Complete genome sequence of lymphocystis disease virus isolated from China

Lymphocystis diseases in fish throughout the world have been extensively described. Here we report the complete genome sequence of lymphocystis disease virus isolated in China (LCDV-C), an LCDV isolated from cultured flounder (Paralichthys olivaceus) with lymphocystis disease in China. The LCDV-C genome is 186,250 bp, with a base composition of 27.25% G+C. Computer-assisted analysis revealed 240 potential open reading frames (ORFs) and 176 nonoverlapping putative viral genes, which encode polypeptides ranging from 40 to 1,193 amino acids. The percent coding density is 67%, and the average length of each ORF is 702 bp. A search of the GenBank database using the 176 individual putative genes revealed 103 homologues to the corresponding ORFs of LCDV-1 and 73 potential genes that were not found in LCDV-1 and other iridoviruses. Among the 73 genes, there are 8 genes that contain conserved domains of cellular genes and 65 novel genes that do not show any significant homology with the sequences in public databases. Although a certain extent of similarity between putative gene products of LCDV-C and corresponding proteins of LCDV-1 was revealed, no colinearity was detected when their ORF arrangements and coding strategies were compared to each other, suggesting that a high degree of genetic rearrangements between them has occurred. And a large number of tandem and overlapping repeated sequences were observed in the LCDV-C genome. The deduced amino acid sequence of the major capsid protein (MCP) presents the highest identity to those of LCDV-1 and other iridoviruses among the LCDV-C gene products. Furthermore, a phylogenetic tree was constructed based on the multiple alignments of nine MCP amino acid sequences. Interestingly, LCDV-C and LCDV-1 were clustered together, but their amino acid identity is much less than that in other clusters. The unexpected levels of divergence between their genomes in size, gene organization, and gene product identity suggest that LCDV-C and LCDV-1 shouldn't belong to a same species and that LCDV-C should be considered a species different from LCDV-1.

Development of a rapid, sensitive and specific diagnostic assay for fish Aquareovirus based on RT-PCR

A rapid, sensitive and highly specific detection method for Aquareovirus based on reverse-transcription polymerase chain reaction (RT-PCR) was developed. Based on multiple sequence alignment of the cloned sequences of a local isolates, the Threadfin reovirus (TFV) and Guppy reovirus (GPV) with Grass carp reovirus (GCRV), a pair of degenerate primers was selected carefully and synthesized. Using this primer combination, only one specific product, approximately 450 bp in length was obtained when RT-PCR was carried out using the genomic double-stranded RNA (dsRNA) of TFV, GPV and GCRV. Similar results were also obtained when Chum salmon reovirus (CSRV) and Striped bass reovirus (SBRV) dsRNA were used as templates. No products were observed when nucleic acids other than the dsRNA of the aquareoviruses described above were used as RT-PCR templates. This technique could detect not only TFV but also GPV and GCRV in low titer virus-infected cell cultured cells. Furthermore, this method has also been shown to be able to diagnose GPV-infected guppy (Poecilia reticulata) that exhibit clinical symptoms as well as GPV-carrier guppy. Collectively, these results showed that the RT-PCR amplification method using specific degenerate primers described below is very useful for rapid and accurate detection of a variety of aquareovirus strains isolated from different host species and origin. (C) 2004 Elsevier B.V. All rights reserved.

Sequence analysis of cytochrome b gene indicates that East Asian group of cyprinid subfamily Leuciscinae (Teleostei : Cyprinidae) evolved independently

The sequences of mitochondrial cytochrome b gene of cyprinid subfamily Leuciscinae are analyzed. Phylogenetic trees generated with methods of neighbor-joining, maximum likelihood and maximum parsimony with Phenacogrammus as an outgroup indicate that Leuciscinae is not a monophyletic group but includes two discrete subgroups. The East Asian group of the subfamily Leuciscinae, including the genera Ctenopharyngodon, Elopichthys, Luciobrama, Mylopharyngodon, Ochetobius, and Squaliobarbus, is close to Aristichthys and Hypophthalmichthys, and they form a monophyletic group which is distant from the leuciscine genera in Europe, Siberia and North America, such as Phoxinus, Leuciscus, Abramis, Rutilus, Chondrostoma, Alburnus, Opsopoedus, Lythrurus, and Pimephales. Our study suggests that the diversified East Asian group of the subfamily Leuciscinae should have an independent origination.

Mitochondrial d-loop sequence variation and phylogeny of gobiobotine fishes

The mitochondrial DNA control region is amplified and sequenced from 8 genera and 10 species of gobiobotine fishes. The phylogenetic tree of Gobiobotinae and some representative species of other Cyprinid subfamilies obtained by the method of neighborhood joining, maximum likelihood and maximum parsimony with Danio rerio as an outgroup indicates that Gobiobotinae fishes are a monophyletic group which is close to Gobioninae subfamily. Gobiobotinae should be included into subfamily Gobioninae in terms of phylogenetic analysis. The research result supports that Gobiobotinae can be divided into genus Xenophysogobio and Gobiobotia. Xenophysogabio is the most primitive genera in the subfamily.

Sequence variations of the S7 ribosomal protein gene in primitive cyprinid fishes: Implication on phylogenetic analysis

Cyprinidae is the largest fish family in the world and contains about 210 genera and 2010 species. Appropriate DNA markers must be selected for the phylogenetic analyses of Cyprinidae. In present study, the 1st intron of the S7 ribosomal protein (r-protein) gene is first used to examine the relationships among cyprinid fishes. The length of the 1st intron obtained by PCR amplification ranges from 655 to 859 by in the 16 cyprinid species investigated, and is 602 by in Myxocyprinus asiaticus. Out of the alignment of 925 nucleotide sites obtained, the parsimony informative sites are 499 and occupy 54% of the total sites. The results indicate that the 1st intron sequences of the S7 r-protein gene in cyprinids are rich in informative sites and vary remarkably in sequence divergence from 2.3% between close species to 66.6% between distant species. The bootstrap values of the interior nodes in the NJ (neighbor-joining) and MP (most-parsimony) trees based on the present S7 r-protein gene data are higher than those based on cytochrome b and the d-loop region respectively. Therefore, the 1st intron sequences of the S7 r-protein gene in cyprinids are sensitive enough for phylogenetic analyses, and the 1st intron is an appropriate genetic marker for the phylogenetic reconstruction of the taxa in different cyprinid subfamilies. However, attempts to discuss whether the present S7 r-protein gene data can be applied to the phylogeny of the taxa at the level of the family or the higher categories in Cypriniformes need further studies.

Mitochondrial DNA sequence variations and systematics of the genus Distoechodon (Teleostei : Cyprinidae)

In the present paper, nucleotide sequences (925-929 bases) of the mitochondrial D-loop region and complete cytochrome b gene (1140 bases) were determined and analysed to investigate the systematic status of the genus Distoechodon . CSB1, CSB2, CSB3, CSB-D and ETAS were successfully identified in the D-loop region. The sequence variations among different samples suggest that Distoechodon compressus is a valid species and has its distribution in Taiwan, and that D. tumirostris multispinnis does not seem to be a valid species.

Development of polymorphic EST markers in Penaeus monodon: applications in penaeid genetics

The objective of this study was to develop type I markers for genome mapping and other genetic studies of Penaeus monodon. Primers were designed based on expressed sequence tags (ESTs) from a P monodon cephalothorax cDNA library to amplify 100-300 bp products. 34 of the primer pairs successfully amplified PCR products from genomic DNA. Single-strand conformation polymorphism analysis showed that similar to 30% of the ESTs tested exhibit polymorphism in a test panel of P monodon individuals. Mendelian inheritance of the EST-derived markers has been established in two international reference mapping families of P monodon, and mapping of these markers is in progress. Some ESTs were successfully amplified from other Penaeus species (P. chinensis, P japonicus and P vannamei), indicating that the markers are applicable in cross-species comparison. Two populations of P. japonicus could be differentiated using one of the ESTS. In conclusion, the polymorphic EST markers developed in this study are applicable in genome mapping and population genetic studies of penaeid shrimp. (C) 2002 Elsevier Science B.V. All rights reserved.

Mitochondrial cytochrome b sequence variations and phylogeny of the East Asian bagrid catfishes

The mitochondrial DNA cytochrome b gene was sequenced from 8 bagrid catfishes in China. Aligned with cytochrome b sequences from 9 bagrid catfishes in Japan, Korea and Russia retrieved from GenBank, and selected Silurus meridionalis, Liobagrus anguillicauda, Liobagrus reini and Phenacogrammus interruptus as outgroups, we constructed a matrix of 21 DNA sequences. The Kimura's two-parameter distances were calculated and molecule phylogenetic trees were constructed by using the maximum parsimony (MP) and neighbor-joining (NJ) methods. The results show that (i) there exist 3-bp deletions of mitochondrial cytochrome b gene compared with cypriniforms and characiforms; (ii) the molecular phylogenetic tree suggests that bagrid catfishes form a monophyletic group, and the genus Mystus is the earliest divergent in the East Asian bagrid catfishes, as well as the genus Pseudobagrus is a monophyletic group but the genus Pelteobagrus and Leiocassis are complicated; and 60 the evolution rate of the East Asian bagrids mitochondrial cytochrome b gene is about 0.18%-0.30% sequence divergence per million years.

Sequence variations in the mitochondrial DNA control region and their implications for the phylogeny of the Cypriniformes

The mitochondrial DNA control region of six cobitids and two catostomids was sequenced and compared with sequences of other cypriniforms to study their sequence variations. The extended termination associated sequence (ETAS) domain, central domain, and conserved sequence block (CSB) domain were partitioned and the ETAS sequence, CSB-D, CSB-E, ECSB-F, CSB1, CSB2, and CSB3 were identified. It is suggested that the "hairpin" TACAT-ATGTA is the key sequence of ETAS and GACATA is the symbol of CSB1. Phylogenetic analysis based on the CSB domain showed that all cyprinids evolved as one monophyletic group, while the non-cyprinid Cypriniformes could be another monophyly that is in accordance with the hypothesis proposed by Siebert. Further analysis of the phylogeny of the Cobitoidei was also conducted and it is tentatively suggested that their relationships are Catostomidae + (Gyrinocheilidae + (Botiinae + (Homalopteridae + (Cobitinae + Nemacheilinae)))).

Genome segment S8 of grass carp hemorrhage virus encodes a virion protein

The complete nucleotide sequence of the genome segment S8 of grass carp hemorrhage virus (GCHV) was determined from cDNA corresponding to the viral genomic RNA. It is 1,287 nucleotides in length and contains a large open reading frame that could encode a protein of 409 amino acids with a predicted molecular mass of 44 kD. The S8 was expressed using the pET fusion protein vector and detected by Western blotting analysis using the chicken egg IgY against intact GCHV particles, indicating that S8 encodes a virion protein. Amino acid sequence comparisons revealed that the protein encoded by S8 is closely related to protein alpha2 of mammalian reovirus, suggesting that the deduced protein of S8 is an inner capsid protein. Copyright (C) 2001 S. Karger AG, Basel.

Retroposon analysis of major cetacean lineages: The monophyly of toothed whales and the paraphyly of river dolphins

SINE (short interspersed element) insertion analysis elucidates contentious aspects in the phylogeny of toothed whales and dolphins (Odontoceti), especially river dolphins. Here, we characterize 25 informative SINEs inserted into unique genomic loci during evolution of odontocetes to construct a cladogram. and determine a total of 2.8 kb per taxon of the flanking sequences of these SINE loci to estimate divergence times among lineages. We demonstrate that: (i) Odontocetes are monophyletic; (ii) Ganges River dolphins, beaked whales, and ocean dolphins diverged (in this order) after sperm whales; (iii) three other river dolphin taxa, namely the Amazon, La Plata, and Yangtze river dolphins, form a monophyletic group with Yangtze River dolphins being the most basal; and (iv) the rapid radiation of extant cetacean lineages occurred some 28-33 million years B.P., in strong accord with the fossil record. The combination of SINE and flanking sequence analysis suggests a topology and set of divergence times for odontocete relationships, offering alternative explanations for several long-standing problems in cetacean evolution.

Complete nucleotide sequence of the S10 genome segment of grass carp reovirus (GCRV)

Hemorrhagic disease, caused by the grass carp reovirus (GCRV), is one of the major diseases of grass carp in China. Little is known about the structure and function of the gene segments of this reovirus. The S10 genome segment of GCRV was cloned and the complete nucleotide sequence is reported here. The S10 is 909 nucleotides long and contains a large open reading frame (ORF) encoding a protein of 276 amino acids with a deduced molecular weight of approximately 29.7 kDa. Comparisons of the deduced amino acid sequence of GCRV S10 with those of other reoviruses revealed no significant homologies. However, GCRV S10 shared a putative zinc-finger sequence and a similar distribution of hydrophilic motifs with the outer capsid proteins encoded by Coho salmon aquareovirus (SCSV) S10, striped bass reovirus (SBRV) S10, and mammalian reovirus (MRV) S4. It was predicted that this segment gene encodes an outer capsid protein.

Molecular phylogenetic systematics of twelve species of acipenseriformes based on mtDNA ND4L-ND4 gene sequence analysis

Acipenseriformes is an endangered primitive fish group, which occupies a special place in the history of ideas concerning fish evolution, even in vertebrate evolution. However, the classification and evolution of the fishes have been debated. The mitochondrial DNA (mtDNA) ND4L and partial ND4 genes were first sequenced in twelve species of the order Acipenseriformes, including endemic Chinese species. The following points were drawn from DNA sequences analysis: (i) the two species of Huso can be ascribed to Acipenser; (ii) A. dabryanus is the mostly closely related to A. sinensis, and most likely the landlocked form of A. sinensis; (iii) genus Acipenser in trans-Pacific region might have a common origin; (iv) mtDNA ND4L and ND4 genes are the ideal genetic markers for phylogenetic analysis of the order Acipenseriformes.

Periodic bubble emission and appearance of an ordered bubble sequence (train) during condensation in a single microchannel

Condensation of steam in a single microchannel, silicon test section was investigated visually at low flow rates. The microchannel was rectangular in cross-section with a depth of 30 pm, a width of 800 mu m and a length of 5.0 mm, covered with a Pyrex glass to allow for visualization of the bubble formation process. By varying the cooling rate during condensation of the saturated water vapor, it was possible to control the shape, size and frequency of the bubbles formed. At low cooling rates using only natural air convection from the ambient environment, the flow pattern in the microchannel consisted of a nearly stable elongated bubble attached upstream (near the inlet) that pinched off into a train of elliptical bubbles downstream of the elongated bubble. It was observed that these elliptical bubbles were emitted periodically from the tip of the elongated bubble at a high frequency, with smaller size than the channel width. The shape of the emitted bubbles underwent modifications shortly after their generation until finally becoming a stable vertical ellipse, maintaining its shape and size as it flowed downstream at a constant speed. These periodically emitted elliptical bubbles thus formed an ordered bubble sequence (train). At higher cooling rates using chilled water in a copper heat sink attached to the test section, the bubble formation frequency increased significantly while the bubble size decreased, all the while forming a perfect bubble train flowing downstream of the microchannel. The emitted bubbles in this case immediately formed into a circular shape without any further modification after their separation from the elongated bubble upstream. The present study suggests that a method for controlling the size and generation frequency of microbubbles could be so developed, which may be of interest for microfluidic applications. The breakup of the elongated bubble is caused by the large Weber number at the tip of the elongated bubble induced by the maximum vapor velocity at the centerline of the microchannel inside the elongated bubble and the smaller surface tension force of water at the tip of the elongated bubble.