Pei-Yu Lee

Real-time target-specific detection of loop-mediated isothermal amplification for white spot syndrome virus using fluorescence energy transfer-based probes.

Aiming to establish a target amplicon-specific detection system for loop-mediated isothermal amplification (LAMP), the fluorescent resonance energy transfer (FRET) probe technology was applied to develop the FRET LAMP platform. This report describes the development of the first FRET LAMP assay targeting white spot syndrome virus (WSSV) of penaeid shrimp. A successful accelerated WSSV LAMP was assembled first in a conventional oven and confirmed by gel electrophoresis and dot-blot hybridization. Subsequently, two additional FRET probes designed to target one loop region within WSSV LAMP amplicons were added to the same LAMP reaction. The reactions were carried out in a LightCycler (Roche) and significant FRET signals were detected in real time. Optimization of the reaction using plasmid DNA shortened the time for the detection of 10(2) copies of the target DNA to less than 70min. Cross reactivity was absent with WSSV-free or infectious hypodermal and hematopoietic necrosis virus-infected Penaeus vannamei samples. The performance of this system was comparable with that of a nested PCR assay from 21 WSSV-infected shrimp. Specifically detecting target amplicons and requiring no post-amplification manipulation, the novel FRET LAMP assay should allow indisputable detection of pathogens with minimized risks of amplicon contamination.

Detection of white spot syndrome virus by polymerase chain reaction performed under insulated isothermal conditions.

Aiming to develop a rapid, low-cost, and user-friendly system for the diagnosis of white spot syndrome virus (WSSV), a PCR assay performed in capillary tubes under insulated isothermal conditions (iiPCR assay) was established on the basis of Rayleigh-Benard convection. WSSV amplicons were generated reproducibly within 30 min from a target sequence-containing plasmid in an iiPCR device, in which a special polycarbonate capillary tube (R-tube™) was heated isothermally by a copper ring attached to its bottom and shielded by a thermal baffle around its upper half. Furthermore, WSSV-specific amplicons were produced from nucleic acid extracts of WSSV-infected Penaeus vannamei in the WSSV iiPCR assay, with sensitivity comparable to that of an OIE-certified commercial nested PCR kit (IQ2000™ WSSV Detection and Prevention System). Specificity of the WSSV iiPCR assay was demonstrated as no amplicons were generated from shrimp genomic DNA, and IHHNV, MBV, and HPV DNA. iiPCR has a potential as a low-cost method for sensitive, specific and rapid detection of pathogens.

The amino-terminal domain of ORF149 of koi herpesvirus is preferentially targeted by IgM from carp populations surviving infection

Recombinantly expressed fragments of the protein encoded by ORF149 (pORF149), a structural protein from the common- and koi-carp-infecting cyprinid herpesvirus-3 (CyHV-3) that was previously shown to be antigenic, were used to obtain evidence that its amino-terminal part contains immunodominant epitopes in fish populations that survived the infection. To obtain such evidence, nonspecific binding of carp serum tetrameric IgM had to be overcome by a novel ELISA protocol (rec2-ELISA). Rec2-ELISA involved pre-adsorption of carp sera with a heterologous recombinant fragment before incubation with pORF149 fragments and detection with anti-carp IgM monoclonal antibodies. Only in this way was it possible to distinguish between sera from uninfected and survivor carp populations. Although IgM from survivors recognised pORF149 fragments to a lesser degree than whole virus, specificity was confirmed by correlation of rec2- and CyHV-3-ELISAs, inhibition of rec2-ELISA by an excess of frgIIORF149, ELISA using IgM-capture, Western blotting, and reduction of reactivity in CyHV-3-ELISA by pre-adsorption of sera with frgIIORF149. The similarity of IgM-binding profiles between frgIORF149 (amino acid residues 42-629) and frgIIORF149 (42-159) and their reactivities with previously described anti-CyHV-3 monoclonal antibodies confirmed that most pORF149 epitopes were localised in its amino-terminal part.

Genetic Variability of Koi Herpesvirus In vitro—A Natural Event?

Worldwide koi herpesvirus (KHV) causes high mortalities in Cyprinus carpio L. aquaculture. So far, it is unknown how the different variants of KHV have developed and how they spread in the fish, but also in the environmental water bodies. Therefore, a phylogenetic method based on variable number of tandem repeats (VNTR) was improved to gain deeper insights into the phylogeny of KHV and its possible worldwide distribution. Moreover, a VNTR-3 qPCR was designed which allows fast virus typing. This study presents a useful method for molecular tracing of diverse KHV types, variants, and lineages.

Generation of a potential koi herpesvirus live vaccine by simultaneous deletion of the viral thymidine kinase and dUTPase genes

Koi herpesvirus (KHV, Cyprinidherpesvirus 3) causes a fatal disease of koi and common carp. To obtain safe and efficacious live vaccines, we generated deletion mutants of KHV lacking the nonessential genes encoding two enzymes of nucleotide metabolism, thymidine kinase (TK, ORF55) and deoxyuridine-triphosphatase (DUT, ORF123). Since single-deletion mutants based on a KHV isolate from Israel (KHV-I) only exhibited partial attenuation (Fuchs W, Fichtner D, Bergmann SM, Mettenleiter TC. Arch Virol 2011;156 : 1059-1063), a corresponding double mutant was generated and tested in vivo, and shown to be almost avirulent but still protective. To overcome the low in vitro virus titres of KHV-I (≤105 p.f.u. ml-1), single and double TK and DUT deletions were also introduced into a cell culture-adapted KHV strain from Taiwan (KHV-T). The deletions did not affect in vitro virus replication, and all KHV-T mutants exhibited wild-type-like plaque sizes and titres exceeding 107 p.f.u. ml-1, as a prerequisite for economic vaccine production. Compared to wild-type and revertant viruses, the single-deletion mutants of KHV-T were significantly attenuated in vivo, and immersion of juvenile carp in water containing high doses of the double mutant caused almost no fatalities. Nevertheless, the deletion mutants induced similar levels of KHV-specific serum antibodies to the parental wild-type virus, and conferred solid protection against disease after challenge with wild-type KHV. For the convenient differentiation of DNA samples prepared from gill swabs of carp infected with wild-type and TK-deleted KHV we developed a triplex real-time PCR. Thus, KHV-TΔDUT/TK might be suitable as a genetic DIVA vaccine in the field.