Cyprinid Herpesvirus Research Articles

Development of an Immunoassay Detection System for Koi Herpesvirus Using Recombinant Single-Chain Variable Fragments

Koi herpesvirus (KHV) is a highly contagious virus that causes high mortality in koi and common carp, leading to a reduction in production worldwide. Recent diagnostic tests based on molecular methods alone (nucleic acid amplification) and indirect immunoassay methods (antibody detection) can be confirmed over KHV infections or prior exposure and latent infections. Unfortunately, there is no established method to detect KHV virus particles, especially when virus titers are low. Therefore, we propose an alternative, direct immunoassay method for viral detection using a single-chain variable fragment (scFv), a specific region of IgG antibodies that binds specifically to KHV particles. The results of functional analyses indicated that four putative scFv candidates, C5, F8, F6, and E4, were specific to KHV, but only F6 and C5 had a high binding affinity. The binding characteristics were confirmed by indirect competitive and sandwich enzyme-linked immunosorbent assays, which indicated that F6 and C5 have a broad penetration area to the binding region and share a similar epitope with commercial KHV monoclonal antibodies. These characteristics were further confirmed by their interactions with purified KHV coat protein by indirect ELISA and Western blot analyses. In conclusion, the F6 and C5 scFvs have adequate binding affinity to KHV particles to permit their use in immunoassays.

Efficacy of a multidose acyclovir protocol against cyprinid herpesvirus 3 infection in koi (Cyprinus carpio).

To evaluate the effect of a multidose acyclovir protocol on koi herpesvirus (KHV) viral load and mortality in a cohabitation challenge. 180 koi fish. Forty fish (shedders) were immersed in a 0.5 KHV plaque-forming units/mL static bath for 8 hours. Mock shedders were treated similarly but exposed to cell culture media. KHV shedders were then transferred into 8 tanks (5 shedders per tank) containing 10 naïve fish (cohabitants) each. Fish in the acyclovir group (AT) received a 10 mg/kg acyclovir intracoelomic injection 1, 3, and 6 days after the first confirmed KHV mortality. Positive controls (PC) were treated similarly but received sterile saline injections. Negative controls (NC) were exposed to mock shedders. Morbidity and mortality were evaluated daily for 50 days post-challenge. Quantitative PCR was used to determine viral load in the gill biopsies of shedders and cohabitants collected at days 19 (T1), 22 (T2), 25 (T3), 34 (T4), and 50 (T5) post-challenge. Survival curves analyzed by the Gehan-Breslow-Wilcoxon method revealed a delayed onset of mortalities and a significantly lower KHV load at T2 and T3 detected in AT cohabitant fish (P = .042) compared to PC group. However, there were no significant differences in overall mortality or viral loads at T5. The acyclovir protocol used in this study did not control viral infection or mortality at the end of the 50-day trial. Shorter intervals between injections could improve outcomes, but the additional stress inflicted by handling should be considered. Exploring other therapeutic alternatives and doses is warranted.

Genome-Wide Analysis of Differentially Expressed mRNAs and lncRNAs in Koi Carp Infected with Koi Herpesvirus.

Long noncoding RNAs (lncRNAs) constitute an emerging group of ncRNAs that modulate gene expression at the transcriptional or translational level. Koi herpesvirus (KHV), also known as Cyprinus herpesvirus type 3 (CyHV-3) and characterized by high pathogenicity and high mortality, has caused substantial economic losses in the common carp and koi carp fisheries industry. In this work, we sequenced the lncRNA and mRNA of host koi carp infected with KHV. A total of 20,178 DEmRNAs were obtained, of which 5021 mRNAs were upregulated and 15,157 mRNAs were downregulated. Both KEGG pathways and GO terms were enriched in many important immune pathways. The KEGG analysis showed that DEGs were significantly enriched in many important immune pathways, such as apoptosis, NOD-like receptor signaling pathway, Jak-STAT signaling pathway, TNF signaling pathway, IL-17 signaling pathway, NF-kappa B signaling pathway, and so on. Furthermore, a total of 32,697 novel lncRNA transcripts were obtained from koi carp immune tissues; 9459 of these genes were differentially expressed. Through antisense, cis-acting, and trans-acting analyses, the target genes of differentially expressed lncRNAs (DElncRNAs) were predicted. Gene ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses showed that the DElncRNA expression pattern was consistent with the differential mRNA expression pattern. The lncRNA-mRNA network analysis, which included many immune pathways, showed that after KHV infection, the expression of most lncRNAs and their target mRNAs were downregulated, suggesting that these lncRNAs engage in a positive regulatory relationship with their target mRNAs. Considering that many studies have shown that herpesviruses can escape the immune system by negatively regulating these immune pathways, we speculated that these lncRNAs play a significant role in KHV's escape from host immunity. Furthermore, 10 immune-related genes and 20 lncRNAs were subsequently verified through RT-qPCR, to confirm the accuracy of the high-throughput sequencing results. In this study, we aimed to explore lncRNA functions in the immune response of lower vertebrates and provide a theoretical basis for the study of noncoding RNAs in teleosts. Therefore, exploring lncRNA expression in KHV-infected koi carp helped us better understand the biological role played by lncRNA-dependent pathways in aquaculture animal viral infection.

MicroRNA miR-722 Inhibits Cyprinid Herpesvirus 3 Replication via Targeting the Viral Immune Evasion Protein ORF89, Which Negatively Regulates IFN by Degrading IRF3.

Cyprinid herpesvirus 3 (CyHV-3) has caused severe economic losses to carp culture, but its pathogenicity is far from clear. Our previous study has revealed that microRNA (miR)-722 was upregulated during CyHV-3 infection, indicating that miR-722 might play an important role in CyHV-3 replication. In this study, we found that overexpression of miR-722 inhibited CyHV-3 replication and promoted IFN expression. The putative target gene of miR-722 was searched over the CyHV-3 genome, and ORF89 was identified and validated as a target gene of miR-722. Overexpression of ORF89 markedly reduced the expression of IFN and IFN-stimulated genes. Mechanistically, ORF89 interacted with and degraded IFN regulatory factor 3 (IRF3), and inhibited the entry of IRF3 into the nucleus by suppressing the dimerization of IRF3. Moreover, ORF89-mediated suppression of IFN expression could be restored by adding miR-722. To our knowledge, our findings confirm a novel virus-host combat, in which CyHV-3 evades host antiviral immunity by its ORF89 protein, whereas host miR-722, upregulated on CyHV-3 infection, targets ORF89 to impede CyHV-3 replication.

Fish herpesvirus KLP manipulates Beclin1 to selectively degrade MITA through a precise autophagic manner for immune evasion

Control of host autophagy acceleration or attenuation has been confirmed in multiple terrestrial animal viruses. Little is known about such mechanisms in aquatic viruses. Here, we report a selective and ingenious autophagy modulation regulated by kinase-like protein (KLP) of cyprinid herpesvirus 2 (CyHV2) to restrict interferon (IFN) production by degrading IFN regulatory factor (IRF) 3 activation (MITA). First, exogenous DNA and RNA-mediated IFN activation were both abrogated by CyHV2 KLP. The common intersection point of MITA with these two signaling pathways was the interaction with KLP. The C terminus of MITA was indispensable for the interaction and was recruited by KLP in subcellular colocalization analysis. Subsequently, we found that KLP degraded MITA in an autophagy-lysosome-dependent manner and, interestingly, individual KLP could not launch host autophagic flow except in the presence of MITA. KLP was also colocalized with the autophagy components Beclin1 and ATG14 and enhanced Beclin1 stability, but not ATG14, through K63-linked polyubiquitination. Finally, KLP significantly decreased the normal state or MITA-enhanced cellular antiviral capacity. These data demonstrated an elaborate autophagic process manipulated by a fish virus only in the presence of the host target, illuminating a mechanism of aquatic viral immune evasion.

Establishment and susceptibility to cyprinid herpesvirus 2 of Carassius carassius gill cell line

Haematopoietic necrosis in Carassius auratus is one of the most serious diseases in recent years caused by CyHV-2 (cyprinid herpesvirus 2) which has caused huge economic losses in aquaculture. Currently, the isolation and propagation of CyHV-2 in vitro is difficult due to the lack of sensitive cell lines, which hinder further studies on the pathogenesis and gene functions of the virus. In this study, Carassius carassius gill cell line (CCG) was constructed and had been passaged up to 90 passages at 25 ℃ in Medium199 (M199) with 15–20% Foetal bovine serum (FBS). Cellular karyotype analysis showed that the chromosome number was 46 at passage 50. CCG could be successfully transfected with GFP reporter gene with an efficiency of more than 60% suggesting that CCG could be used for exogenous gene expression in vitro. CCG infected with CyHV-2 displayed typical cytopathic effects (CPE), and viral replication was confirmed by PCR, indirect immunofluorescence assay (IFA) and transmission electron microscopy (TEM) assay, respectively. In conclusion, the CCG cell line can be used as a useful tool to study the propagation and pathogenesis of CyHV-2.

Combination of iron flocculation and qPCR for quantitative evaluation of virus-shedding intensity of goldfish Carassius auratus infected with cyprinid herpesvirus 2 in the water and the effect of sodium chlorite powder in blocking waterborne horizontal viral transmission

Cyprinid herpesvirus 2 (CyHV-2), a member of the Alloherpesviridae family belonging to the genus Cyprinivirus, was initially isolated from goldfish (Carassius auratus) and has been recently emerging as a virulent pathogen for cultured prussian carp (Carassius auratus gibelio) world-wide. In this study, a novel and effective method for concentration and quantification of live CyHV-2 virions from water was successfully established through coupling the iron flocculation with real time qPCR assay. Then, the shedding intensity of CyHV-2 in fish-tank water from artificially-challenged goldfish (25 fish/20 L) was monitored continuously for 7 days on a daily basis through quantitating viral genomic copy numbers by qPCR, and the maximum shedding level was determined to be 105 copies/L. Horizontal transmission research system was established by inoculating healthy goldfish in water spiked with serial dilution of CyHV-2 virions ranging from 107 to 103 copies/L. Our results indicated that water-borne CyHV-2 efficiently caused the infection of tested goldfish even in a concentration of 103 copies/L, and the overall transmission efficacy was not linearly correlated with the level of input virus in the fish tank. Commercial disinfectant Composite Sodium Chlorite Powder (CSCP) has been widely applied in aquaculture to control microbial infection through direct spill in the water, and its effect in inactivating the CyHV-2 infectivity remains unknown. We further determined that the EC50 of CSCP against 3.89 TCID50/mL CyHV-2 was close to 15.625 μg/mL in vitro, and application of CSCP in a level as high as 60 μg/mL (the safety concentration of CSCP for goldfish) couldn't protect goldfish from CyHV-2 challenge through immersion. Thus, the disinfectant CSCP was regarded as none-effective for blocking CyHV-2 transmission in water during epidemic. Overall, our data provided quantitative data to demonstrate the shedding intensity of CyHV-2 in water, and CSCP was shown to be not effective in blocking water-borne horizontal transmission of CyHV-2 in goldfish. The virus-concentration protocol and virus-inhibition assay established here also paved the way for evaluating more commercial disinfectants in their effects in blocking water-borne horizontal transmission of CyHV-2.

Development of a droplet digital PCR method for the sensitive detection and quantification of largemouth bass ranavirus.

Largemouth bass ranavirus (LMBRaV), also known as largemouth bass virus (LMBV), is a high mortality pathogen in largemouth bass. A rapid, sensitive, specific and convenient diagnosis method is an urgent requirement for the prevention of virus transmission. In the present study, a droplet digital PCR (ddPCR) method based on the major capsid protein (mcp) gene was established to detect and quantify the virus genome copy number. Oligonucleotide primers were designed based on the LMBRaV mcp gene sequence. The specificity and sensitivity of ddPCR assay were analysed. The other aquatic virus including Chinese giant salamander iridovirus (GSIV), Cyprinid herpesvirus II (CyHV-2) and infectious spleen and kidney necrosis virus could not be detected by LMBRaV ddPCR assay. The detection limit of ddPCR assay was 2 ± 0.37 copies/μl DNA sample. And this ddPCR assay had great repeatability and reproducibility. In clinical diagnosis of 50 largemouth bass, 43 positive samples were detected by ddPCR, whereas only 34 positive samples were detected by quantitative PCR (qPCR). This LMBRaV detection assay provided a specific and sensitive method for the rapid diagnosis of LMBRaV infection in largemouth bass as well as quantification of the virus load.

The application of exopolysaccharides (EPS) can prevent viral disease of fish

In the frame of investigations on the use of exopolysaccharides (EPS) from Arthrospira platensis in carp and koi cultures, two animal experiments were carried out to assess their efficiency as prophylactic and metaphylactic (therapeutic) measures against koi herpesvirus disease (KHVD). In experiment 1, carp were treated with algae biomass (BM) and EPS before and after infection with European lineage koi herpesvirus (KHV-E). In experiment 2, carp were treated with EPS prior and after the infection with Taiwan isolate of KHV (KHV-T), only prior to infection with higher concentration of EPS or only after the infection with KHV-T. No conclusive protection against KHV was observed in experiment 1 in carp treated with BM. In groups where EPS was applied, carp were protected to a certain extent. In experiment 2, carp were protected significantly against a severe KHVD outbreak. In the prophylactic group, which received a double EPS concentration for six weeks, and in the metaphylactic group, KHVD was stopped. Fish developed antibodies against EPS as well as against KHV at day 30 post infection.

A fish herpesvirus highlights functional diversities among Zα domains related to phase separation induction and A-to-Z conversion.

Zalpha (Zα) domains bind to left-handed Z-DNA and Z-RNA. The Zα domain protein family includes cellular (ADAR1, ZBP1 and PKZ) and viral (vaccinia virus E3 and cyprinid herpesvirus 3 (CyHV-3) ORF112) proteins. We studied CyHV-3 ORF112, which contains an intrinsically disordered region and a Zα domain. Genome editing of CyHV-3 indicated that the expression of only the Zα domain of ORF112 was sufficient for normal viral replication in cell culture and virulence in carp. In contrast, its deletion was lethal for the virus. These observations revealed the potential of the CyHV-3 model as a unique platform to compare the exchangeability of Zα domains expressed alone in living cells. Attempts to rescue the ORF112 deletion by a broad spectrum of cellular, viral, and artificial Zα domains showed that only those expressing Z-binding activity, the capacity to induce liquid-liquid phase separation (LLPS), and A-to-Z conversion, could rescue viral replication. For the first time, this study reports the ability of some Zα domains to induce LLPS and supports the biological relevance of dsRNA A-to-Z conversion mediated by Zα domains. This study expands the functional diversity of Zα domains and stimulates new hypotheses concerning the mechanisms of action of proteins containing Zα domains.

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.

Quick detection of Carassius auratus herpesvirus (CaHV) by recombinase-aid amplification lateral flow dipstick (RAA-LFD) method.

Crucian carp (Carassius auratus) is one of the major freshwater species and is also a common food fish in China. Recently, Carassius auratus herpesvirus (CaHV) could induce fatal viral disease with high mortality of crucian carp, which had caused huge economic losses. In this study, we described a rapid and simple recombinase-aid amplification (RAA) assay coupled with lateral flow dipstick (LFD), which could achieve sensitive diagnosis of tumor necrosis factor receptor (TNFR) of CaHV within 35 min at 40°C. Our RAA-LFD method had a satisfactory detection limit of 100 gene copies per reaction, which was 100-fold more sensitive than traditional PCR. In addition, no cross-reaction was observed with other viral pathogens, including koi herpesvirus (KHV), cyprinid herpesvirus 2 (CyHV-2), infectious hematopoietic necrosis virus (IHNV), spring viremia of carp virus (SVCV) and grass carp reovirus (GCRV). Furthermore, the overall cost of the method was cut in half compared to previous studies. In conclusion, RAA-LFD assay is therefore, a promising alternative for point-of-care testing (POCT) of CaHV, which is feasible and of certain value in application of aquatic disease control.

Andrographolide loaded Pickering emulsion: A bioactive component for improved growth, digestibility, and haematological properties in cultured common carp Cyprinus carpio

In aquaculture, the therapeutic potential of andrographolide had been beneficially documented but still restricted owing to its high dosage and low bioavailability. To maximize andrographolide's potential value in carps, we want to create an effective delivery mechanism. During the 8-week Cyprinus carpio feeding trial, we aimed to test a low dose (0.05 g kg−1 or 0.005%) andrographolide-supplemented diet with or without a novel delivery system (chitosan particles stabilized Pickering emulsion). The experiment included four treatment groups, having a commercial carp feed as basal diet (control), diet containing Pickering emulsion without andrographolide encapsulation (PE), diet with free andrographolide (without Pickering emulsion) (AGL), diet with andrographolide encapsulated Pickering emulsion (AGL-PE). A significantly improved (p < 0.05) body weight, thermal growth coefficient and feed conversion ratio of the AGL-PE group was observed than control or AGL alone. It demonstrated improvement of AGL's efficacy may be brought by a proper delivery system. However, the delivery system alone (PE) did not significantly interfere with the results, as the differences of PE group were non-significant (p > 0.05) with either control or AGL group. The apparent digestibility coefficient of dietary nutrients, most notably protein, fiber, carbohydrates, and energy, had improved significantly (p < 0.05) by AGL-PE treatment compared to control. This most likely resulted in improved feed utilization and growth. AGL-PE addition in carp feed did not alter the ionic concentration of common blood electrolytes or metabolites, with the exception of nitrogen factors such as NH3, urea, and creatinine, possibly because of a higher digestible intake of nitrogen. Haemato-biological parameters showed a significant increase in lymphocyte, albumin, globulin, total protein, triacylglycerol with AGL-PE group (p > 0.05) than control supporting a stress free and strengthened immune system. Additionally, AGL-PE significantly improved survivability of common carp when challenged with koi herpes virus (KHV) due to immunostimulant effect of andrographolide (and chitosan in PE). Neutrophil left shift, a marker we used for establishing immunostimulant effect, was persistently high when either PE or andrographolide was introduced in diet; even maximum when they formed a hurdle system in AGL-PE diet. Modulation of Neutrophil left shift was possibly associated with amplified gut microbiota activity that coincided with higher fiber (and NFE) digestibility of AGL-PE group. The results firmly establish application of AGL-PE system as prebiotic additive in commercial aquafeed to simultaneously improve digestibility, feed utilization, growth, immunomodulatory and haematological properties in cultured fish.

Development of an attenuated vaccine against Koi Herpesvirus Disease (KHVD) suitable for oral administration and immersion

Since the end of the1990ies, Cyprinid herpesvirus 3 (also known as koi herpesvirus, KHV) has caused mass mortality events of koi and common carp all over the globe. This induced a high economic impact, since the KHV disease cannot be cured up to now, but only prevented by vaccination. Unfortunately, there is only one commercial vaccine available which is not approved in most countries. Therefore, there is an urgent need for new, safe and available vaccines. In this study, a live attenuated vaccine virus was generated by cell culture passages of virulent KHV, and shown to protect carp or koi after immersion or oral application against wild type challenge. An advantage of boost immunization was demonstrated, especially after oral application. Vaccination induced no or mild clinical signs and protecting antibodies have been measured. Additionally, the vaccine virus allowed differentiation of infected from vaccinated animals (DIVA) by PCR. The attenuation of the newly generated vaccine was tracked down to a partial deletion of open reading frame 150. This was confirmed by the generation of engineered ORF150 deletion mutants of wild-type KHV which exhibited a similar attenuation in vivo.

Comparative analysis of differential gene expression in two species of crucian carps in response to Cyprinid herpesvirus 2 (CyHV-2) infection

We assessed the expressions of MHCI, LYZC, keratin8, MPO, DUSP1, IκBα, Rab21, and Rac2 between two species of carps (Erqisi river crucian carp and allogynogenetic crucian carp) after Cyprinid herpesvirus 2 (CyHV-2) infection. The relative expressions of MHCI, LYZC, and keratin8 in the virus-challenged groups were significantly higher than control groups. Moreover, the expression of IκBα in the virus-challenged groups was significantly lower than in the control groups. Compared with the virus-challenged ERO group, the expression of IκBα in the virus-challenged ZHO group decreased. The expression of Rab21 in the virus-challenged groups gradually increased and was significantly higher than in the control groups, and then its expression began to decrease after 24 h. At 72 h, the expression of IκBα in both virus-challenged groups was significantly lower than in the control groups. In addition, the expression of Rab21 in the virus-challenged ZHO group was significantly higher than the virus-challenged ERO group at all time points except for 72 h. Before 24 h, the expression of Rac2 remained unchanged in these four groups, and its expression in the virus-challenged ZHO group was significantly higher than in the other three groups. Nevertheless, its expression began to decrease after 24 h but was still slightly higher than the control group at 72 h. MPO showed a similar expression pattern as Rac2. The expression of DUSP1 in the four groups was the same at 0 h. However, its expression in the virus-challenged ZHO group was significantly higher than in the other three groups at other time points.

The influence of viral infection on cell line characteristics: Lessons learned from working with new cell lines from common carp.

Several factors influence the susceptibility of cell lines to infection by different viruses. These can be related to tissue specificity of the viruses, physiological status of the cells, their differentiation level and their capacity to mount immune responses to combat viral infection. To study the influence of cell characteristics and immune responses on their susceptibility on virus infection, newly developed cell lines from common carp brain (CCAbre), fins (CCApin), gills (CCAgill), and heart (CCAcar) and the established common carp brain (CCB) cells were exposed to the carp infecting viruses cyprinid herpesvirus 3 (CyHV-3), carp oedema virus (CEV), and the yet not fully characterized common carp paramyxovirus (CCPV). The susceptibility of these cells to viral infection was measured by formation of a cytopathic effect (CPE), estimation of viral particles produced by the cells and presence of viral mRNA in the cells. Viral susceptibility of the cells was compared to cell characteristics, measured by mRNA expression of the epithelial cell markers cadherin 1, occludin, and cytokeratin 15 and the mesenchymal cell marker vimentin, as well as to the level of type I interferon (IFN) responses. All cell lines were susceptible to CyHV-3 and CCPV but not to CEV infection. The cell lines had different levels of type I IFN responses towards the viruses. Typically, CyHV-3 did not induce high type I IFN responses, while CCPV induced high responses in CCAbre, CCAcar, CCApin cells but no response in CCAgill cells. Consequently, the type I IFN response modulated cell susceptibility to CCPV but not to CyHV-3. Interestingly, when the three different passage levels of CCB cells were examined, the susceptibility of one passage was significantly lower for CyHV-3 and higher for CCPV infection. This coincided with a loss of epithelial markers and lower type I IFN responses. This study confirms an influence of cell characteristics and immune responses on the susceptibility of carp cell lines for virus infection. Depending on the vulnerability of the virus to type I IFN responses, cells with a lower IFN-response can be superior for replication of some viruses. Batches of CCB cells can differentiate and thus may have significantly different levels of susceptibility to certain viruses.

Early immune response of two common carp breeds to koi herpesvirus infection

Economic importance of common carp (Cyprinus carpio L.) increases every year. Viral diseases are major threat for carp aquaculture and cause significant economic losses. Koi herpesvirus (KHV) is one of the most serious carp diseases. Current study is focused on confirmation of possible differences in early immune response to KHV depending on level of resistance. Class I interferon signalling, complement cascade and cell-mediated cytotoxicity are hypothesized as major mechanisms of early innate immune response against KHV. Different breeds of common carp show distinct level of resistance to KHV. Two breeds of common carp with completely different susceptibility to KHV were chosen for current research: amur wild carp (AS) as highly resistant and koi carp (KOI) as very susceptible breed. KHV infection caused no mortalities, but the viral load in selected tissues increased during infection. Levels of expressions of chosen genes was examined using qRT-PCR and overall change in protein expression profiles was analysed by mass spectrometry. Significant differences in immune response between AS and KOI were detected mostly at the level of protein expression. Although cell-mediated cytotoxicity showed minimal influence during KHV infection, many immune response parameters related to class I interferon signalling pathway and complement cascade were increased earlier during KHV infection in AS comparing to KOI.

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.

Development and characterization of monoclonal antibodies specific for cyprinid herpesvirus 2

Infections of Cyprinid herpesvirus 2 in goldfish and farmed crucian carp (Carassius auratus gibelio) are still an urgent problem worldwide. Detection and prevention are necessary for the control of haematopoietic necrosis disease caused by CyHV-2. Although many sensitive molecular diagnostic methods have been developed, effective immunodiagnosis and neutralization approaches based on monoclonal antibodies (MAbs) against CyHV-2 are still important to CyHV-2 study. In this experiment, purified CyHV-2 was used as antigens to produce monoclonal antibodies (Mabs). Six Mabs bound to different proteins were selected by Dot-blot screening and Western-blot analysis, and no one had cross-reactivity with closely related koi herpesvirus. Among them, Mabs 2E1-B10, 1F5-A3 and 4C4-A7 belonged to IgG1 isotype, while other three Mabs 3G9-B11, 3B4-G5 and 4F4-B7 belonged to IgM isotype. These six Mabs all could specifically detect CyHV-2 in CyHV-2 infected caudal fin of Carassius auratus gibelio (GiCF) cells by immunofluorescence assays. Then, the neutralization ability was tested in vitro, and the result showed that all six Mabs can attenuate CPE by CyHV-2 in vitro among which 2E1-B10 had the best neutralization ability. The virus proteins recognized by these six Mabs were identified by mass spectrometry identification, and the result showed they probably were ORF88, ORF55R, ORF115 and ORF151R. This study is the first to prepare Mabs by purifying CyHV-2, which will provide a practical basis for the in-depth study of CyHV-2 virus.