In vitro inhibition of Cyprinid herpesvirus-3 replication by RNAi

In 2 week-old fry infected experimentally by bath, HSC appeared after 1 week of incubation at 20°C (Sano et al., 1991). Internal signs of KHVD are variable and non-specific but may include greater than normal adhesions in the body cavity and enlargement and/or mottled appearance of internal organs (Hedrick et al. Het gevaar voor het besmetten van andere vijvers is groot. Also, the phylogenetic analysis was conducted using Bioedit software and Molecular Evolutionary Genetics Analysis (MEGA) 5 software, with bootstrap values calculated from 1000 replicates. Plugs were then transferred to 5 volumes of buffer L supplemented with 0.1 mg/ml proteinase K (Sigma-Aldrich) and 1% (wt/vol) Sarkosyl (Sigma-Aldrich) and incubated for 16 h at 50°C. In the FL BAC revertant and the FL BAC revertant ORF134 Rev, the ORF134Del probe led to a single band corresponding to a 6.3 kb restriction fragment consistent with the sequence of this region (6333kb). Y.

CCB and KF-1 cell lines.Common carp brain cell line (CCB) and koi fin cell line (KF-1) (gift from Ronald Hedrick, University of California, Davis) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (Gemini Bio-Products, West Sacramento, CA), penicillin (100 U/ml), and streptomycin (100 μg/ml) (Sigma-Aldrich, Inc., St. The clinical signs of CyHV-2 infection include lethargy, development of pale skin with white, mucoid, blister-like projections on tissue, and ultimately mortality (Jeffery et al. Taking mock-infected fish as a reference, expression of several cytokines (IFN-γ2, IL-1β, IL-6, and IL-10) was up-regulated as early as day 3 post-infection. The first PCR was carried out with an outer primer set (JF1 and JR1), yielding a PCR product with 716 bp. Half of the colonies were then transferred to 500 ml LB medium supplemented with ampicillin and chloramphenicol and incubated shaking at 200 rpm at 37°C until an OD600 of 0.6–0.8 was reached. Several recent studies addressed the host–pathogen interactions of CyHV-3 on the protein level (Gotesman et al., 2013a, 2013b; Ouyang et al., 2013) or on the transcriptional level (Adamek et al., 2012, 2013; Rakus et al., 2012). These results suggest that the early stages of carp development are resistant to CyHV-3.


Purified viral pellets were suspended and the viral DNA was purified as described by Hutoran et al. B/For each analysed organ, the number of positive fish is presented according to time post-infection. Preparation of infected-cell RNA.Total cell RNA was isolated from AngHV1-infected cells at 12 h postinfection (p.i.). Mucus samples were pooled and stored on ice. We produced a LUC-expressing recombinant strain by intergenic insertion of a LUC expression cassette. We also evaluated the latency of CyHV-3 in carriers by assessing the expression of genes that are stably transcribed without virus replication (Ilouze et al., 2012b) and by detecting circular and linear CyHV-3 genome. A PCR primer set (JF/JR) was designed to amplify the complete cds of the viral putative DNA helicase gene (GenBank accession EU349287).

(A) Study sites. Alternatively, PCR-based methods have become prominent recently by virtue of their sensitivity, specificity, and rapidity (Fong & Lipp, 2005). Crucially, there is no evidence that CyHV-3 causes clinical, or histological, signs of disease in any non-target species (McColl et al. Proteins in purified virions, capsids and virion fractions were analyzed by SDS-PAGE to check purity and analyze the protein content of the different virion compartments. Cell lines derived from goldfish and ginbuna C. Herpesviral hematopoietic necrosis (HVHN), caused by Cyprinid herpesvirus 2 (CyHV-2), is a disease of goldfish Carassius auratus auratus (Linnaeus, 1758) that was first reported in juvenile goldfish in Japan in the spring and autumn of 1992 and the spring of 1993 and later in Australia, Taiwan, and the USA [1, 2]. These results indicate that the LAMP assay is simple, sensitive, and specific and has a great potential use for CyHV-2 detection in the laboratory and field.

Our results also suggest that an activation of type I IFN in CyHV-3 infected cells can limit the spread of the virus in cell culture. Our results revealed that the life cycle of CyHV-3 may fit perfectly into the ecology of its host, resulting in the long-term persistence of this emerging virus in wild common carp populations. The authors thank Dr. Here, bioinformatic analyses of the CyHV-3 genome suggested the presence of non-conserved precursor miRNA (pre-miRNA) genes. IFNγ-1, IFNγ-2, IL-12 and IL-10 host genes, and the CyHV-3 vIL-10 gene (khvIL-10) were highly significantly up-regulated in different phases of CyHV-3 infection. KHV thus has the largest genome reported to date for this family. Virol.

cKey Laboratory of Freshwater Animal Breeding, Ministry of Agriculture, Wuhan 430070, People’s Republic of China. Recently, the virus has also been detected from Prussian carp (C. Cyprinid herpesvirus-3 (CyHV-3) is a highly infectious pathogen that causes fatal disease in common and koi carp Cyprinus carpio L. ABSTRACT: In this study, we examined the influence of water temperature on the development of herpesviral haematopoietic necrosis (HVHN) in goldfish Carassius auratus after experimentally induced infection with cyprinid herpesvirus 2 (CyHV-2).

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