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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 (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.

We have previously observed that in common carp (Cyprinus carpio), administration of β-glucan (MacroGard™) as feed additive leads to a lower expression of pro-inflammatory cytokines suggesting that this immunostimulant may be preventing an acute and potentially dangerous response to infection. However, in general, mechanisms to detect and eliminate pathogens must also be induced in order to achieve an efficient clearance of the infection. Protection against viral diseases acquired through β-glucan-supplemented feed has been extensively reported for several experimental models in fish but the underlining mechanisms are still unknown. Thus, in order to better characterize the antiviral action induced by β-glucans in fish, MacroGardTM was administered daily to common carp in the form of supplemented commercial food pellets. Carp were fed for a period of 25 days prior to intra-peritoneal injection with polyinosinic:polycytidylic acid (poly I:C), a well-known double-stranded RNA mimic that triggers a type-I interferon (IFN) response and a set of immune related genes, including Mx, were analysed by real-time PCR in liver, spleen, head-kidney and mid-gut. Results obtained confirmed that treatment with β-glucan alone generally down-regulated the mRNA expression of pro-inflammatory cytokines when compared to untreated fish, while Mx gene expression remained stable. A similar expression pattern was observed for cytokines in samples obtained from β-glucan fed fish 24 h after injection with poly I:C. However, poly I:C injection markedly increased Mx gene expression but mainly in the group fed with β-glucan. Toll-like receptor 3 (TLR3) is the candidate pattern recognition receptor possibly responsible also in fish for the binding of viral double-stranded RNA and triggering of a type-I IFN response. Through a carp genome data mining, two sequences for carp TLR3 were retrieved (ccTLR3.1 and ccTLR3.2) and characterized. Constitutive gene expression of both genes was detected by rea

The use of immunomodulating substances such as β-glucan has been shown to increase survival during viral infections in fish. Various underlying mechanisms to explain this effect have been proposed, however the potential role of apoptosis, i.e. a form of programmed cell death, which is an important factor in the immune response against viral infections, has been neglected. The anti-viral response often includes the induction of apoptosis in infected cells in order to limit the reproduction and distribution of the virus. The influence of β-glucan on the apoptotic process is largely unknown, although it can influence apoptosis-related gene expression following exposure to LPS and Poly(I:C). Hence we aimed to ascertain the effects of immunomodulation on the apoptotic process during viral infections to understand if apoptosis plays a role in disease protection. We used spring viremia of carp virus (SVCV) and Cyprinid herpesvirus 3 (CyHV-3) in an in vitro model, as they cause extensive mortalities in aquaculture during disease outbreaks, and β-glucan, in the form of the feed supplement MacroGard®, as an immunomodulator. Levels of apoptosis were assessed through microscopy using acridine orange staining, and genetic analysis targeting the pro-apoptotic genes p53, caspase 9, apaf-1 and the anti-apoptotic gene IAP, as well as the nitric oxide producing enzyme iNOS. The results indicated that the effect of the immunomodulant on apoptosis varied depending on the infectious agent. During the SVCV infection, viral-induced apoptosis and some apoptosis related genes seemed to be enhanced in the infected cell cultures by previous exposure to MacroGard®. However, no such effect was observed with CyHV-3 infection. These results could explain some of the protective effects of β-glucan against viral infections, but also suggest the role of immunomodulants on apoptosis may not be consistent across all disease systems. We will discuss these findings in the context of our recent research

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