterless pCAT-Basic. Deletion analysis defined a positive regulatory region between -1,023 and -528. A smaller region (-181 to +59) without any typical TATA-box sequences, G + C-rich sequences, or other binding sequences for known transcription factors still had promoter activity. Constructs without this region did not have detectable promoter activity. This suggests that this region of DNA may play an important role in the expression of carp JAK1 gene.

Genomic structure of carp mitogen-activated protein kinase kinase 1 gene.

Carp mitogen-activated protein kinase kinase 1 (cMKK1) gene was isolated from a liver genomic library. The sequence around the exon-intron boundaries and 2 kb of the promoter region were determined. Our data indicate that this gene is composed of 11 exons and 10 introns spanning about 9 kb. Multiple potential transcription initiation sites were located by primer extension analysis. Examination of 2 kb of 5'-flanking sequence revealed potential binding sites for a variety of transcription factors such as E2F, Ets-1, GATA-1, Myb, NF-IL6, Sp1, and NF-kB.

Comparison of three different immunoassays and PCR for the detection of hepatitis C virus infection in pregnant women in Taiwan.

To compare different hepatitis C virus (HCV) immunoassays and HCV-RNA in pregnant women, we investigated two independent groups: 1,687 cases without screening for serum alanine aminotransferase (ALT) (group A) and 333 cases with elevated ALT (> 45 IU/l) (group B), after screening 21,459 pregnant women. In group A, 11 (0.65%) and 21 (1.24%) were anti-HCV-positive by first- and second-generation tests, respectively, while in group B 8 (2.40%) and 19 (5.71%) were positive, respectively. The results revealed by second-generation assays based on either recombinant protein or synthetic peptides were identical, as were the anti-HCV titers in group B. Among 40 second-generation anti-HCV-positive cases, 18 (86%) of 21 in group A and 17 (89%) of the 19 in group B contained serum HCV-RNA by RT-PCR. Thus the prevalence of anti-HCV in Taiwanese pregnant women is 1.24% versus 5.71% in those with elevated ALT level.