Actin, myosin, and tubulin are ubiquitous components of the fibrous network known as the cytoskeleton. Cytoskeletal proteins are involved in a plethora of intracellular processes such as maintenance of cellular organization, organelle translocation, and various nuclear roles including chromosome separation during mitosis. Early methods for protein extraction primarily relied on the salting-out method which was performed in conjunction with biochemical assays. Since the advent of recombinant molecular biology, protein tagging has been coupled with chromatography to obtain highly purified proteins required for sensitive assays. This chapter provides a general standard operating procedure (SOP) for using the ÄKTA™ Start System controlled by UNICORN software for fast protein liquid chromatography (FPLC) of 6× his-tagged cytoskeletal proteins. The protocol can readily be modified for affinity and non-affinity purification techniques using the various ÄKTA™ Chromatography Systems.

Cyprinid herpesvirus 3 (CyHV-3) was identified by Bretzinger, Fischer-Scherl, Oumouma, Hoffmann, and Truyen (1999) and Hedrick et al. (2000) as the aetiological agent of a viral disease termed KHVD, which can cause mortality as high as 80%–100% in common carp Cyprinus carpio (Reviewed in Gotesman, Kattlun, Bergmann, & El-Matbouli, 2013). CyHV-3 is a double-stranded DNA virus consisting of a 295 kB genome encoding164 putative open reading frames (ORFs), and mass spectrometry analysis of viral particles has identified 40 proteins packaged in a mature virion including 22 structural, 3 capsid, 2 tegument and 13 envelope proteins (Michel, Leroy, et al., 2010). Furthermore, the immunogenic and vaccine potentials of several epitopes of CyHV-3 have been investigated, including Orf12 (Kattlun, Menanteau-Ledouble, & El-Matbouli, 2014) which is readily recognized by the immune system of carp and Orf81 for which conflicting evidence exists (Kattlun et al., 2016; Zhou et al., 2014), although little such research has been conducted in goldfish (Carassius auratus). In previous reports, our group elucidated pathogen–host interactions in CyHV-3-infected C. auratus through the use of monoclonal antibody-linked pulldown assay followed by electro-spray ionization mass spectrometry (ESI-MS) as described in Gotesman, Menanteau-Ledouble, and El-Matbouli (2016). C. auratus is a non-symptomatic carrier of CyHV-3, and previous studies demonstrated that in C. auratus, several host defence proteins interact with CyHV-3 (Bergmann et al., 2010; Gotesman, Abd-Elfattah, Kattlun, Soliman, & El-Matbouli, 2014). Interestingly, several of these proteins were not found to interact in the common carp, the susceptible carp host for CyHV-3 (Gotesman, Soliman, & El-Matbouli, 2013). A recent study by Torrent et al. (2016) has demonstrated that the IgMs of asymptotic CyHV-3 surviving carp recognize an epitope derived from the amino-terminal of the glycoprotein coded by the ORF149 of CyHV-3 (Orf149). Monoclonal antibodies (mAbs) were generated by immunizing mice with purified CyHV-3 particles (Cabon et al., 2017), and these mAbs were used to detect the virus in common carp brain cells by enzyme-linked immunosorbent assay (Bergmann et al., 2017). Pulldown assays use antibodies to capture a “bait” protein in an affinity resin, and in the present study, we applied a pulldown assay to investigate the proteins interacting with the Orf149 epitope: Orf149 mAbs were linked to N-hydroxysuccinimide (NHS)-activated agarose columns (Gotesman et al., 2016) to capture and identify host proteins interacting with CyHV-3. Kidney samples of C. auratus previously infected by intraperitoneal injection with 200 µl of CyHV-3 virus at a concentration of 104 TCID50 (Kattlun et al., 2014, 2016) were lysed using a Tissue Lyser (Qiagen). The sample were resuspended in non-denaturing buffer (Gotesman et al., 2014; Gotesman, Soliman, et al., 2013) and passed through the columns by gravity filtration to expose the extracted host proteins to the agarose-linked mAbs. The columns were rinsed 8 times with phosphate-buffered saline (PBS) to ensure that unbound extracts were washed away as measured by spectrophotometry280 (OD = 0) and the bound proteins were eluted from the column using glycine (pH 3) into microcentrifuge tubes containing neutralizing Tris base (pH 8). The entire eluted fraction was analysed by liquid chromatography tandem mass spectrometry LC-MS/MS analysis (performed at the VetMedUni VetCore facilities) to elucidate host proteins that putatively interact with Orf149. The majority of the proteins identified (Table 1A) were identical to proteins previously identified in C. carpio (Gotesman, Soliman, et al., 2013) as well as using a different epitope of CyHV3 species (Gotesman et al., 2014), including cytoskeletal, elongation factors and enzymatic proteins (Table 1A). The cytoskeletal protein actin (Sandquist, Kita, & Bement, 2011), which was detected in both CyHV-3-positive and CyHV-3-negative samples, serves as a track for both conventional and unconventional myosins (Moen, Johnsrud, Thomas, & Titus, 2011) and plays a role in intracellular translocation and cell remodelling (Gotesman, Hosein, & Gavin, 2010, 2011). Another protein identified was the eukaryotic elongation factor 1 alpha (eEF1A) which has a diverse set of functions in the cell including interactions with the cytoskeleton (reviewed in Sasikumar, Perez, & Kinzy, 2012). Interestingly, certain RNA viruses interact with eF1A directly to aid in viral replication (Sasikumar et al., 2012) and this could explain the recovery of cytoskeletal protein actin by the pulldown assay. Because eEF1A’s activity is hijacked for viral propagation, it is plausible that an antibody targeting the glycoprotein Orf149 that interacts with the cell membrane of the host protein would also detect this cytoskeletal protein. This explanation is further supported by the fact that eEF1A was also pulled down by this assay. Myeloid protein 1 is the final member of previously identified proteins. It is no surprise that a haemoglobin protein was recovered in the CyHV-3-positive samples (Table 1A) because CyHV-3 is detectable in various regions of the circulatory system (Reviewed by Michel, Fournier, Lieffrig, Costes, & Vanderplasschen, 2010) including the hematopoietic tissue in the spleen (Lee et al., 2016). The results from this trial also suggested that the mAb was able to capture the same metalloendopeptidase (metalloendopeptidase 042326) in both the infected and non-infected samples (Table 1B). More importantly, a unique, mitochondrial cytochrome c protein (mitochondrial cytochrome C X4Z1X5) was also detected in the CyHV-3-positive samples. Intriguingly, this protein had not been previously implicated in CyHV-3 infections (Table 1C). In our previous study, we identified interactions of CyHV-3 with mitochondrial enzymes involved in ATP synthesis (Gotesman et al., 2014; Gotesman, Soliman, et al., 2013), and in this study, another mitochondrial protein was shown to interact with CyHV-3. Cytochrome c-like metalloendopeptidase is known to coordinate with metal ions for correct functions and has implications in immune function and disease (Bond & Jiang, 1997), and it is interesting to speculate what role the mitochondria plays in CyHV-3 infection. Whether CyHV-3 alters the activity of the mitochondrial machinery to produce higher amounts of ATP (Murata et al., 2000) or modulates apoptosis factors (Cotter & Blaho, 2009) to either increase viral replication (Aubert, Pomeranz, & Blaho, 2007; Zhou & Roizman, 2000) or the release of mature viruses via apoptosis (Zhang, Tang, & Xu, 2014), respectively, remains unclear. Interestingly, some viruses such as spring viremia of carp virus (SVCV) are known to modulate ROS (reactive oxidative species) production (Liu et al., 2017; Shao et al., 2016). Antimycin A (a small molecule inhibitor of cellular respiration) is known to inhibit the mitochondrial complex III, reducing ROS production in SVCV-infected cells and inhibiting the transcription of SVCV glycoprotein and viral replication (Zhao et al., 2018). Alternatively, CyHV-3 may curtail the production or release of ROS by the mitochondria to reduce the cell's natural viral defence mechanism (Gonzalez-Dosal, Horan, & Paludan, 2012; Gonzalez-Dosal et al., 2011). Such viral strategies have been previously reported, for example, among the important viral diseases that affect domesticated poultry, the fusogenically activated F and HN glycoproteins of Newcastle disease perturb mitochondrial fusion/fission haemostasis (Ren et al., 2019). The fact that CyHV-3 can putatively interact with this aforementioned enzyme and other mitochondrial components raises interesting questions regarding how this virus modulates the natural host immune response and mitochondria for increased viability. Pulldown assays have demonstrated good specificity in the past, and, because of the extensive cleaning steps, it is unlikely that our assay would have detected proteins that did not interact with the Orf149 protein. Indeed, using a different bait protein resulted in a different set of purified proteins. This confirmed that interactions between bait and prey proteins were critical in the purification process. In future research, it would be interesting to further investigate the interactions of CyHV-3 with host proteins in different species, for example using other immunoprecipitation methods such as co-immunoprecipitation. The aquamedicine field is rapidly adapting unconventional approaches for the detection, characterization and treatment of emergent threats to marine and aquaculture industries (Reviewed by Gotesman, Menanteau-Ledouble, Saleh, Bergmann, & El-Matbouli, 2018). Interactions with host cells are one of the most critical aspects of viral infections; therefore, such studies can greatly improve our understanding of the disease. Moreover, such studies could potentially suggest new therapeutic possibilities. Antibodies were obtained from the Freidrich Loefler Institute. This Research was partially funded by a 2018 PSC-CUNY Adjunct/CET Professional Development Fund Award granted to MG. This research was also supported by the Austrian Science Funds (Fonds zur Förderung der wissenschaftlichen Forschung), project P28837-B22. The funding body did not contribute to the study's design or analysis of the data. None. The data sets used and/or analysed during the current study are available from the corresponding author on reasonable request.

BACKGROUND:Marine and aquaculture industries are important sectors of the food production and global trade. Unfortunately, the fish food industry is challenged with a plethora of infectious pathogens. The freshwater and marine fish communities are rapidly incorporating novel and most up to date techniques for detection, characterization and treatment strategies. Rapid detection of infectious diseases is important in preventing large disease outbreaks.MAIN TEXT:One hundred forty-six articles including reviews papers were analyzed and their conclusions evaluated in the present paper. This allowed us to describe the most recent development research regarding the control of diseases in the aquatic environment as well as promising avenues that may result in beneficial developments. For the characterization of diseases, traditional sequencing and histological based methods have been augmented with transcriptional and proteomic studies. Recent studies have demonstrated that transcriptional based approaches using qPCR are often synergistic to expression based studies that rely on proteomic-based techniques to better understand pathogen-host interactions. Preventative therapies that rely on prophylactics such as vaccination with protein antigens or attenuated viruses are not always feasible and therefore, the development of therapies based on small nucleotide based medicine is on the horizon. Of those, RNAi or CRISPR/Cas- based therapies show great promise in combating various types of diseases caused by viral and parasitic agents that effect aquatic and fish medicine.CONCLUSIONS:In our modern times, when the marine industry has become so vital for feed and economic stability, even the most extreme alternative treatment strategies such as the use of small molecules or even the use of disease to control invasive species populations should be considered.

Megalocytiviruses, such as infectious spleen and kidney necrosis virus (ISKNV), induce lethal systemic diseases in both ornamental and food fish species. In this study, we investigated an epizootic affecting Nile tilapia Oreochromis niloticus cultured in the US Midwest. Diseased fish displayed lethargy, gill pallor, and distension of the coelomic cavity due to ascites. Histopathological examination revealed a severe systemic abundance of intravascular megalocytes that were especially prominent in the gills, kidney, spleen, liver, and intestinal submucosa. Transmission electron microscopic examination revealed abundant intracytoplasmic polygonal virions consistent with iridovirus infection. Comparison of the full-length major capsid protein nucleotide sequences from a recent outbreak with a remarkably similar case that occurred at the same facility many years earlier revealed that both epizootics were caused by ISKNV. A comparison of this case with previous reports suggests that ISKNV may represent a greater threat to tilapia aquaculture than previously realized.

In this chapter, we describe laboratory protocols for rearing fish and a simple and efficient method of extracting and identifying pathogen and host proteins that may be involved in entry and replication of commercially important fish viruses. We have used the common carp (Cyprinus carpio L.) and goldfish (Cyprinus auratus) as a model system for studies of proteins involved in viral entry and replication. The chapter describes detailed protocols for maintenance of carp, cell culture, antibody purification of proteins, and use of electrospray-ionization mass spectrometry analysis to screen and identify cytoskeleton and other proteins that may be involved in viral infection and propagation in fish.

The bdelloid rotifer Brachionus plicatilis Muller is an aquatic invertebrate that predominately populates freshwater lakes and ponds. It is a pseudocoelomate which consists of approximately 1000 cells. B. plicatilis exhibits both asexual reproduction in which parthenogenetically derived eggs quickly mature into new progeny (~12 hours) or sexual reproduction in which fertilized eggs diapause for 2-3 months before reinitiating progression into organismic development based on environmental cues, such as water conditions and sunlight. Because B. plicatilis shares many of the conserved genes responsible for dormancy survival such as genes that code for late embryogenesis abundant (LEA), trehalose metabolism and small heat shock proteins (sHSP), it has become an excellent model organism for studies investigating dormancy and developmental biology. Several recent reports have developed procedures in quiescent B. plicatilis resting eggs for molecular manipulation of gene transcription, such as RNAi. However, the dechorionation procedure employed for those previous studies was not effective in our hands to penetrate dessicated resting eggs for nuclei staining. Therefore, we developed a procedure described in this manuscript for decapsulating encysted resting eggs for staining with Hoechst dye. This method may ultimately be used to track phenotypic aspects of entry into and exit from dormancy by determining whether there is a correlation in nuclei number and developmental progress in the Monogonont Rotifer, Brachionus plicatilis Muller.

Cyprinid herpesvirus 3 (CyHV-3) is the causative agent of koi herpesvirus diseases (KHVD), a notifiable condition associated with mass mortalities in koi and common carp (Cyprinus carpio L.). Open reading frame 81 (ORF 81) encodes a well characterized nonglycosylated membrane protein of CyHV-3 that is thought to be highly immunogenic due to its C-terminal hydrophilic domain. To test its immunogenic potential, ORF 81 was cloned into a DNA vaccine vector and administered to common carp by intramuscular injection. To assess the protective immunity of the vaccinated carps, antibody titres against CyHV-3 following immunization were measured by ELISA and fish were submitted to a viral challenge. Additionally, a Western Blot was carried out using purified recombinant pORF81 and sera from vaccinated, infected and uninfected fish. Although the gene was found to be reliably transcribed in vitro and in vivo, no antibody response could be detected in vivo following vaccination using the existing method. These results call into question the immunogenicity of the protein derived from ORF 81 of CyHV-3 or suggest that this protein only induces antibody titres that remain below the limit of detection.

This chapter describes protocols for using a handheld gene gun to deliver transformation vectors for overexpression of genes or gene replacement in the macronucleus of Tetrahymena thermophila. The protocols provide helpful information for preparing Tetrahymena for biolistic bombardment, preparation of vector-coated microcarriers, and basic gene gun operating procedures.

Tetracapsuloides bryosalmonae (Myxozoa) is the causative agent of proliferative kidney disease in various species of salmonids which are found in Europe and North America. Less information about the interactions of T. bryosalmonae proteins with salmonid proteins during parasite development is known. In this study, anti-T. bryosalmonae monoclonal antibody-linked to N-hydroxysuccinimide-activated spin columns were used to purify parasite and host proteins from the kidneys of infected and non-infected brown trout (Salmo trutta) Linnaeus, 1758. The samples were next analyzed by electrospray ionization coupled to mass spectrometry to identify proteins that may be involved in the infection and proliferation of T. bryosalmonae within the brown trout host. A total of 6 parasite proteins and 40 different host proteins were identified in this analysis. The identified host proteins function in various processes, which include host defense, enzymatic, and structural components. In conjunction with modern molecular based tools, such siRNA, gene replacement, or gene disruption, this data can ultimately be used to develop novel control methods for T. bryosalmonae, based on the proteins or pathways identified in this study.

Ranaviruses are globally distributed pathogens in amphibian, fish, and reptile communities that appear to be emerging. Cases of ranavirus infection or disease have been confirmed in at least 105 amphibian species (18 families), 41 fish species (22 families), and 29 reptile species (12 families). Ranaviruses have been documented on all continents except Antarctica, and are frequently associated with mass die-offs. Host susceptibility differs among species, with some species harboring subclinical infections and likely serving as reservoirs for the virus, and other highly susceptible species amplifying the virus. Currently, there are six recognized species of ranavirus, and all are not equally pathogenic among hosts. Frog virus 3 (FV3) is the type species of the genus Ranavirus, and appears to be the most globally distributed species infecting ectothermic taxonomic across three vertebrate classes. International commerce involving subclinically infected ectothermic vertebrates undoubtedly has contributed to the global distribution and emergence of ranaviruses. Herein, we describe the global distributed species infecting ectothermic vertebrates across three taxonomic classes.

The genus Dermocystidium comprises fungal pathogens of the order Dermocystida in the class Ichthyosporea (http://www.ncbi.nlm.nih.gov/taxonomy). Several different Dermocystidium species have been described, infecting a variety of fish hosts and producing gill infections, skin lesions and visceral disease all over the world (Wildgoose 1995; Pekkarinen & Lotman 2003; Pekkarinen et al. 2003; Feist et al. 2004; Zhang & Wang 2005). To our knowledge, there are two reports on Dermocystidium infections in cardinal tetra, Paracheirodon axelrodi and neon tetra, Paracheirodon innesi (Reichenbach-Klinke 1982; Lewisch 2010). However, these reports did neither include histological and ultrastructural examinations nor molecular genetic investigations to confirm the diagnosis and identify the aetiologic species. In January 2013, increased mortalities of cardinal tetra, P. axelrodi of a 350-L aquarium were reported, occurring after purchase of additional fish. During the clinical examination, most of the fish were swimming in normal active condition, but some were lethargic or displayed a transparent mass on the skin of the head or body. The masses were up to 5 mm in diameter and contained a central, white tubular structure. Similar masses were located on the fins of some fish, but were considerably smaller in these locations. A parasitic infestation of the skin of these fish was excluded via microscopic examination, and normal results were obtained after analysis of the water values and inspection of the pump and filter system. Six cardinal tetra, P. axelrodi and two firehead tetra, Hemigrammus bleheri were submitted for pathological examination in formalin and for molecular genetic examination in ethanol. Only two cardinal tetra displayed skin lesions. Before embedding, one tetra was post-fixed in Davidson’s fixative and sections of glycolmethacrylate/methylmethacrylate-embedded samples were routinely processed for histological examination and stained with haematoxylin and eosin (HE), Giemsa, silver impregnation and periodic acid Schiff (PAS) reaction according to standard protocols. Samples of the mass were also routinely processed for transmission and scanning electron microscopy on a transmission electron microscope (Zeiss EM 10) or on a digital scanning electron microscope (Zeiss DSM 950), respectively. The macroscopic examination of the cardinal tetra revealed a focally extensive, bulging, hemispherical, transparent oedema (4 mm in diameter) of the ventral skin on the head. Central, within Correspondence M C Langenmayer, Institute of Veterinary Pathology at the Centre for Clinical Veterinary Medicine, LMU Munich, Veterin€ arstrasse 13, 80539 Munich, Germany (e-mail: langenmayer@patho.vetmed.uni-muenchen.de) Langenmayer and Lewisch contributed equally to this work.

Cyprinid herpesvirus-3 (CyHV-3) is an etiological agent of a notifiable disease that causes high mortality rates affecting both the common and koi carp Cyprinus carpio L. There is no current treatment strategy to save CyHV-3 infected fish. RNA mediated interference (RNAi) is an emerging strategy used for understanding gene function and is a promising method in developing novel therapeutics and antiviral medications. For this study, the possibility of activating the RNAi pathway by the use of small interfering (si)RNAs was tested to inhibit in vitro viral replication of CyHV-3 in common carp brain (CCB) cells. The siRNAs were designed to target either thymidine kinase (TK) or DNA polymerase (DP) genes, which both code for transcripts involved in DNA replication. The inhibition of viral replication caused by the siRNAs was measured by a reporter gene, termed ORF81. Treatment with siRNA targeting either TK or DP genes reduced the release of viral particles from infected CCB cells. However, siRNA targeting DP was most effective at reducing viral release as measured by qPCR.

Spring viraemia of carp virus (SVCV) is an aetiological agent of a serious disease affecting carp farms in Europe and is a member of the Rhabdoviridae family of viruses. The genome of SVCV codes for five proteins: nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G) and RNA-dependent RNA polymerase (L). RNA-mediated interference (RNAi) by small interfering RNAs (siRNAs) is a powerful tool to inhibit gene transcription and is used to study genes important for viral replication. In previous studies regarding another member of Rhabdoviridae, siRNA inhibition of the rabies virus nucleoprotein gene provided in vitro and in vivo protection against rabies. In this study, synthetic siRNA molecules were designed to target SVCV-N and SVCV-P transcripts to inhibit SVCV replication and were tested in an epithelioma papulosum cyprini (EPC) cell line. Inhibition of gene transcription was measured by real-time quantitative reverse-transcription PCR (RT-qPCR). The efficacy of using siRNA for inhibition of viral replication was analysed by RT-qPCR measurement of a reporter gene (glycoprotein) expression and by virus endpoint titration. Inhibition of nucleoprotein and phosphoprotein gene expression by siRNA reduced SVCV replication. However, use of tandem siRNAs that target phosphoprotein and nucleoprotein worked best at reducing SVCV replication.

Keywords: antibody, electrospray ionization mass spec-trometry, koi herpesvirus disease, protein purification.Cyprinid herpesvirus-3 (CyHV-3) is the aetiologi-cal agent of a serious and deadly disease, termed‘koi herpesvirus disease’ (KHVD), that causes highmortality in koi and common carp Cyprinus carpioL. (Pikarsky et al. 2004; Ilouze et al. 2008). Sincethe initial identification of CyHV-3 in Europe(Bretzinger et al. 1999), the USA and Israel (Hed-rick et al. 2000), and Japan (Sano et al. 2004),there is a great effort to limit the spread ofCyHV-3. Recently, the focus of prevention hasshifted to understanding how the virus propagatesand what carriers exist for the virus. Environmen-tal factors such as mating (Uchii et al. 2011),feeding activities (Kielpinski et al. 2010; Minam-oto et al. 2011; Fournier et al. 2012) and proxim-ity in aquaculture (Dishon et al. 2005) arecontributing factors in the spread of CyHV-3.Earlier studies suggested that immunization withattenuated virus may prevent the spread ofCyHV-3 (Ronen et al. 2003; Perelberg et al.2005), but current studies suggest that CyHV-3spread is due to subclinical carriers (Bergmann K Eide et al. 2011), which can infectna€ive fish (St-Hilaire et al. 2005). Recent studieshave shown that CyHV-3 DNA exists indifferent species of healthy looking Cyprinid fishthat include grass carp Ctenopharyngodon idella,goldfish Carassius auratus and blue back ideLeuciscus idus, as well as in non-Cyprinid fish thatinclude Russian sturgeons Acipenser gueldenstaedtii,Atlantic sturgeons A. oxyrinchus and Ancistrus sp.,suggesting that these fish may be potential carriersfor the CyHV-3 (Bergmann et al. 2009; Kempteret al. 2009). In addition, cohabitation studies haveshown that CyHV-3 can be transferred to goldfishfrom infected koi fish (El-Matbouli, Saleh & Soli-man 2007), but goldfish do not show clinicalsigns of the disease (Bergmann et al. 2010a).The genome of CyHV-3 consists of 295 kB(Aoki et al. 2007) that codes for 156 unique openreading frames (ORFs) that are all transcribed intomRNA transcripts (Ilouze, Dishon & Kotler2012). Mass spectrometry (MS) identified 40CyHV-3 proteins incorporated into mature virions(Michel et al. 2010). Some of the CyHV-3 pro-teins have been further characterized, such asORF81, a component of the viral envelope(Rosenkranz et al. 2008) and genes involved inDNA synthesis (Ilouze et al. 2006; Fuchs et al.2011). Recently, Sunarto et al. (2012) showedthat CyHV-3 transcribes a potential immune sup-pression gene, ORF134, a homolog of interleu-kin-10 (IL-10). CyHV-3 has also been shown tomodulate the innate immune response pathway invitro (Adamek et al. 2012) and in vivo (Adameket al. 2013). Gene expression analysis usingmicroarray chips of two common carp lines thatdiffer in mortality rates during CyHV-3 infectionidentified 581 genes in line ‘R3’ and 107 genes inline ‘K’ that are differentially transcribed duringCyHV-3 infection (Rakus et al. 2012). The eluci-dation of interaction partners is often used to

Common carp (including ornamental koi carp) Cyprinus carpio L. are ecologically and economically important freshwater fish in Europe and Asia. C. carpio have recently been endangered by a third cyprinid herpesvirus, known as cyprinid herpesvirus-3 (CyHV-3), the etiological agent of koi herpesvirus disease (KHVD), which causes significant morbidity and mortality in koi and common carp. Clinical and pathological signs include epidermal abrasions, excess mucus production, necrosis of gill and internal organs, and lethargy. KHVD has decimated major carp populations in Israel, Indonesia, Taiwan, Japan, Germany, Canada, and the USA, and has been listed as a notifiable disease in Germany since 2005, and by the World Organisation for Animal Health since 2007. KHVD is exacerbated in aquaculture because of the relatively high host stocking density, and CyHV-3 may be concentrated by filter-feeding aquatic organisms. CyHV-3 is taxonomically grouped within the family Alloherpesviridae, can be propagated in a number of cell lines, and is active at a temperature range of 15 to 28°C. Three isolates originating from Japan (KHV-J), USA (KHV-U), and Israel (KHV-I) have been sequenced. CyHV-3 has a 295 kb genome with 156 unique open reading frames and replicates in the cell nucleus, and mature viral particles are 170 to 200 nm in diameter. CyHV-3 can be detected by multiple PCR-based methods and by enzyme-linked immunosorbent assay. Several modes of immunization have been developed for KHVD; however, fish immunized with either vaccine or wild-type virus may become carriers for CyHV-3. There is no current treatment for KHVD.

Cyprinid herpesvirus 3 (CyHV-3) is the aetiological agent of a serious and notifiable disease afflicting common and koi carp, Cyprinus carpio L., termed koi herpesvirus disease (KHVD). Significant progress has been achieved in the last 15 years, since the initial reports surfaced from Germany, USA and Israel of the CyHV-3 virus, in terms of pathology and detection. However, relatively few studies have been carried out in understanding viral replication and propagation. Antibody-based affinity has been used for detection of CyHV-3 in enzyme-linked immunosorbent assay and PCR-based techniques, and immunohistological assays have been used to describe a CyHV-3 membrane protein, termed ORF81. In this study, monoclonal antibodies linked to N-hydroxysuccinimide (NHS)-activated spin columns were used to purify CyHV-3 and host proteins from tissue samples originating in either CyHV-3 symptomatic or asymptomatic fish. The samples were next analysed either by polyacrylamide gel electrophoresis (PAGE) and subsequently by electrospray ionization coupled to mass spectrometry (ESI-MS) or by ESI-MS analysis directly after purification. A total of 78 host proteins and five CyHV-3 proteins were identified in the two analyses. These data can be used to develop novel control methods for CyHV-3, based on pathways or proteins identified in this study.

Karpfen und Koi (Cyprinus carpio) werden weltweit intensiv gezuchtet, einerseits als Nutzfische und andererseits als Zierfische fur einen wachsenden Liebhaberkreis. Seit den spaten 90er Jahren des letzten Jahrhunderts wird diese Spezies von einem neuartigen Virus bedroht, welches mittlerweile als Herpesvirus klassifiziert wurde und jahrlich massive Verluste in beiden Wirtschaftszweigen verursacht: Das Cyprinide Herpesvirus 3 (CyHV-3), auch bezeichnet als Koi Herpesvirus (KHV). Dieser Artikel gibt eine Ubersicht uber Wissen und Wissenslucken die Erkrankung und den Erreger selbst betreffend und beschreibt, welche Umstande dazu fuhren, dass auch 14 Jahre nach seiner Entdeckung CyHV-3 noch immer ein schwerwiegendes Problem in der Karpfen- Aquakultur und in Koihaltungen darstellt.

Infection of carp (Cyprinus carpio) with Cyprinid Herpesvirus 3 (CyHV-3) - an update Common carp and koi (Cyprinus carpio) are bred intensively worldwide, both as farm animals and as pet fish with an ever growing circle of enthusiasts. Since the late 90s of the last century, the species has been threatened by a novel virus, recently classified as a herpesvirus. The virus, now known as Cyprinid Herpesvirus 3 (CyHV-3) or Koi Herpesvirus (KHV), causes massive losses in both industrial branches each year. This review aims to provide a general overview of current knowledge concerning the disease and the pathogen, as well as describing why, 14 years since its discovery, CyHV-3 still poses a serious problem in both carp and koi aquacultures.