ABSTRACT
Clonorchiasis caused by the oriental liver fluke Clonorchis sinensis is a fish-borne zoonosis endemic in a number of countries. This article describes the development of a TaqMan based real-time PCR assay for detection of C. sinensis DNA in human feces and in fishes. Primers targeting the first internal transcribed spacer (ITS-1) sequence of the fluke were highly specific for C. sinensis, as evidenced by the negative amplification of closely related trematodes in the test with the exception of Opisthorchis viverrini. The detection limit of the assay was 1pg of purified genomic DNA, 5EPG (eggs per gram feces) or one metacercaria per gram fish filet. The assay was evaluated by testing 22 human fecal samples and 37 fish tissues microscopically determined beforehand, and the PCR results were highly in agreement with the microscopic results. This real-time PCR assay provides a useful tool for the sensitive detection of C. sinensis DNA in human stool and aquatic samples in China and other endemic countries where O. viverrini and Opisthorchis felineus are absent.
Subject(s)
Clonorchiasis/diagnosis , Clonorchis sinensis/isolation & purification , DNA, Helminth/isolation & purification , Feces/parasitology , Parasitology/methods , Real-Time Polymerase Chain Reaction/methods , Animals , Cat Diseases/parasitology , Cats , China , Clonorchis sinensis/genetics , Clonorchis sinensis/growth & development , DNA, Helminth/genetics , DNA, Ribosomal Spacer , Fish Diseases/parasitology , Fishes , Humans , Ovum/growth & development , Sensitivity and SpecificityABSTRACT
A large number of O-linked N-acetylglucosamine (O-GlcNAc) residues have been mapped in vertebrate proteins, however targets of O-GlcNAcylation in plants still have not been characterized. We show here that O-GlcNAcylation of the N-terminal region of the capsid protein of Plum pox virus resembles that of animal proteins in introducing O-GlcNAc monomers. Thr-19 and Thr-24 were specifically O-GlcNAcylated. These residues are surrounded by amino acids typical of animal O-GlcNAc acceptor sites, suggesting that the specificity of O-GlcNAc transferases is conserved among plants and animals. In laboratory conditions, mutations preventing O-GlcNAcylation of Thr-19 and Thr-24 did not have noticeable effects on PPV competence to infect Prunus persicae or Nicotiana clevelandii. However, the fact that Thr-19 and Thr-24 are highly conserved among different PPV strains suggests that their O-GlcNAc modification could be relevant for efficient competitiveness in natural conditions.
Subject(s)
Capsid Proteins/chemistry , Plum Pox Virus/chemistry , Amino Acid Sequence , Amino Acid Substitution , Base Sequence , Binding Sites , Capsid Proteins/genetics , Capsid Proteins/metabolism , DNA, Viral/genetics , Glycosylation , Molecular Sequence Data , Mutagenesis, Site-Directed , Plant Diseases/virology , Plum Pox Virus/genetics , Plum Pox Virus/pathogenicity , Protein Processing, Post-Translational , Prunus/virology , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Threonine/chemistry , Nicotiana/virologyABSTRACT
The capsid protein of Plum pox virus (PPV-CP) is modified with O-linked GlcNAc (O-GlcNAc). While Arabidopsis has two O-GlcNAc transferases, SECRET AGENT (SEC) and SPINDLY (SPY), previous work suggests that SEC modifies PPV-CP and that the modification plays a role in the infection process. Here, we show that when co-expressed in Escherichia coli SEC modifies PPV-CP. Deletion mapping and site-directed mutagenesis identified three threonine and a serine located near the N-terminus of PPV-CP that are modified by SEC. Two of these threonines have recently been shown to be modified in virus from plants suggesting that SEC has the same specificity in plants and E. coli.
