ABSTRACT
Nodaviruses are widespread causative agents of viral nervous necrosis in fish. Based on the coat protein sequence, fish nodaviruses are categorized into four different genotypes. In this study, we present data on the phylogenetic and antigenic characterization of 12 new isolates, eight European and four of Asian origin, from farmed and wild species of fish. Phylogenetic analysis based on the nucleotide sequence (688 bases) of the coat protein classified the majority of these new isolates to the RGNNV genotype. Geographic or host-species specificities were not revealed by this study. Neutralizing assay experiments, further confirmed the genotypic classification, supporting the possibility that the different nodavirus genotypes can also be serologically distinguishable.
Subject(s)
Capsid/genetics , Fishes/virology , RNA Viruses/classification , Amino Acid Sequence , Animals , Antigens, Viral/genetics , Antigens, Viral/immunology , Asia , Capsid/immunology , Cloning, Molecular , Europe , Genome, Viral , Genotype , Immune Sera , Molecular Sequence Data , Neutralization Tests , Phylogeny , RNA Viruses/genetics , RNA Viruses/immunology , Rabbits , Sequence AlignmentABSTRACT
Two overlapping clones encoding for a ribonuclease from six-day-old larvae of the insect Ceratitis capitata (Cc-RNase) have been isolated by immunoscreening a cDNA library and by 5' RACE. The sequence of the Cc-RNase cDNA contains an open reading frame of 414 nucleotides encoding for a precursor protein of 138 amino acids long with a putative signal peptide consisting of 19 amino acids. The calculated M(r) of the mature protein was found to be 13.7 kDa. Multiple alignments of the deduced amino acid Cc-RNase sequence with other ribonucleases revealed an approximate 25% average identity. Despite the low percentage of identity, histidine and lysine residues which are essential for its catalytic activity, were found to be completely conserved. Furthermore, expression of the clone in E. coli resulted in the production of a recombinant product that showed strong immunoreactivity with anti-RNase specific antibodies. These results support the hypothesis that the identified clone encodes for a protein which is a new member of the RNase superfamily.
Subject(s)
DNA, Complementary/genetics , DNA, Complementary/isolation & purification , Diptera/enzymology , Diptera/genetics , Ribonucleases/genetics , Amino Acid Sequence , Animals , Base Sequence , Cloning, Molecular , DNA Primers/genetics , Escherichia coli/genetics , Genes, Insect , Larva/enzymology , Molecular Sequence Data , RNA, Messenger/genetics , RNA, Messenger/metabolism , Sequence Homology, Amino AcidABSTRACT
The coat protein gene from encephalitis virus infecting Dicentrarhus labrax (DIEV) has been cloned by gene amplification, sequenced and expressed in Escherichia coli. DNA sequencing has revealed an open reading frame of 1017 bases encoding a polypeptide of 338 amino acids. The sequence similarities between the DIEV coat protein gene and the same gene in five encephalitis viruses infected other fish species were over 71.5% at the nucleotide level and over 79.5% at the amino acid level. These results indicate that the nodaviruses that cause encephalopathy and retinopathy in fishes are very closed related. E. coli cells harbouring the plasmid containing the DIEV gene can produce the viral coat protein. An efficient purification scheme using a Sepharore-Ni+2 column is presented. This, gives approx. 10 mg of more than 95% pure protein per gr of E. coli culture.
Subject(s)
Bass/virology , Capsid Proteins , Capsid/genetics , Capsid/metabolism , Encephalitis Viruses/chemistry , Amino Acid Sequence , Animals , Base Sequence , Capsid/isolation & purification , Chromatography, Affinity/methods , Cloning, Molecular , Encephalitis Viruses/genetics , Molecular Sequence Data , RNA Viruses/chemistry , RNA Viruses/genetics , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Sequence Analysis , Sequence Homology, Amino Acid , Sequence Homology, Nucleic AcidABSTRACT
The cDNA encoding the L-dopa decarboxylase (ddc) from the eclosion stage of the insect Ceratitis capitata was isolated by PCR and a molecular cloning strategy. The isolated cDNA clone encoded a protein of 431 amino acids with a calculated molecular weight of 47,843 Da. Northern blot analysis of poly(A)+ RNA showed an approximately 2 kb transcript. The deduced protein sequence shares a high percentage of homology with Ddc protein sequences of other species. Furthermore, the molecular weight of the deduced protein agreed well with that of the purified Ddc from the same insect. Data base search revealed significant and extensive sequence similarities among prokaryotic and eukaryotic PLP-dependent decarboxylases including Ceratitis capitata and bacterial histidine decarboxylase (HDC), strongly suggesting an ancient and common origin for all PLP-dependent decarboxylases.
Subject(s)
Diptera/enzymology , Dopa Decarboxylase/genetics , Insect Proteins/genetics , Amino Acid Sequence , Animals , Base Sequence , Cloning, Molecular , DNA, Complementary , Diptera/genetics , Diptera/growth & development , Evolution, Molecular , Humans , Molecular Sequence Data , Sequence Homology, Amino AcidABSTRACT
A poly(U), poly(C) specific RNase of apparent MW 34 kDa has recently been purified from 6 day old larvae of the insect Ceratitis capitata. Two monoclonal antibodies were obtained by immunizing mice with this protein. Immunoblot analysis of the RNase revealed that both antibodies recognize the 34 kDa protein. Furthermore, immunoprecipitation experiments show that both antibodies were capable of precipitating the ribonuclease without affecting its catalytic activity.
Subject(s)
Antibodies, Monoclonal/immunology , Poly C/metabolism , Poly U/metabolism , Ribonucleases/immunology , Animals , Antibody Specificity , Blotting, Western , Diptera/enzymology , Mice , Precipitin Tests , Ribonucleases/isolation & purification , Ribonucleases/metabolism , Tumor Cells, CulturedABSTRACT
A specific ribonuclease was detected and purified to homogeneity from six-day-old larvae of the insect Ceratitis capitata and its homogeneity was checked by analysis in polyacrylamide gels in the presence of sodium dodecyl sulfate. The nuclease specifically degrades poly(U) and poly(C) whilst it fails to do so with other single-stranded homopolyribonucleotides. The enzyme has a pH optimum in the region 7-9 and relative molecular mass of about 25,000. The effect of this ribonuclease on the integrity of RNAs isolated from six-day-old larvae or rat liver was also studied.
