Herpesviral Hematopoietic Necrosis Research Articles

Genomic and molecular characterization of a cyprinid herpesvirus 2 YC-01 strain isolated from gibel carp

Cyprinid herpesvirus 2 (CyHV-2) is the pathogen of herpesviral hematopoietic necrosis (HVHN), causing the severe economic losses in farmed gibel carp (Carassius gibelio). Further exploration of the genome structure and potential molecular pathogenesis of CyHV-2 through complete genome sequencing, comparative genomics, and molecular characterization is required. Herein, the genome of a CyHV-2 YC-01 strain isolated from diseased gibel carp collected in Yancheng, Jiangsu Province, China was sequenced, then we analyzed the genomic structure, genetic properties, and molecular characterization. First, the complete YC-01 genome comprises 275,367 bp without terminal repeat (TR) regions, with 151 potential open reading frames (ORFs). Second, compared with other representative published strains of the genus Cyvirus, several evident variations are found in YC-01, particularly the orientation and position of ORF25 and ORF25B. ORF107 and ORF156 are considered as potential molecular genetic markers for YC-01. ORF55 (encoding thymidine kinase) might be used to distinguish YC-01 and ST-J1 from other CyHV-2 isolates. Third, phylogenetically, YC-01 clusters with the members of the genus Cyvirus (together with the other six CyHV-2 isolates). Fourth, 43 putative proteins are predicted to be functional and are mainly divided into five categories. Several conserved motifs are found in nucleotide, amino acid, and promoter sequences including cis-acting elements identification of YC-01. Finally, the potential virulence factors and linear B cell epitopes of CyHV-2 are predicted to supply possibilities for designing novel vaccines rationally. Our results provide insights for further understanding genomic structure, genetic evolution, and potential molecular mechanisms of CyHV-2.

Effect of temperature on the protective efficacy of a live attenuated vaccine against herpesviral haematopoietic necrosis in goldfish.

The live attenuated vaccine P7-P8 strain against herpesviral haematopoietic necrosis, which is caused by cyprinid herpesvirus 2 (CyHV-2), exhibits high protective efficacy in goldfish at 25°C, the predominant temperature for this disease; however, the effect of water temperature during the vaccination period on efficacy has not been determined. In this study, an in vitro experiment revealed that the vaccine strain grew between 15 and 30°C in the goldfish cell line RyuF-2. Subsequent in vivo efficacy tests were conducted with vaccination temperatures ranging from 15 to 30°C. During the vaccination period, organs were sampled to determine the vaccine growth dynamics. Blood plasma was collected to assess anti-CyHV-2 antibody titres. The protective efficacy of the vaccine at 15, 20, 25, and 30°C after subsequent virulent CyHV-2 challenge resulted in a relative percentage survival of 73.3%, 77.8%, 100%, and 77.8%, respectively, which indicated that the vaccine is effective over this temperature range. The vaccine virus load in the spleen was lowest at 15°C (103.7 DNA copies/mg) and highest at 25°C (106.5 DNA copies/mg). This indicates that the vaccine virus load over 104 DNA copies/mg may elicit sufficient acquired immunity. No significant differences in antibody titre were observed between groups, which suggests that cell-mediated immunity can be fundamentally involved in protection.

Alterations of Plasma Biochemical and Immunological Parameters and Spatiotemporal Expression of TLR2 and TLR9 in Gibel Carp (Carassius auratus gibelio) after CyHV-2 Infection.

Cyprinid herpesvirus II (CyHV-2), a highly contagious pathogen of gibel carp (Carassius auratus gibelio), causes herpesviral hematopoietic necrosis disease (HVHND) and enormous financial losses. However, there is limited information available regarding the changes in plasma biochemical and immunological parameters and the response characteristics of Toll-like receptor 2 (TLR2) and Toll-like receptor 9 (TLR9) in gibel carp after CyHV-2 infection. To address this knowledge gap, a sub-lethal CyHV-2 infection was conducted in gibel carp, and the sample was collected daily from 1 to 7 days post infection. The plasma biochemical analyses showed significant decreases in the content of glucose, total cholesterol (TCHO), and total protein (TP), along with marked increases in the level of uric acid, urea, creatinine (CREA), Complement 3 (C3), immunoglobulin D (IgD), and immunoglobulin M (IgM) as well as in the activity of alanine aminotransferase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP), and lactate dehydrogenase (LDH) in the infected group. Compared with the control group, the concentration of cortisol, triglyceride (TG), and Complement 4 (C4) had no noticeable alterations in the infected group. Real-time quantitative PCR analysis showed significant upregulation of TLR2 and TLR9 mRNA expression in the spleen, kidney, brain, liver, intestine, and gill post CyHV-2 infection. Interestingly, a time- and tissue-dependent expression profile has been comparatively observed for TLR2 and TLR9 in the above tissues of gibel carp after CyHV-2 infection, suggesting distinct roles between TLR2 and TLR9 in antiviral response to CyHV-2 infection. Overall, our results demonstrated that CyHV-2 infection led to the disruption of the physiological metabolic process and damage to the liver and kidney, and induced different spatiotemporal expression patterns of TLR2 and TLR9, ultimately stimulating antiviral response via innate and adaptive immune system. These findings may provide a deeper understanding of the host immunity response to CyHV-2 infection and offer novel perspectives for the prevention and treatment and therapeutic drug development against CyHV-2.

Efficient showering vaccination with a live attenuated vaccine against herpesviral hematopoietic necrosis in goldfish

Herpesviral hematopoietic necrosis caused by cyprinid herpesvirus 2 (CyHV-2) frequently affects farmed goldfish Carassius auratus and gibel carp C. auratus gibelio, leading to significant economic damage. In our previous study, a live attenuated vaccine, called the P7-P8 vaccine strain, was developed by propagating virulent CyHV-2 in two non-natural host cell lines. The vaccine demonstrated high protective efficacy in goldfish with no signs of virulence reversion. Methods that reduce the costs of the procedure, including labor costs, are warranted for the practical application of this vaccine in goldfish aquaculture. The aim of this study was to explore alternative vaccination protocols for the P7-P8 strain in goldfish. First, we tested the previous standard administration protocol which is the bath vaccination with 1: 1000 diluted vaccine cultures for 2 h. In the simple first step to reduce vaccine virus culture, we examined the bathing protocol with more-diluted virus culture (ranging from 1: 1000 to 1: 81,000). The relative percentage survival (RPS) after the virulent virus immersion challenge for 2 h at 21 days post-vaccination was remarkably decreased in a dose-dependent manner; a dilution of 1: 3000 resulted in a 19% reduction from that used in the standard bathing protocol. Therefore, alternative methods (dipping and showering vaccination protocols) were tested using a 1: 100 dilution vaccine culture (10-fold more concentrated than the standard protocol) with short exposure times. Dipping vaccination for 10 s, 1 min, 2 min, and 5 min showed sufficient RPS of 82.4%–100%. The 10 s process was repeated six times using the same vaccine solution, resulting in an RPS >89%. In the showering vaccination protocol, the vaccine solution (1: 100 dilution; 1 mL, 3 mL, or 5 mL) was showered onto 20 goldfish on a net and held for 10 s, resulting in an RPS of >89%. We selected the showering vaccination as the suitable vaccination protocol. The effectiveness of this vaccination protocol was tested using the virus challenge by employing the cohabitation method with infected fish. The results showed high protective efficacy with an RPS of 65%–100%. This study demonstrated that the showering vaccination protocol, which uses small amounts of vaccine culture and produces less waste vaccine solution with short labor time, may be considered as a suitable alternative to the standard bath method in goldfish aquaculture.

Adoptive transfer of sensitized leukocytes confers partial protection against herpesviral hematopoietic necrosis (HVHN) in ginbuna crucian carp Carassius auratus langsdorfii immunized with a live attenuated vaccine

Adoptive transfer of sensitized leukocytes confers partial protection against herpesviral hematopoietic necrosis (HVHN) in ginbuna crucian carp Carassius auratus langsdorfii immunized with a live attenuated vaccine

An attenuated strain of cyprinid herpesvirus 2 as a vaccine candidate against herpesviral hematopoietic necrosis disease in gibel carp, Carassius auratus gibelio.

Herpesviral hematopoietic necrosis disease causes by cyprinid herpesvirus 2 (CyHV-2) infection is a high mortality disease that leads to great economic damage to gibel carp, Carassius auratus gibelio aquaculture. In this study, an attenuated strain of CyHV-2 G-RP7 was achieved by subculture on RyuF-2cells derived from the fin of Ryukin-variety goldfish and GiCF cells derived from fin of gibel carp. As the attenuated vaccine candidate, there are no clinical symptoms of gibel carp that immersion or intraperitoneal injection with G-RP7 strain. The protection rates of G-PR7 to gibel carp by immersion and intraperitoneal injection were 92% and 100%, respectively. In the test for virulence reversion, the candidate was propagated through gibel carp six times by intraperitoneal injection with kidney and spleen homogenate of the inoculated fish. During in vivo passages in gibel carp, no abnormality and mortality of the inoculated fish were observed, and the virus DNA copies maintain a low level from the first passage to the sixth passage. The dynamic of virus DNA in each tissue of G-RP7 vaccination fish increased within 1, 3, and 5 days post-immunization, and subsequently decreased and stabilized within 7 and 14 days. In addition, the increase of anti-virus antibody titer was detected both immersion and injection immunization fish 21 days after vaccination by ELISA. These results demonstrated that G-RP7 can be a promising live attenuated vaccine candidate against the disease.

Protective effects of egg yolk immunoglobulins (IgY) against CyHV-2 infection in gibel carp (Carassius gibelio)

Herpesviral hematopoietic necrosis caused by cyprinid herpesvirus-2 (CyHV-2) is highly prevalent in gibel carp (Carassius gibelio Bloch) and is associated with high mortality and great economic loss. Currently, no efficient methods of prevention and control of CyHV-2 infection are available. Increasing evidence suggests that antigen-specific egg yolk immunoglobulins (IgY) are highly effective against various pathogens in farmed fish. Therefore, the aim of this study was to develop a specific IgY against CyHV-2 infection in gibel carp. We first produced a recombinant D4ORFs protein that encodes four open reading frames of CyHV-2 and then generated specific IgY by immunizing laying hens with this protein. The eggs laid by immunized hens were processed into spray-dried egg powder containing D4ORFs IgY. The D4ORFs IgY showed high neutralizing activity in vitro against CyHV-2, as evidenced by a significant reduction in cytopathic effect in gibel carp brain cells cultured in the presence of the IgY antibodies. Whole-egg powder containing IgY specific to D4ORFs protein was incorporated into the basal diet at levels of 0, 0.1, 0.3, and 0.6% to prepare experimental diets for use in intervention studies in gibel carp. Another group received non-specific IgY. For preventive intervention, all fish were fed these experimental diets for 25 days before being immersion challenged with CyHV-2, after which the fish were switched to the basal diet for 21 days. For therapeutic intervention, fish were first challenged with CyHV-2 and then fed the diet with 0.6% D4ORFs IgY. Fish that received D4ORFs IgY prior to CyHV-2 challenge had significantly higher survival rates and significantly lower viral load in the trunk kidney compared to those that received non-specific IgY. Improved survival and reduced viral load were also observed in CyHV-2-challenged fish that received dietary supplementation of D4ORFs IgY. Hematological and serum biochemical analysis also showed improved health and enhanced antiviral defense in fish that received D4ORFs IgY. Overall, these findings demonstrated both preventive and therapeutic effects of anti-D4ORFs IgY against CyHV-2 infection.

Astragalus polysaccharide or β-glucan combined with inactivated vaccine markedly prevent CyHV-2 infection in Carassius auratus gibelio

Herpesviral hematopoietic necrosis disease caused by cyprinid herpesvirus 2 (CyHV-2) results in huge economic losses for the gibel carp (Carassius auratus gibelio) aquaculture industry. A vaccination strategy is a feasible method to prevent CyHV-2 infection and the resulting economic losses. In this study, inactivators (including β-propiolactone, formaldehyde and binary ethylenimine) were used to prepare an inactivated vaccine. The optimal inactivated CyHV-2 vaccine (0.2% β-propiolactone inactivates CyHV-2 for 48 h) mixed with β-glucan, anisodamine, chitosan, and astragalus polysaccharide (APS) adjuvants were injected into gibel carp. The β-glucan and APS combined with inactivated vaccine significantly improved the relative immune protection rate of gibel carp against CyHV-2 infection. The highest specific antibody levels in β-glucan and APS groups were found in serum by ELISA assay and antibody neutralization titer. Furthermore, adjuvant addition groups produced better results for lysozyme, total superoxide dismutase, and complement C3 in serum. β-glucan and APS combined with vaccine treatment effectively enhanced mRNA expressions of important immune genes (IL-1β, IgM, IL-2 and IFN-γ2). Minimal tissue lesions (gill, spleen, and head kidney) and virus replication were seen in the β-glucan and APS groups by histopathological examination and expression analysis of CyHV-2 TK gene. These results indicated that the combination of inactivated vaccine and adjuvant β-glucan or APS is an effective method against CyHV-2 infection and provided a case study reference for prevention of fish viral diseases using inactivated vaccines.

Oral Vaccination of Recombinant Saccharomyces cerevisiae Expressing ORF132 Induces Protective Immunity against Cyprinid Herpesvirus-2.

Cyprinid herpesvirus 2 (CyHV-2) is the etiological agent of herpesviral hematopoietic necrosis (HVHN) disease, which causes serious economic losses in the crucian carp culture industry. In this study, by displaying ORF132 on the surface of Saccharomyces cerevisiae cells (named EBY100/pYD1-ORF132), we evaluated the protective efficacy of oral administration against CyHV-2 infection. Intense innate and adaptive immune responses were evoked in both mucosal and systemic tissues after oral vaccination with EBY100/pYD1-ORF132. Importantly, oral vaccination provided significant protection for crucian carp post CyHV-2 infection, resulting in a relative percent survival (RPS) of 64%. In addition, oral administration suppressed the virus load and relieved histological damage in selected tissues. Our results indicated that surface-displayed ORF132 on S. cerevisiae could be used as potential oral vaccine against CyHV-2 infection.

Evaluation of protective effects of heat-inactivated cyprinid herpesvirus-2 (CyHV-2) vaccine against herpesviral hematopoietic necrosis disease (HVHND) in goldfish (Carassius auratus)

Cyprinid herpesvirus-2 (CyHV-2) is an important virus that causes herpesviral hematopoietic necrosis disease (HVHND) leading to huge economic losses in goldfish (Carassius auratus). However, until now no proper prophylactic measure or treatment is available for CyHV-2 infection in goldfish. Hence, in this experiment, we developed a heat-inactivated CyHV-2 vaccine and evaluated its performance in goldfish. Initially, CyHV-2 was propagated in the fantail goldfish fin (FtGF) cell line and the titer of the viral inoculum was 107.8 TCID50/ml. Subsequently, various temperatures (40 °C, 50 °C, 60 °C, 70 °C, and 80 °C) were evaluated to achieve the complete inactivation of CyHV-2. Only the viral inoculum inactivated at 80 °C for 1 h did not show any cytopathic effect in the FtGF cell line after five blind passages. Hence the heat-inactivated CyHV-2 vaccine developed at 80 °C was further used for immunization trials in goldfish. The experimental goldfish were intraperitoneally immunized with 300 μL of the heat-inactivated CyHV-2 vaccine. Subsequently, the kidney and spleen tissues were sampled at various time points post-vaccination (6th hr, 2nd day, 4th day, 6th day, 10th day, 16th day, and 30th day) to evaluate the expression of immune genes (IL-12, IL-10, IFN-γ, CD8, and CD4). A significant upregulation of immune genes was observed at various time points in the kidney and spleen tissue of the vaccinated goldfish. Furthermore, in order to study the efficacy of the vaccine, the experimental fish were challenged with CyHV-2 (107.8 TCID50/ml) after the 30th day post-vaccination. The survival of the fish in the vaccine group (86.7%) was significantly higher compared to the non-vaccinated group (20%). Moreover, the relative percentage survival of the vaccinated group was 83.34%. In spite of the single dose, the heat-killed vaccine developed in the present study elicited the immune response and offered better protection in goldfish against CyHV-2. However, further large-scale field performance evaluation studies are necessary to develop this vaccine on a commercial scale.

Investigation of the latency of Cyprinid herpesvirus 2 in apparently healthy farmed gibel carp, Carassius auratus gibelio

Cyprinid herpesvirus 2 (CyHV-2) is a highly contagious pathogen that causes herpesviral haematopoietic necrosis (HVHN) disease to goldfish, Carassius auratus and gibel carp, C. auratus gibelio. In this study, the virus persistence or latency in apparently healthy farmed gibel carp and reactivation in vitro were investigated. All of the freshly dissected kidney tissues of 42 individuals of asymptomatic farmed gibel carp from six culture regions tested positive in PCR, indicating all fish were CyHV-2 carriers. The viral mRNA was detected in two of 42 fish: one fish expressed virus immediate-early (IE) gene ORF147 only, and another fish expressed IE gene ORF147, early (E) gene ORF35, and late (L) gene ORF72. Following the explant cultivation, the viral DNA copies in the kidney significantly increased in three fish including two individuals that expressed IE gene before explant cultivation. After explant cultivation, the expression of both the E and L genes was detected in the two fish in which active viral gene transcription was detected initially, and the infectious virus in those two fish was confirmed by an infection experiment. The study suggests CyHV-2 can establish persistent or latent infection in asymptomatic farmed gibel carp, and the different viral mRNA expression cascades were detected in different individuals. The persistent and latent virus can reactivate and be infectious, indicating that the apparently healthy gibel carp can be a source of infection.

Complete Genome and Molecular Characterization of a New Cyprinid Herpesvirus 2 (CyHV-2) SH-01 Strain Isolated from Cultured Crucian Carp.

Cyprinid herpesvirus 2 (CyHV-2) is a causative factor of herpesviral hematopoietic necrosis (HVHN) in farmed crucian carp (Carassius carassius) and goldfish (Carassius auratus). In this study, we analyzed the genomic characteristics of a new strain, CyHV-2 SH-01, isolated during outbreaks in crucian carp at a local fish farm near Shanghai, China. CyHV-2 SH-01 exhibited a high sensitivity to goldfish and crucian carp in our previous research. The complete genome of SH-01 is 290,428 bp with 154 potential open reading frames (ORFs) and terminal repeat (TR) regions at both ends. Compared to the sequenced genomes of other CyHVs, Carassius auratus herpesvirus (CaHV) and Anguillid herpesvirus 1 (AngHV-1), several variations were found in SH-01, including nucleotide mutations, deletions, and insertions, as well as gene duplications, rearrangements, and horizontal transfers. Overall, the genome of SH-01 shares 99.60% of its identity with that of ST-J1. Genomic collinearity analysis showed that SH-01 has a high degree of collinearity with another three CyHV-2 isolates, and it is generally closely related to CaHV, CyHV-1, and CyHV-3, although it contains many differences in locally collinear blocks (LCBs). The lowest degree of collinearity was found with AngHV-1, despite some homologous LCBs, indicating that they are evolutionarily the most distantly related. The results provide new clues to better understand the CyHV-2 genome through sequencing and sequence mining.

Gut mucosal immune responses and protective efficacy of oral yeast Cyprinid herpesvirus 2 (CyHV-2) vaccine in Carassius auratus gibelio

Cyprinid herpesvirus 2 (CyHV-2) causes herpesviral hematopoietic necrosis (HVHN) disease outbreaks in farmed Cyprinid fish, which leads to serious economic losses worldwide. Although oral vaccination is considered the most suitable strategy for preventing infectious diseases in farmed fish, so far there is no commercial oral vaccine available for controlling HVNN in gibel carp (C. auratus gibelio). In the present study, we developed for the first time an oral vaccine against CyHV-2 by using yeast cell surface display technology and then investigated the effect of this vaccine in gibel carp. Furthermore, the protective efficacy was evaluated by comparing the immune response of a single vaccination with that of a booster vaccination (booster-vaccinated once 2 weeks after the initial vaccination). Critically, the activities of immune-related enzymes and genes expression in vaccine group, especially in the booster vaccine group, were higher than those in the control group. Moreover, strong innate and adaptive immune responses could be elicited in both mucosal and systemic tissues after receipt of the oral yeast vaccine. To further understand the protective efficacy of this vaccine in gibel carp, we successfully developed the challenge model with CyHV-2. Our results showed the relative percent survival was 66.7% in the booster vaccine group, indicating this oral yeast vaccine is a promising vaccine for controlling CyHV-2 disease in gibel carp aquaculture.

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.

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.

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.

Susceptibility of Goldfish to Cyprinid Herpesvirus 2 (CyHV-2) SH01 Isolated from Cultured Crucian Carp.

Cyprinid herpesvirus 2 (CyHV-2), a member of the Alloherpesviridae family belonging to the genus Cyprinivirus, is a fatal contagious aquatic pathogen that affects goldfish (Carassius auratus) and crucian carp (Carassius carassius). Although crucian carp and goldfish belong to the genus Carassius, it is unclear whether they are susceptible to the same CyHV-2 isolate. In addition, the origin of the crucian carp-derived CyHV-2 virus isolate remains unclear. CyHV-2 SH01 was isolated during herpesviral hematopoietic necrosis disease (HVHN) outbreaks in crucian carp at a local fish farm near Shanghai. CyHV-2 SH01 was confirmed by PCR and Western blot analysis of kidney, spleen, muscle, and blood tissue from the diseased crucian carp. Moreover, histopathological and ultra-pathological analyses revealed pathological changes characteristic of CyHV-2 SH01 infection in the tissues of the diseased crucian carp. In the present study, goldfish and crucian carp were challenged with CyHV-2 SH01 to elucidate viral virulence. We found that CyHV-2 SH01 could cause rapid and fatal disease progression in goldfish and crucian carp 24 h post-injection at 28 °C. Experimental infection of goldfish by injection indicated that the average virus titer in the kidney of the goldfish was 103.47 to 103.59 copies/mg. In addition, tissues exhibited the most prominent histopathological changes (cellular wrinkling and shrinkage, cytoplasmic vacuolation, fusion of the gill lamellae, and hepatic congestion) in CyHV-2 SH01-infected goldfish and crucian carp. Thus, crucian carp and goldfish showed a high sensitivity, with typical symptoms, to HVHN disease caused by CyHV-2 SH01.

Comparative sensitivity of three new cell lines developed from gill, liver and brain tissues of goldfish, Carassius auratus (L.) to cyprinid herpesvirus-2 (CyHV-2)

Cyprinid herpesvirus 2 (CyHV-2) is the etiological agent of Goldfish herpesviral haematopoietic necrosis (GHVHN) in goldfish. In this study, three new cell lines including Fantail goldfish gill (FtGG), Fantail goldfish liver (FtGL) and Fantail goldfish brain (FtGB) had been established and characterized from the gill, liver and brain tissue of C. auratus respectively. Cell lines were optimally grown at 28 °C in Leibovitz-15 (L-15) medium supplemented with 10 % fetal bovine serum (FBS). The PDT during exponential growth of FtGG, FtGL and FtGB cells were determined to be 41.47 h, 63.43 h and 79.79 h respectively. Karyotyping analysis of cell lines remained diploid (2n = 100). The revival rate was 82 %, 72 % and 70 % in FtGG, FtGL and FtGB cells respectively after 6 months of cryopreservation. All the three cells showed similar cytopathic effect (CPE) between 3–5 days post-infection (dpi) with CyHV-2 and complete destruction of the monolayer was observed at 8–10 dpi. The viral titers of CyHV-2 in FtGG, FtGL and FtGB reached 107.375±0.35 TCID50 ml−1, 104·55±0.070 TCID50 ml−1 and 106.45±0.070 TCID50 ml−1 respectively. These newly established cell lines will be a useful diagnostic tool for viral diseases in this fish species and also for the isolation and study of goldfish viruses in future.