Cyprinid Herpesvirus Research Articles

Molecular detection and phylogenetic analysis of Cyprinid herpesvirus 3 in Brazilian ornamental fish.

Cyprinid herpesvirus 3 has a worldwide distribution and presents high mortality rates in species of Cyprinus carpio, causing serious economic loss to the global aquaculture industry. The description of this infection in other ornamental fish species is still limited. For this purpose, 100 ornamental fish from 24 different species were tested by PCR for Cyprinid hespesvirus 3 and the positive samples represented 6% of the tested samples. Phylogenetic reconstruction, based on the Thymidine Kinase gene, revealed the existence of two distinct clades. One clade grouped a Brazilian sample with European and Asian genotypes of CyHV-3 and a second clade, containing only Brazilian sequences described in this study. All of the Brazilian sequences showed identity values greater than 97.7% when compared to each other. This is the first report of the occurrence of Cyprinid herpesvirus 3 in ornamental fish species in Brazil. These results in association with further studies of viral isolation and characterization can help in establishing effective surveillance and disease control program.

Molecular cloning and functional analysis of common carp (Cyprinus carpio) integrin α6 and the correlation with the resistance to CyHV-3 infection

Integrin α6 (ITGα6) is involved in a variety of cellular physiological responses and plays an important role in the immune response. Currently, common carp is severely affected by the virus of Cyprinid herpesvirus 3 (CyHV-3) in aquaculture. Studies have shown that ITGα6 is associated with herpesvirus infection. However, the role of ITGα6 in the response to CyHV-3 virus infection is still unclear. In this paper, we cloned the ITGα6 from common carp (CcITGα6) and studied its expression profile after infection with CyHV-3. Two transcripts of CcITGα6 were cloned, named CcITGα6a and CcITGα6b. The full-length cDNA of the CcITGα6a transcript was 3867 bp, the ORF was 3273 bp, and 1090 amino acids were encoded. The full-length cDNA of the CcITGα6b transcript was 3368 bp, the ORF was 3189 bp, and 1062 amino acids were encoded. Two transcripts of CcITGα6 consist of four parts: the signal peptide, extracellular region, transmembrane region, and cytoplasmic region. The difference between the two CcITGα6 transcripts lies in the cytoplasmic region and no homologous sequence. Similarly, the secondary structure, glycosylation sites, and phosphorylation sites were different in the cytoplasmic region. Phylogenetic analysis showed that CcITGα6 was highly conserved and clustered with zebrafish ITGα6b. The two CcITGα6 transcripts were constitutively expressed in ten sample tissues but displayed distinct expression patterns. The highest relative expression levels of the two transcripts of CcITGα6 were observed in the head kidney, whereas the lowest was observed in the heart. The relative expression levels of the CcITGα6b transcript in the five tissues were significantly higher than the relative expression levels of the CcITGα6a transcript (p < 0.05), and they were significantly lower in the brain (p < 0.05). After CyHV-3 infection, the relative expression levels of two transcripts of CcITGα6 were higher in the breeding strain than in the non-breeding strain and reached the highest at 48 h and the lowest at 144 h. However, the expression of CcITGα6b in the breeding strain was significantly higher than the expression of CcITGα6b in the non-breeding strain at all infection stages (p < 0.05). This result indicated that CcITGα6b might play an important role in resistance to CyHV-3 in common carp, which provides a theoretical basis for the study of the CyHV-3 resistance mechanism of common carp and lays the foundation for guiding breeding.

Understanding the in vitro evolution of Cyprinid herpesvirus 3 (CyHV-3), a story of structural variations that can lead to the design of attenuated virus vaccines

A recommendation of: Nurul Novelia Fuandila, Anne-Sophie Gosselin-Grenet, Marie-Ka Tilak, Sven M Bergmann, Jean-Michel Escoubas, Sandro Klafack, Angela Mariana Lusiastuti, Munti Yuhana, Anna-Sophie Fiston-Lavier, Jean-Christophe Avarre, Emira Cherif Structural variation turnovers and defective genomes: key drivers for the in vitro evolution of the large double-stranded DNA koi herpesvirus (KHV) https://doi.org/10.1101/2022.03.10.483410

Potential of (-)-epigallocatechin-3-gallate against bacterial and viral pathogens isolated from gibel carp (Carassius auratus gibelio)

Gibel carp (Carassius auratus gibelio) is an important fish species cultured worldwide. Bacterial septicemia and herpesviral hematopoietic necrosis disease (HVHND) were caused by Aeromonas hydrophila and cyprinid herpesvirus 2 (CyHV-2) respectively, which were the main causes of mortality and economical loss in gibel carp production. With known pharmacological activities against certain gram-negative bacteria and herpesvirus, (−)-Epigallocatechin-3-gallate (EGCG) was tested here to evaluate its inhibitory activity against A. hydrophila and CyHV-2 pathogens isolated from gibel carp. The minimum inhibitory concentration (MIC) of EGCG against four A. hydrophila strains ranged from 256 to 512 μg/mL and the minimum bactericidal concentration (MBC) was determined to be 1024 μg/mL. The growth curves of A. hydrophila A56 in the presence of various amounts of EGCG indicated that the concentration and treatment time of EGCG served as the two dominant factors for EGCG-mediated inhibitory effects in vitro. The bacterial load in liver, spleen and kidney tissues of gibel carps infected with 0.1 × LD50 A56 was significantly reduced after oral administration of EGCG (40 mg/kg body weight) 8 h in advance. The inactivation effect of CyHV-2 virus treated with 50 μg/mL EGCG for 2 h could reach 99.99%, and 70 μg/mL of EGCG could completely inactivate CyHV-2 in our infection and inhibition assays in vitro. These results collectively revealed the potential and broad-spectrum useage of EGCG in gibel carp cultivation against either bacterial or viral pathogen.

Proteomic and Phosphoproteomic Analyses Reveal Gibel Carp Responses to Cyprinid Herpesvirus 2 Infection.

Cyprinid herpesvirus 2 (CyHV-2) is a typical linear double-stranded DNA virus, which can induce severe herpesviral hematopoietic necrosis disease (HVHND) in gibel carp. However, the CyHV-2 infection mechanisms still remain unresolved till now. Here, we combined the isobaric tag for relative absolute quantitation (iTRAQ)-labeled quantitative proteomic and phosphoproteomic analysis enriched by Ti4+-immobilized titanium ion affinity chromatography (IMAC) to uncover the host responses to CyHV-2 infection in the kidneys of symptomatic and diseased gibel carp. We totally identified 192 differential expression proteins and 951 high-confident phosphopeptides involved in 657 proteins. After being infected with CyHV-2, the proteins involved in energy generation and ion balance were significantly downregulated in the host, and the phosphorylated proteins induced by viral infection mainly participated in the regulation for RNA processing, translation, cytoskeleton organization, immunoreaction, etc. Furthermore, 11 phosphorylated CyHV-2 viral proteins were found in the diseased group by the host proteome. The virus-host protein-protein interactions were investigated, in which the potential host kinases casein kinase II (CK-II) and cyclin-dependent kinase (CDK) that interacted with viral ORF88 or ORF89 were identified and can serve as candidate targets for disease treatment in the future. Overall, our study provides a comprehensive understanding of CyHV-2-induced perturbations at the protein and phosphorylation levels in gibel carp, forming a base for the treatment of HVHND.

Detection of herpesviral hematopoietic necrosis virus (Cyprinid herpesvirus 2, CyHV-2) from goldfish, Carassius auratus (L.) in Korea

In April and June 2014, mortalities of goldfish occurred at Korea. The principal signs included pale gills, severely enlarged and softened spleen and kidney and red liver. Moreover, the histopathological characteristics observed mainly in hematopoietic tissues of kidney, gill lamellae and intestine. The predominant histopathological changes were severe necrosis of hematopoietic tissue. In addition, nucleus exhibiting peripherally displaced chromatin were particularly abundant in the kidney of affected fish. The histological examination led to hypothesize the implication of a virus in the mortality. The hematopoietic necrosis herpes-virus (Cyprinid herpesvirus 2, CyHV-2) DNA polymerase gene successfully detected in DNA extracted from kidney and spleen of affected fish using PCR assay and showed 100% identity with already deposited CyHV-2 DNA polymerase gene in NCBI. Artificial infection trials using affected tissues filtrates gave cumulative mortalities of 30% for virus injected goldfish. In the present study, CyHV-2 was detected and identified as the causative pathogen of the epizootic in goldfish in Korea.

Circ-Udg Derived from Cyprinid Herpesvirus 2 Promotes Viral Replication.

ABSTRACTCyprinid herpesvirus 2 (CyHV-2) has caused great losses to the gibel carp (Carassius auratus gibelio) industry. Previous studies showed that certain DNA viruses can encode circular RNAs (circRNAs) to regulate virus infection, which provides new clues for the treatment of viral disease. Whether CyHV-2 can encode circRNAs is still unknown. Here, 10 CyHV-2-derived circRNAs were identified, and the function of circ-udg, a circRNA derived from the CyHV-2 uracil DNA glycosylase (udg) gene, was studied. Although the expression level of circ-udg was lower than that of the parental gene, udg, its expression level was elevated in tandem with the proliferation of CyHV-2 and udg. In vitro experiments confirmed that circ-udg could promote the proliferation of CyHV-2. Moreover, circ-udg could encode a truncated UDG protein consisting of 147-amino-acid residues (termed circ-udg-P147). Both UDG and circ-udg-P147 were found to promote CyHV-2 proliferation, but the promoting effect of circ-udg on CyHV-2 proliferation was attenuated after circ-udg lost the ability to encode circ-udg-P147. Also, circ-udg-P147 could not change the transcription level of the udg gene. Interestingly, the UDG protein level was increased by circ-udg-P147. These results deepen the understanding of the genetic information carried by the genome of CyHV-2 and provide a new target for the treatment of gibel carp bleeding disease caused by CyHV-2.IMPORTANCE The outbreak of C. auratus gibelio gill hemorrhagic disease caused by CyHV-2 brought great losses to the gibel carp industry. Therefore, exploring the interaction between CyHV-2 and host and the molecular mechanism of viral infection is of great significance in preventing and treating the gibel carp gill hemorrhagic disease. Although some progress has been made in the study of CyHV-2, the mechanism of interaction between CyHV-2 and crucian carp is still unclear. In this study, we found that CyHV-2 can encode circRNA to regulate virus replication. Our study provides novel information on CyHV-2 functional genomics, a reference for research into the circRNA of other viruses, and theoretical guidance for preventing and treating gibel carp bleeding disease.

First report of cyprinid herpesvirus 2 isolated from the golden crucian carp in China

In this study, a virus strain designated GCC2021 was isolated from the golden crucian carp (Carassius auratus red var. ♂ × Cyprinus carpio L. mirror ♀) in Jiangsu Province, China. GCC2021 was propagated in the Gibel carp brain (GiCB) cell line with a typical cytopathic effect. Healthy golden crucian carp was injected with GCC2021 virus solution intraperitoneally, and the symptoms of the artificially infected fish were consistent with those of the naturally diseased golden crucian carps. The mortality was 6.67 ± 3.33% (kidney homogenate group) and 4.44 ± 1.93% (cell culture virus group). The histopathological results indicated systemic inflammation and changes in multiple organs. Electron microscopy observation using ultra-thin sections of the diseased fish kidney revealed a number of spherical particles with capsid that were approximately 110–120 nm in diameter, and the mature virus particle was about 170–220 nm in diameter. The helicase (Hel) gene of GCC2021 was amplified using PCR and sequenced. PCR amplification showed positive results for GCC2021 in the diseased fish, artificially infected fish, and infected GiCB cells. Sequence analysis of the amplification products showed 99.68% identity with the helicase gene of CyHV-2 SY-C1 strain. Phylogenetic analysis of the CyHV-2 ORF25B non-coding region revealed that the isolate clustered with CyHV-2 1301 strain, SY strain, and SY-C1 strain. CyHV-2 copies in different tissues were determined using digital PCR. Viral loads were detected in the liver, spleen, kidney, brain, intestine, and gill, with the highest in the kidney and lowest in the brain. In this study, it was provided solid evidence that CyHV-2 could infect golden crucian carp.

Impact of climate-driven temperature increase on inland aquaculture: Application to land-based production of common carp (Cyprinus carpio L.).

Climate change will expose the food-producing sector to a range of challenges. Inland aquaculture farms are particularly vulnerable, due to the difficulty in changing their location, and therefore require specific tools to predict the influence of direct and indirect effects on production, environment and economic feasibility. The objective of our study was to apply a simple set of models to produce a set of growth, risk and suitability maps for stakeholders within the common carp sector in Poland, to assist decision-making under two different scenarios of climate change: a moderate situation (RCP 4.5) and an extreme situation (RCP 8.5). We used present (2000-2019) and future projections (2080-2099) for water surface temperature based on land surface temperature data from regionally downscaled climate models to draw maps to: (i) show optimal temperature conditions for carp growth, (ii) assess risk of disease outbreak caused by three important common carp pathogens: Cyprinid herpesvirus 3 (CyHV-3), carp oedema virus (CEV) and spring viremia of carp (SVCV) and (iii) predict potential suitability changes of carp farming in Poland. The study identified areas with the most and least favourable temperature conditions for carp growth, as well as those areas with the highest/lowest number of days with suitable temperatures for virus infection. These suitability maps showed the combined effect of direct and indirect effects of climate change projections under RCP 8.5 and RCP 4.5 scenarios. The approach applied herein will be of use worldwide for analysing the risks of temperature increase to land-based aquaculture, and the results presented are important for carp farmers in Poland and elsewhere, industry in general, and government stakeholders, to understand the direct and indirect effects of climate change on the triple bottom line of people, planet, and profit.

First report on the occurrence of cyprinid herpesvirus 3 in koi carp (Cyprinus carpio koi) in India.

This study reports the occurrence of cyprinid herpesvirus 3 (CyHV-3) in koi carp (Cyprinus carpio koi) for the first time in India. The koi carp, with clinical signs of ulcer with haemorrhage on body surface, necrosis of fin and discolouration of gill associated with huge mortality, were observed in aquarium shops, rearing tanks and grow-out ponds located in Chennai, India. The PCR assay carried out on infected fish samples using different primer sets specific to CyHV-3 confirmed its presence in the infected fish. Sequence analysis of partial thymidine kinase gene revealed 100% similarity with the sequence of CyHV-3 available in GenBank. Cell lines of koi carp and catla were found to be susceptible to CyHV-3 and its replication was confirmed by viral-specific cytopathic effect, PCR and bioassay. The CyHV-3 infection was reproduced by intramuscular injection of inoculum prepared from CyHV-3-infected fish to satisfy Koch's postulates. Tissue tropism of CyHV-3 in infected fish by PCR assay revealed the presence of CyHV-3 in all vital organs with prominent band in gill and gut tissue.

Optimum Koi Herpesvirus DNA Extraction from the Aquaculture Water

Koi Herpesvirus (KHV) is one of the viral fish disease in the common carp aquaculture. At present, KHV diagnoses is performed using Polymerase Chain Reaction (PCR) method. However, the effort to extract DNA viruses from environmental samples depends on two fundamental problems: inhibition of the extraction and amplification processes and a very small number of viruses. The small amount of virus load in the water requires a concentration process before further molecular analysis. In this study, the sensitivity of three methods of extraction and purification of viral genetic material from the water, namely deposition with PEG, filtration-elution, and flocculation with antibodies for the detection of KHV from the water with the PCR method was performed. Water samples were collected from Cirata reservoir, West Java which is a site location for carp aquaculture. The organic flocculation cannot be carried out in the present study because anti-KHV antibodies cannot be produced. This is because the amount of virus in the sample used by the vaccine is low. Meanwhile, the precipitation method with PEG cannot produce DNAextract’s although quantity and purity are good enough to be amplified. For the filtration-elution method, both with and without the reconcentration process, a clear band of DNA can be produced after amplification. In conclusion, the filtration-elution method is considered as the most suitable method in obtaining KHV DNA’s taken from the water samples.

Safe management of Cyprinid herpesvirus 3-induced mortalities of common carp (Cyprinus carpio) by silaging process

In open farming systems, fish losses are unfortunate daily finding, hence a simple, safe and highly adaptable method is needed to manage dead fish. The aim of this study was to develop three silaging methods of common carp (Cyprinus carpio) carcasses to identify cost-effective, eco-friendly, and efficient method to turn dead fish into organic fertiliser and to determine whether Cyprinid herpesvirus 3 (CyHV-3) can be inactivated during a 34-weeks trial. In our study, dead fish were chopped, minced and mixed with starter bacteria culture and wheat bran (Basic), water and wheat bran starter (Hydrated) or Bokashi commercial mix (Commercial), and placed in a 1 L jars (in quadruplicates). The CyHV-3 infectivity potential was assessed by two cell cultures and expression of the virus genes classified into three temporal kinetic classes. The feasibility of the silaging method and selection of the best method assessed by pH monitoring, characterisation of the odour profile, elemental analysis and calculating the cost of the in-farm silaging. Cell cultures and the subsequent gene expression analyses showed that the virus was successfully inactivated in the Basic, Hydrated and Commercial silages, confirming their safety. Among the three silaging methods, the Hydrated was the cost-effective one; however, concerning the other features (odour profile, feasibility and final pH level), the Basic was selected as the most promising for implementation. Additionally, elemental analysis showed that the level of nutrients in Basic silage was higher than in commonly used natural fertilisers, while the content of heavy metals (Pb, Cd, Zn) met the official recommendations for organic fertilisers. The study provides premise of an effective method of silaging of dead carp that offers pathogen inactivation (via combination of decreased pH and microbial activity), turns common by-stream into a valuable product and increases profitability of the farm in a sustainable and cost-effective way.

Immune gene expression and protective effects in goldfish (Carassius auratus L.) immunized with formalin-inactivated cyprinid herpesvirus-2 (CyHV-2) vaccine

The goldfish hematopoietic necrosis viral disease (GHNVD) has led to worldwide economic losses in goldfish aquaculture. The present study has focused on the development of an inactivated vaccine for the cyprinid herpesvirus (CyHV-2) and to check the immunogenicity of the vaccine in the host. The fantail goldfish fin (FtGF) cell line was used in the propagation of the CyHV-2 and the viral titer obtained were of 107.8 TCID50/ml. Followed by the virus was inactivated using 0.1% formalin for 2 days. Various concentrations of formalin-inactivated CyHV-2 (1%, 0.7%, 0.5%, 0.3% and 0.1%) were studied in the FtGF cell line. Morphological changes were observed in the FtGF cell line in all other concentrations of formalin except 0.1% formalin-inactivated CyHV-2 vaccine. The goldfishes were intraperitoneally injected with 300 μl of vaccine and various immune gene responses were studied for a period of 30 days. The gene expression of the adaptive markers CD8, CD4, IFN-ϒ, the cytokines (IL-10, IL-12) was studied in kidney and spleen tissues. Formalin-inactivated CyHV-2 vaccine showed a significant up-regulation of the genes CD8 and IFN-ϒ by the 6th hr post-vaccination onwards. The experimental fish were challenged intraperitoneally with CyHV-2 virus of concentration 107.8 TCID50/ml after 30 days of post-vaccination. A significant difference in cumulative mortality rate was observed for the vaccinated fishes from the unvaccinated fishes. The relative percent survival for formalin immunized fish was 74.03%. Our results have proven that the formalin-inactivated vaccines were efficient and it resulted in triggering the immune gene expression in goldfish. The development and further enhanced studies for this vaccine will lead to a promising low-cost commercial vaccine for CyHV-2 viral infection.

MicroRNA (miR-KT-635) encoded by Cyprinid herpesvirus 2 regulates the viral replication with targeting to the ORF23.

Herpesviruses have been reported to be able to encode and express functional viral microRNAs that target both viral and cellular transcripts. In our previous studies, we found a new miRNA miR-KT-635 encoded by Cyprinid herpesvirus 2, which is predicted to target viral genes and cellular genes involved in innate immune signalling pathway and apoptosis. However, the function and target gene of miR-KT-635 are not proved. In this study, the regulating target gene of miR-KT-635 was proved as the viral gene ORF23 directly, the target point sequence on gene was verified and miR-KT-635 was identified to regulate the expression of ORF23 protein. According to the bioinformatics analysis, the tRNA domain and ribosome domain in the protein sequence of ORF23 were found to share high homology with R2i and P53R2i, which are related to the ribonucleotide reductase small subunit in the host (transform NTP to dNTP). Within expectations, silencing of viral ORF23 or transfecting miR-KT-635 mimics in Carassius auratus gibelio caudal fin cell line (GiCF) could suppress viral propagation significantly.

Quick hassle-free detection of cyprinid herpesvirus 2 (CyHV-2) in goldfish using recombinase polymerase amplification-lateral flow dipstick (RPA-LFD) assay.

Cyprinid herpesvirus 2 (CyHV-2) is the etiological agent of herpesviral hematopoietic necrosis disease (HVHND), which causes severe mortality in ornamental goldfish (Carassius auratus), crucian carp (Carassius auratus), and gibel/prussian carp (Carassius gibelio). Quick and hassle-free point-of-care detection of CyHV-2 is vital for the maintenance of ornamental fish health. In this manuscript, we describe the development of a rapid and sensitive RPA (recombinase polymerase amplification) assay, coupled with lateral flow dipsticks (LFD), that can achieve sensitive diagnosis of CyHV-2 in goldfish within 20 min at 36 °C with the satisfactory detection limit of 102 gene copies per reaction. This is the first report wherein major capsid protein (MCP) of CyHV-2 was targeted for RPA-LFD assay development. The assay did not show any cross-reactivity with other viral pathogens like cyprinid herpesvirus 3 (CyHV-3), spring viremia of carp virus (SVCV), infectious spleen and kidney necrosis virus (ISKNV), and viral nervous necrosis virus (VNNV). Furthermore, screening of CyHV-2 infection in CyHV-2-infected goldfish did not yield any false positive/negative results. In short, the RPA-LFD assay developed in this study presents a simple, rapid, and sensitive method for point-of-care diagnosis of CyHV-2, especially under resource-limited conditions.

Don't Let It Get Under Your Skin! - Vaccination Protects the Skin Barrier of Common Carp From Disruption Caused by Cyprinid Herpesvirus 3.

Vaccination is the best form of protecting fish against viral diseases when the pathogen cannot be contained by biosecurity measures. Vaccines based on live attenuated viruses seem to be most effective for vaccination against challenging pathogens like Cyprinid herpesvirus 3. However, there are still knowledge gaps how these vaccines effectively protect fish from the deadly disease caused by the epitheliotropic CyHV-3, and which aspects of non-direct protection of skin or gill integrity and function are important in the aquatic environment. To elucidate some elements of protection, common carp were vaccinated against CyHV-3 using a double deletion vaccine virus KHV-T ΔDUT/TK in the absence or presence of a mix of common carp beta-defensins 1, 2 and 3 as adjuvants. Vaccination induced marginal clinical signs, low virus load and a minor upregulation of cd4, cd8 and igm gene expression in vaccinated fish, while neutralisation activity of blood serum rose from 14 days post vaccination (dpv). A challenge infection with CyHV-3 induced a severe disease with 80-100% mortality in non-vaccinated carp, while in vaccinated carp, no mortality was recorded and the virus load was >1,000-fold lower in the skin, gill and kidney. Histological analysis showed strongest pathological changes in the skin, with a complete destruction of the epidermis in non-vaccinated carp. In the skin of non-vaccinated fish, T and B cell responses were severely downregulated, inflammation and stress responses were increased upon challenge, whereas vaccinated fish had boosted neutrophil, T and B cell responses. A disruption of skin barrier elements (tight and adherence junction, desmosomes, mucins) led to an uncontrolled increase in skin bacteria load which most likely exacerbated the inflammation and the pathology. Using a live attenuated virus vaccine, we were able to show that increased neutrophil, T and B cell responses provide protection from CyHV-3 infection and lead to preservation of skin integrity, which supports successful protection against additional pathogens in the aquatic environment which foster disease development in non-vaccinated carp.

Development of a live attenuated vaccine candidate against herpesviral hematopoietic necrosis of goldfish

Herpesviral hematopoietic necrosis causes huge economic loss in goldfish Carassius auratus and gibel carp C. auratus gibelio farming. To develop a live attenuated vaccine as a preventive measure against the disease, the causative virus cyprinid herpesvirus 2 (CyHV-2) was attenuated by serial subculture in two cell lines. The CyHV-2 SaT-1 isolate routinely propagated in the goldfish cell line RyuF-2 was serially propagated in the CFS cell line derived from ginbuna C. auratus langsdorfii, a non-natural host. Virus propagated in CFS showed no lethality in goldfish by immersion administration and provided high protective efficacy in subsequent virulent virus challenge, but virus propagated once in RyuF-2 caused skin hemorrhage in some fish after administration. The virus was further attenuated by subculturing several times in the KF-1 cell line from carp Cyprinus carpio, also a non-natural host. Viruses subcultured 7 or 10 times in CFS were propagated 5 or 8 times in KF-1 to obtain further attenuated viruses abbreviated as P7-P5, P7-P8, P10-P5 and P10-P8 (Passage number in CFS – KF-1). P7-P5, P7-P8 and P10-P5 showed high protective efficacy of >80% relative percentage survival in the vaccine trial, but P10-P8 was not highly effective. P7-P8 was selected as a vaccine candidate for further virulence reversion tests. In the in vitro reversion test, the candidate subcultured 5 times in RyuF-2 caused no pathogenicity in goldfish, revealing no signs of virulence reversion. Furthermore, in the in vivo test for virulence reversion, the candidate was propagated through goldfish 6 times by intraperitoneal injection with kidney homogenate of the inoculated fish. During in vivo passages in goldfish, no abnormality and mortality of the inoculated fish were observed, and virus DNA copy number in the kidney and caudal fin decreased from 104 to 108 copies/mg at the first passage to ~103 copies/mg at the second passage. These results demonstrated that P7-P8 can be a promising live attenuated vaccine candidate against the disease.

Induction of Reactive Oxygen Species Is Necessary for Efficient Onset of Cyprinid Herpesvirus 2 Replication: Implications for Novel Antiviral Strategy With Antioxidants

Cyprinid herpesvirus 2 (CyHV-2) has caused great economic loss to the crucian carp breeding industry. Upon viral stimulation, eukaryotic cells generally activate the expression of anti-oxidative genes to maintain the intracellular oxidative balance and resist viral infection. Here, intracellular reactive oxygen species (ROS) levels in CyHV-2-infected cells were monitored to show that CyHV-2 induced the increase of intracellular ROS during early infection, and intracellular excessive accumulation of ROS was ameliorated during late infection, which was accompanied by activated expression of genes related to Nrf2 signaling pathway. In order to explore the interaction between CyHV-2 infection and ROS production, RyuF-2 cells were treated with either antioxidant epigallocatechin-3-gallate (EGCG) or berberine hydrochloride (BBH) and then infected with CyHV-2. Both BBH and EGCG could effectively inhibit the amplification of CyHV-2 while inhibiting the accumulation of intracellular ROS. Consistent with this, the oxidant stress-related genes were up-regulated by CyHV-2 infection and down-regulated in cells treated with either BBH or EGCG, through which the production of intracellular ROS was modulated. These results collectively demonstrated that early ROS accumulation favored the replication of CyHV-2, while antioxidants (BBH and EGCG) could inhibit the amplification of CyHV-2 by inhibiting ROS induction.

Cyclophilin A is incorporated into cyprinid herpesvirus 3 (CyHV-3) virion via interaction with envelope VP32 and facilitates CyHV-3 replication in in vitro

Cyprinid herpesvirus 3 (CyHV-3), a large double-stranded nucleocytoplasmic DNA virus, is the major viral pathogen for Cyprinus carpio distributed worldwide. Cyclophilin A (CypA) is a well-characterized ubiquitous cellular immunosuppressor possessing peptidyl-prolyl cis-trans isomerase activity in many species and plays an important role in regulating the replication of several well-known viruses. In our previous study, C. carpio CypA was detected as a high confident cellular protein involvement in purified extracellular virion of CyHV-3, however, the exact role of C. carpio CypA in CyHV-3 remains unclear. Here we identified that C. carpio CypA was incorporated into mature CyHV-3 virion and located in envelope fraction. Co-Immunoprecipitation and subcellular co-localization showed that C. carpio CypA interacted with CyHV-3 VP32, an essential viral envelope protein for CyHV-3 replication. Drug inhibition assay showed that knocking down of C. carpio CypA significantly reduced CyHV-3 proliferation in KCF-1 cells, whereas overexpression of C. carpio CypA enhanced CyHV-3 replication. Subcellular locations of C. carpio CypA showed that both exogenous and endogenous CypA were found to translocate from cytoplasm to whole cell upon CyHV-3 infection. Collectively, our results confirmed that C. carpio CypA was incorporated into CyHV-3 virion via interaction with CyHV-3 VP32 and played an essential role in CyHV-3 replication. C. carpio CypA is suggested as a potential target cellular protein for developing antiviral agents against CyHV-3 infection.

Valuing uncertain environmental outcomes from herpes virus control of the European Carp

ABSTRACT European Carp is a species of freshwater fish that live in Australia's freshwater rivers, wetlands and lakes. It is a dominant pest species in Australia. As part of investigations into the potential release of the Cyprinid Herpes Virus 3 to control Carp numbers, a Choice Modelling (CM) study was undertaken to value the potential non-market environmental benefits of such a program. These potential benefits relate to increases in native fish populations, the area of healthy wetlands and waterbird populations. The CM study explicitly included a probability attribute to reflect the risk that various release options will deliver the identified environmental outcomes. Consequently, the willingness to pay (WTP) estimates derived from the choice models are expressed in terms of the expected values of outcomes: the physical outcomes multiplied by their probabilities of occurring. The preferred model of choice was a Random Parameter Logit model with a non-linear (cubed) transformation of the levels for each probability weighted environmental attribute. Consequently, estimates of WTP derived from the model increase as the level of an expected attribute increases. This non-linear form is consistent with respondents treating risk in a nonlinear fashion.