Grass carp reovirus (GCRV) is the primary cause of grass carp haemorrhagic disease. The major catechin in green tea, (-)-epigallocatechin-3-gallate (EGCG), has been found to have anti-GCRV activity in the C. idellus kidney cell line (CIK). The aim of this study was to test the potential application of EGCG as an anti-GCRV agent in aquaculture. Here, we demonstrate that various concentrations (99%, 50% and 35%) of EGCG could inhibit GCRV infectivity. EGCG (50%) + GCRV treatment significantly reduced the number of dead fish at 1-, 2-, 3-, 4 -and 5-day post-challenge compared with the negative control (GCRV challenge without EGCG treatment). The safety of EGCG compound products on cell survival was studied using four fish cell lines; we did not detect a significant change in cell viability within 24 hours of EGCG incubation. We also evaluated toxicity and concentrations of malondialdehyde (MDA), glutathione (GSH) and lysozyme (LZM) in the grass carp, and the results showed that even a high dose of EGCG did not induce toxicity. Following EGCG compound injection, the concentration of MDA decreased and the concentration of GSH and LZM increased compared with the control groups. We also detected EGCG concentration in grass carp plasma and kidney using HPLC with electrochemical detection after intraperitoneal injection at a dose of 150 mg/kg. The concentration of EGCG in the plasma and kidney reached the highest levels (20 μg/ml and 1.5 μg/ml) about 12 hr after injection and then decreased. Overall, EGCG is a safe, effective product that could inhibit GCRV infection and improve immunoactivity in aquaculture.

Epidemics caused by cyprinid herpesvirus 2 (CyHV-2) in domestic cyprinid species have been reported in both European and Asian countries. Although the mechanisms remain unknown, acute CyHV-2 infections generally result in high mortality, and the surviving carps become chronic carriers displaying no external clinical signs. In this study, in situ hybridization analysis showed that CyHV-2 tended to infect peripheral blood cells during either acute or chronic infections in silver crucian carp, Carassius auratus gibelio (Bloch). Laboratory challenge experiments coupled with real-time PCR quantification assays further indicated that steady-state levels of the viral genomic copy number in fish serum exhibited a typical 'one-step' growth curve post-viral challenge. Transcriptional expression of open reading frames (ORF) 121, which was selected due to its highest transcriptional levels in almost all tested tissues, was monitored to represent the replication kinetics of CyHV-2 in peripheral blood cells. Similar kinetic curve of active viral gene transcription in blood cells was obtained as that of serum viral load, indicating that CyHV-2 replicated in peripheral blood cells as well as in other well-characterized tissues. This study should pave the way for designing non-invasive and cost-effective serum diagnostic methods for quick detection of CyHV-2 infection.

Herpesviral haematopoietic necrosis (HVHN), caused by cyprinid herpesvirus 2 (CyHV-2), causes significant losses in crucian carp (Carassius carassius) aquaculture. Rapid and convenient DNA assay detection of CyHV-2 is useful for field diagnosis. Recombinase polymerase amplification (RPA) is a novel isothermal DNA amplification and detection technology that can amplify DNA within 30 min at ~37°C by simulating in vivo DNA recombination. Herein, a rapid and convenient detection assay based on RPA with a lateral flow dipstick (LFD) was developed for detecting CyHV-2. The highly conserved ORF72 of CyHV-2 was targeted by specific and sensitive primers and probes. The optimized assay takes only 15 min at 38°C using a water bath, with analysis of products by 2% agarose gel electrophoresis within 30 min. A simple lateral flow strip based on the unique probe in reaction buffer was developed for visualization. The entire RPA-LFD assay takes 50 min less than the routine PCR method, is 100 times more sensitive and displays no cross-reaction with other aquatic viruses. The combined isothermal RPA and lateral flow assay (RPA-LFD) provides a simple, rapid, reliable method that could improve field diagnosis of CyHV-2 when resources are limited.

Cyprinid herpesvirus 2 (CyHV-2) is the main pathogen responsible for causing haematopoietic necrosis disease in Carassius auratus gibelio. Although many nucleic acid-based diagnostic methods have been applied, no stable and sensitive immunological diagnostic approaches have been reported. In this study, to detect CyHV-2 in clinical samples using immunological methods, recombinant ORF72 protein (pORF72), encoded by the CyHV-2 ORF72 gene, was used as a capture antigen to identify blood and tissues infected with CyHV-2. First, ORF72 gene was amplified from the CyHV-2 genome and cloned into a PGEX-4t-3 expression vector to produce pORF72 in Escherichia coli. The purified pORF72 was used as an immunogen to prepare monoclonal antibodies. The Western blotting assays revealed that the monoclonal antibody could specifically identify the pORF72. Furthermore, an immunohistochemical protocol and a blood smear method were established to detect CyHV-2 in carps. The results indicate that the monoclonal antibody against pORF72 could be utilized as an effective detection tool for haematopoietic necrosis disease in Carassius auratus gibelio.