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Keywords = VNNV

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16 pages, 3401 KB  
Article
Designing and Validation of a Droplet Digital PCR Procedure for Diagnosis and Accurate Quantification of Nervous Necrosis Virus in the Mediterranean Area
by Sandra Souto, José G. Olveira, Carmen López-Vázquez, Isabel Bandín and Carlos P. Dopazo
Pathogens 2023, 12(9), 1155; https://doi.org/10.3390/pathogens12091155 - 12 Sep 2023
Cited by 4 | Viewed by 3674
Abstract
The viral nervous necrosis virus (VNNV) is the causative agent of an important disease affecting fish species cultured worldwide. Early and accurate diagnosis is, at present, the most effective control and prevention tool, and molecular techniques have been strongly introduced and accepted by [...] Read more.
The viral nervous necrosis virus (VNNV) is the causative agent of an important disease affecting fish species cultured worldwide. Early and accurate diagnosis is, at present, the most effective control and prevention tool, and molecular techniques have been strongly introduced and accepted by official organizations. Among those, real-time quantitative polymerase chain reaction (rt-qPCR) is nowadays displacing other molecular techniques. However, another PCR-based technology, droplet digital PCR (ddPCR), is on the increase. It has many advantages over qPCR, such as higher sensitivity and more reliability of the quantification. Therefore, we decided to design and validate a protocol for the diagnosis and quantification of SJ and RG type VNNV using reverse transcription-ddPCR (RT-ddPCR). We obtained an extremely low limit of detection, 10- to 100-fold lower than with RT-qPCR. Quantification by RT-ddPCR, with a dynamic range of 6.8–6.8 × 104 (SJ type) or 1.04 × 101–1.04 × 105 (RG type) cps/rctn, was more reliable than with RT-qPCR. The procedure was tested and validated in field samples, providing high clinical sensitivity and negative predictive values. In conclusion, we propose this method to substitute RT-qPCR protocols because it exceeds the expectations of qPCR in the diagnosis and quantification of VNNV. Full article
(This article belongs to the Special Issue Diagnostics of Animal Viral Infectious Diseases)
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19 pages, 1782 KB  
Article
Sea Bass Immunization to Downsize the Betanodavirus Protein Displayed in the Surface of Inactivated Repair-Less Bacteria
by Raquel Lama, Patricia Pereiro, Beatriz Novoa and Julio Coll
Vaccines 2019, 7(3), 94; https://doi.org/10.3390/vaccines7030094 - 20 Aug 2019
Cited by 2 | Viewed by 4839
Abstract
This work describes immunization of European sea bass (Dicentrarchus labrax) juveniles against viral nervous necrosis virus (VNNV), a betanodavirus causing worldwide mortalities in many fish species. Protection was obtained with the so-called spinycterin vehicles consisting of irreversibly DNA-damaged DNA-repair-less Escherichia coli [...] Read more.
This work describes immunization of European sea bass (Dicentrarchus labrax) juveniles against viral nervous necrosis virus (VNNV), a betanodavirus causing worldwide mortalities in many fish species. Protection was obtained with the so-called spinycterin vehicles consisting of irreversibly DNA-damaged DNA-repair-less Escherichia coli displaying at their surface a downsized VNNV coat antigen. In this work we have (i) maximized bacterial expression levels by downsizing the coat protein of VNNV to a fragment (frgC91–220) containing most of its previously determined antigenicity, (ii) developed a scalable autoinduction culture media for E. coli based in soy-bean rather than in casein hydrolysates, (iii) enriched surface expression by screening different anchors from several prokaryotic sources (anchor + frgC91–220 recombinant products), (iv) preserved frgC91–220 antigenicity by inactivating bacteria by irreversible DNA-damage by means of Ciprofloxacin, and (v) increased safety using a repair-less E. coli strain as chassis for the spinycterins. These spinycterins protected fish against VNNV challenge with partial (Nmistic + frgC91–220) or total (YBEL + frgC91–220) levels of protection, in contrast to fish immunized with frgC91–220 spinycterins. The proposed spinycterin platform has high levels of environmental safety and cost effectiveness and required no adjuvants, thus providing potential to further develop VNNV vaccines for sustainable aquaculture. Full article
(This article belongs to the Section Veterinary Vaccines)
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