Cyprinid Herpesvirus 3 Il10 Inhibits Inflammatory Activities of Carp Macrophages and Promotes Proliferation of Igm+ B Cells and Memory T Cells in a Manner Similar to Carp Il10.

Cyprinid herpesvirus 3 (CyHV-3) is the causative agent of a lethal disease of carp and encodes for an Il10 homolog (ORF134). Our previous studies with a recombinant ORF134-deleted strain and the derived revertant strain suggested that cyprinid herpesvirus 3 Il10 (CyHV-3 Il10 [cyhv3Il10]) is not essential for viral replication in vitro, or virulence in vivo. In apparent contrast, cyhv3Il10 is one of the most abundant proteins of the CyHV-3 secretome and is structurally very similar to carp Il10 and also human IL10. To date, studies addressing the biological activity of cyhv3Il10 on cells of its natural host have not been performed. To address the apparent contradiction between the presence of a structurally conserved Il10 homolog in the genome of CyHV-3 and the lack of a clear phenotype in vivo using recombinant cyhv3Il10-deleted viruses, we used an in vitro approach to investigate in detail whether cyhv3Il10 exerts any biological activity on carp cells. In this study, we provide direct evidence that cyhv3Il10 is biologically active and, similarly to carp Il10, signals via a conserved Stat3 pathway modulating immune cells of its natural host, carp. In vitro, cyhv3Il10 deactivates phagocytes with a prominent effect on macrophages, while also promoting proliferation of Igm(+) B cells and memory T cells. Collectively, this study demonstrates a clear biological activity of cyhv3Il10 on cells of its natural host and indicates that cyhv3Il10 is a true viral ortholog of carp Il10. Furthermore, to our knowledge, this is the first report on biological activities of a nonmammalian viral Il10 homolog.

Interaction between type I interferon and Cyprinid herpesvirus 3 in two genetic lines of common carp Cyprinus carpio.

Cyprinid herpesvirus 3 (CyHV-3) infection in common carp Cyprinus carpio L. and its ornamental koi varieties can induce the severe systemic disease known as koi herpesvirus disease. This disease is characterised by a rapid replication and spreading of the virus through multiple organs and results in a fast onset of mortality (starting on Day 6 post infection) in up to 100% of infected fish. During the first phase of viral infections, type I interferons (IFNs) have generally been proven to be essential in inducing an innate immune response; however, very little is known about the type I IFN response to herpesviruses in fish. The aim of this work was to study the type I IFN responses during CyHV-3 infection in 2 genetically divergent lines of common carp which presented differing survival rates. Our results show that CyHV-3 induced a systemic type I IFN response in carp, and the magnitude of type I IFN expression is correlated with the virus load found in skin and head kidney. In this in vivo experimental setup, the level of type I IFN response cannot be linked with higher survival of carp during CyHV-3 infection.

C-reactive protein and complement as acute phase reactants in common carp Cyprinus carpio during CyHV-3 infection.

Cyprinid herpesvirus 3 (CyHV-3) is the aetiological agent of a highly virulent and lethal disease of common carp Cyprinus carpio and its ornamental koi varieties. However, specific knowledge about immune mechanisms behind the infection process is very limited. We aimed to evaluate the effect of the CyHV-3 infection on the profile of 2 major components of the common carp immune acute phase response: the C-reactive protein (CRP) and the complement system. Common carp were infected with CyHV-3 by bath immersion. Fish were sampled before the infection and at 6, 12, 24, 72, 120 and 336 h post-infection for serum and head kidney, liver, gill and spleen tissues. CRP levels and complement activity were determined from the serum, whereas CRP- and complement-related genes (crp1, crp2, c1rs, bf/c2, c3, masp2) expression profiles were analysed in the tissues by quantitative PCR. Both CRP levels and complement activity increased significantly up to 10- and 3-fold, respectively, in the serum of infected fish during the challenge. Analysis revealed distinct organ- and time-dependent expression profile patterns for all selected genes. These results suggest that CRP and complement behave as acute phase reactants to CyHV-3 infection in common carp with an organ- and time-dependent response.

Intestinal barrier of carp (Cyprinus carpio L.) during a cyprinid herpesvirus 3-infection: molecular identification and regulation of the mRNA expression of claudin encoding genes.

As a major part of tight junctions in the intestinal epithelium of vertebrates, claudin proteins are crucial to develop a selective permeable function and to maintain integrity of the barrier. The intestine has been reported as one of the targeted tissue of the cyprinid herpesvirus 3 (CyHV-3) or koi herpesvirus (KHV) which causes major disease problems in carp production worldwide. To analyse the impact of the disease on the epithelial barrier of the intestine, carp claudin encoding genes were cloned, their tissue expression was described, and the abundance of gene transcripts in the intestine of carp under CyHV-3 infection was determined. Some of the carp claudin genes such as claudin-7 and -11 were expressed in various tissues, whilst others, like claudin-2 and -23, showed more tissue-specific expression profiles, which may reflect specific functions of these particular claudins. Along the gut axis, a spatial distribution of claudin gene expressions was found, with a lower abundance of gene transcripts in anterior regions of the intestine and increased expression in the distal section of the intestine, which might indicate specific functions of different regions in the intestinal tract of carp. In carp under CyHV-3 infection, an up-regulation in the expression of IFN-a2, IL-1beta and iNOS was observed, together with an elevation of transcriptional levels of claudin-2, -3c, -11, and -23. The data suggest that expression of claudins is involved in the reorganisation of the intestinal epithelium in CyHV-3-infected carp, which may be responsible for changes in the paracellular permeability. An increased expression of the claudins might be a response to the disturbance of the hydromineral balance in carp under CyHV-3 infection.

Interferon type I responses to virus infections in carp cells: In vitro studies on Cyprinid herpesvirus 3 and Rhabdovirus carpio infections.

Interferons (IFNs) are secreted mediators that play a fundamental role in the innate immune response against viruses among all vertebrate classes. Common carp is a host for two highly contagious viruses: spring viraemia of carp virus (Rhabdovirus carpio, SVCV) and the Cyprinid herpesvirus 3 (CyHV-3), which belong to Rhabdoviridae and Alloherpesviridae families, respectively. Both viruses are responsible for significant losses in carp aquaculture. In this paper we studied the mRNA expression profiles of genes encoding for proteins promoting various functions during the interferon pathway, from pattern recognition receptors to antiviral genes, during in vitro viral infection. Furthermore, we investigated the impact of the interferon pathway (stimulated with poly I:C) on CyHV-3 replication and the speed of virus spreading in cell culture. The results showed that two carp viruses, CyHV-3 and SVCV induced fundamentally different type I IFN responses in CCB cells. SVCV induced a high response in all studied genes, whereas CyHV-3 seems to induce no response in CCB cells, but it induces a response in head kidney leukocytes. The lack of an IFN type I response to CyHV-3 could be an indicator of anti-IFN actions of the virus, however the nature of this mechanism has to be evaluated in future studies. Our results also suggest that an activation of type I IFN in CyHV-3 infected cells can limit the spread of the virus in cell culture. This would open the opportunity to treat the disease associated with CyHV-3 by an application of poly I:C in certain cases.

Resistance of common carp (Cyprinus carpio L.) to Cyprinid herpesvirus-3 is influenced by major histocompatibility (MH) class II B gene polymorphism.

The role of MH class II B (Cyca-DAB1-like) genes in resistance of common carp (Cyprinus carpio L.) to Cyprinid herpesvirus-3 (CyHV-3), also known as koi herpesvirus (KHV) was analysed. The material consisted of 934 fish from six carp crosses. Fish were challenged with CyHV-3 at an age of 7 and 10 months. During challenge experiments the peak of mortality caused by CyHV-3 was observed at days 8-12 p.i. and the overall cumulative mortality reached 79.9%. Among six Cyca-DAB1-like genotypes, revealed by PCR-RF-SSCP analysis, one genotype (E) was found associated with higher resistance to CyHV-3. Three other genotypes (B, H and J) could be linked to higher susceptibility to CyHV-3. Analysis of the alleles that compose the Cyca-DAB1-like genotypes linked one particular allele (Cyca-DAB1*05) to significantly increased, and two alleles (Cyca-DAB1*02 and Cyca-DAB1*06) to significantly decreased resistance to CyHV-3. Our data indicate that MH class II B genes could be used as potential genetic markers in breeding of common carp for resistance to this virus.

Classical crosses of common carp (Cyprinus carpio L.) show co-segregation of antibody response with major histocompatibility class II B genes.

In cyprinids, two paralogous groups of major histocompatibility (MH) class II B genes, DAB1 and DAB3, have been reported but have not been studied in detail. In our study on MH association with immune responsiveness in common carp (Cyprinus carpio L.) we have taken a long-term approach using divergent selection for antibody production. We report the co-segregation of Cyca-DAB1-like and Cyca-DAB3-like genes with antibody response, in backcrosses to high- and low-responsive parental carp lines. We show that the presence of Cyca-DAB1-like, but not Cyca-DAB3-like genes, preferentially leads to a high DNP-specific antibody response in carp. Background genes other than Cyca-DAB genes also influenced the level of antibody response. Our data support the hypothesis of a genetic control by Cyca-DAB genes on the antibody response measured. We could not detect an association of the Cyca-DAB genes with disease resistance to the parasite Trypanoplasma borreli. Sequence information, constitutive transcription levels and our co-segregation data indicate that both paralogous Cyca-DAB1-like and Cyca-DAB3-like groups represent functional MH class II B genes. Previously defined differences in allelic diversity between Cyca-DAB1-like genes, especially, identify Cyca-DAB1 as the most interesting DAB gene for further study in common carp.

Genetic resistance of carp (Cyprinus carpio L.) to Trypanoplasma borreli: influence of transferrin polymorphisms.

In serum most of the iron molecules are bound to transferrin (Tf), which is a highly polymorphic protein in fish. Tf is an essential growth factor for mammalian trypanosomes. We performed a series of experiments with Trypanoplasma borreli to detect putative correlations between different Tf genotypes of common carp (Cyprinus carpio L.) and susceptibility to this blood parasite. Five genetically different, commercially exploited carp lines (Israelian 'D', Polish 'R2' and 'K', Ukrainian 'Ur', Hungarian 'R0') and a reference laboratory cross ('R3xR8') were challenged with T. borreli and parasitaemia measured to determine susceptibility to the parasite. Among the commercial carp lines, Israelian 'D' carp were identified as most and Polish 'R2' carp as least susceptible, and used to produce a next generation and reciprocal crosses. These progenies were challenged with T. borreli and parasitaemia measured. We demonstrated significant effects of genetic background of the carp lines on susceptibility to T. borreli. This genetic effect was preserved in a next generation. We also observed a significant male effect on susceptibility to T. borreli in the reciprocal crosses. Serum samples from a representative number of fish from two infection experiments were used for Tf genotyping by polyacrylamide gel electrophoresis (PAGE), identifying DD, DG and DF as most frequent Tf genotypes. We could detect a significant association of the homozygous DD genotype with low parasitaemia in the least susceptible 'R2' (and 'K') carp lines and the lack of a such an association in the most susceptible 'D' carp line. Upon examination of parasite growth in vitro in culture media supplemented with 3% serum taken from fish with different Tf genotypes, we could show a faster decrease in number of parasites in culture media with serum from DD-typed animals.

Differential transcription of multiple forms of alpha-2-macroglobulin in carp (Cyprinus carpio) infected with parasites.

Alpha-2-macroglobulin (a2M) is a non-specific protease inhibitor involved in host defense mechanisms, inhibiting both endogenous and exogenous proteases. It is unique among the plasma anti-proteases with respect to the diversity of proteases that it can inactivate. Carp a2M consists of an alpha and beta chain of which the first includes the bioactive regions. Previously, three a2M alpha chain sequences were reported for East-Asian common carp. We studied a2M alpha chain variability in European common carp and report the cloning of a fourth a2M alpha chain with distinct sequence diversity in the bait region. The role of a2M in the immune response to parasites was studied in the liver of carp infected with Trypanoplasma borreli or with Ichthyophthirius multifiliis. Quantitative gene transcription analysis showed a differential regulation of the four isoforms, most clearly seen in infections with I. multifiliis. A2M3 was the only a2M isoform with a highly upregulated transcription during infection, suggesting that this particular isoform is of foremost biological importance.

Differential effects of alloherpesvirus CyHV-3 and rhabdovirus SVCV on apoptosis in fish cells.

Whilst Herpesviridae, which infect higher vertebrates, actively influence host immune responses to ensure viral replication, it is mostly unknown if Alloherpesviridae, which infect lower vertebrates, possess similar abilities. An important antiviral response is clearance of infected cells via apoptosis, which in mammals influences the outcome of infection. Here, we utilise common carp infected with CyHV-3 to determine the effect on the expression of genes encoding apoptosis-related proteins (p53, Caspase 9, Apaf-1, IAP, iNOS) in the pronephros, spleen and gills. The influence of CyHV-3 on CCB cells was also studied and compared to SVCV (a rhabdovirus) which induces apoptosis in carp cell lines. Although CyHV-3 induced iNOS expression in vivo, significant induction of the genetic apoptosis pathway was only seen in the pronephros. In vitro CyHV-3 did not induce apoptosis or apoptosis-related expression whilst SVCV did stimulate apoptosis. This suggests that CyHV-3 possesses mechanisms similar to herpesviruses of higher vertebrates to inhibit the antiviral apoptotic process.

Cyprinid herpesvirus 3 infection disrupts the skin barrier of common carp (Cyprinus carpio L.).

Cyprinid herpesvirus-3 (CyHV-3) is recognised as a pathogen which causes mass mortality in populations of carp, Cyprinus carpio. One of the characteristic symptoms of the disease associated with CyHV-3 infection is the occurrence of skin lesions, sloughing off the epithelium and a lack of mucus. Furthermore, fish then seem to be more susceptible to secondary infections by bacterial, parasitic or fungal pathogens which may cause further mortality within the population. The observed pathological alterations lead to the assumption that the carp skin barrier is strongly challenged during CyHV-3 associated disease. Therefore we examined mRNA expression of genes encoding inflammatory mediators, type I interferons, and the following skin defence molecules: antimicrobial peptides, claudins, and mucin. In addition, we monitored changes in the bacterial flora of the skin during disease conditions. Our results show that CyHV-3 associated disease in the skin of common carp leads to a reduction in mRNA expression of genes encoding several important components of the mucosal barrier, in particular mucin 5B, beta defensin 1 and 2, and the tight junction proteins claudin 23 and 30. This caused changes in the bacterial flora and the development of secondary bacterial infection among some individual fish. To our knowledge this is the first report showing that under disease conditions associated with virus infection, the mucosal barrier of fish skin is disrupted resulting in a higher susceptibility to secondary infections. The reported clinical signs of CyHV-3 skin infection can now be explained by our results at the molecular level, although the mechanism of a probable virus induced immunomodulation has to be investigated further.

Gene expression analysis of common carp (Cyprinus carpio L.) lines during Cyprinid herpesvirus 3 infection yields insights into differential immune responses.

Cyprinid herpesvirus 3 (CyHV-3), also known as koi herpesvirus (KHV), is the etiological agent of a virulent and lethal disease in common and koi carp. This study aimed to determine the genetic basis underlying the common carp immune response to the CyHV-3 virus. Two common carp lines (R3 and K) were infected with CyHV-3 by immersion. The R3 line presented a 20% higher survival rate compared to the K line and significantly lower viral loads as measured at day 3 post infection (p.i.). Microarray analysis using a common carp slides containing a number of 10,822 60-mer probes, revealed that 581 genes in line K (330 up-regulated, 251 down-regulated) and 107 genes in line R3 (77 up-regulated, 30 down-regulated), showed at least a 2-fold difference in expression at day 3 p.i. compared to day 0. Genes which showed at least a 4-fold difference in expression in both lines were selected as potential markers of a CyHV-3 infection in common carp. Additionally, 76 genes showed at least 2-fold differentially expression between K and R3 lines at day 3 p.i. Significantly higher expression of several immune-related genes including number of those which are involve in pathogen recognition, complement activation, MHC class I-restricted antigen presentation and development of adaptive mucosal immunity was noted in more resistant R3 line. Further real-time PCR based analysis provided evidence for higher activation of CD8(+) T cells in R3 line. This study uncovered wide array of immune-related genes involved into antiviral response of common carp toward CyHV-3. It is also demonstrated that the outcome of this severe disease in large extent could be controlled by genetic factors of the host.

Application of PCR-RF-SSCP to study major histocompatibility class II B polymorphism in common carp (Cyprinus carpio L.).

A variety of methods have been applied for the characterization of major histocompatibility (MH) polymorphism in fish. We optimized a technique designated polymerase chain reaction-restriction fragments-single strand conformation polymorphism (PCR-RF-SSCP) for screening a large number of individuals for the Cyca-DAB1 and Cyca-DAB2 genes polymorphism in common carp. The advantages of this technique are simplicity, high sensitivity and low costs. PCR-RF-SSCP analysis revealed different genotypes consisting of unique combinations of the Cyca-DAB1 and Cyca-DAB2 sequences with the number of SSCP bands clearly correlating with the degree of heterozygosity of the Cyca-DAB1 and Cyca-DAB2 genes. We found four alleles for Cyca-DAB1 (*02-*05) gene but only one allele for Cyca-DAB2 (*02) and noted that the Cyca-DAB2 gene was either homozygous or absent. PCR-RF-SSCP analysis of n=79 carp individuals challenged with Aeromonas hydrophila indicated that individuals bearing no Cyca-DAB2 gene showed higher cumulative mortality and lower bacterial agglutination titers during the experiment. We suggest that our PCR-RF-SSCP method can be used to study correlations of different MH class II B genotypes/alleles with resistance of common carp to specific pathogens.