workshop novel viral in ections threatening cyprinid sh · pdf filenovel viral in ections...

Bull. Eur. Ass. Fish Pathol., 36(1) 2016, 11

* Corresponding author and workshop leading organizer’s email: [email protected]

WORKSHOP

Novel viral in ections threatening Cyprinid sh

O. Haenen1*, K. Way2*, B. Gorgoglione3, T. Ito4, R. Paley2, L. Bigarré5 and T. Wal ek6*

1 Central Veterinary Institute of Wageningen UR, The Netherlands; 2 Centre for Environment, Fisheries and Aquaculture Science (CEFAS), Weymouth DT4 8UB, England; 3 Clinical Division of Fish Medicine,

4 Tamaki Laboratory, Aquatic Animal Health Division, National Research Institute of Aquaculture (NRIA), Fisheries Research

5 6 College of Veterinary Medicine, Department

* Workshop co-organizer

IntroductionIn 2007 koi herpesvirus disease (KHVD), caused by the alloherpesvirus Cyprinid herpesvirus 3 (CyHV-3), was listed by the World Organiza-tion or Animal Health (OIE) and was quickly ollowed by listing as a non-exotic disease in the

European Union (EU), related to the Directive 2006/88/EC (EC, 2006). Up until the listing o KHVD, Spring Viraemia o Carp (SVC) was the only viral disease o cyprinids listed by the OIE (OIE, 2012).

Apart rom CyHV-3 and SVCV, in the last ve years, several novel non-noti able cyprinid viruses have been detected in Europe and their relation to clinical disease and mortality out-breaks is a cause or concern.

To gain a comprehensive overview o current potential new viral threats or cyprinid sh, and to provide an update on their characterization,

an open workshop was organized at the 17th EAFP International Con erence on Diseases o Fish and Shell sh. Seven short lectures were ol-lowed by a discussion, involving an audience o approximately 60 international experts. New eld observations and preliminary research on novel cyprinid viral in ections were presented, aiming to illustrate their impact in various geographic regions. The main topics presented included issues related to the current emergence and detec-tion o Cyprinid iridoviruses, Carp Edema Virus (CEV), Cyprinid Herpesvirus 2 (CyHV-2), and a novel myxo-like virus. Additionally, the promo-tion o a collaborative approach to be undertaken or the genetic characterization o novel cyprinid

viruses was highlighted. The general aim o the workshop was to identi y potential collaborative approaches to carry out multidisciplinary studies aiming to de ne risks, diagnostic methods and suggest adequate prophylactic measures.

12, Bull. Eur. Ass. Fish Pathol., 36(1) 2016

Overview of Carp Edema Virus in EuropeK Way and DM Stone

Carp Edema Virus (CEV) is the etiological agent o Koi Sleepy Disease (KSD), originally de-scribed in Japan in the 1970’s as a viral edema o juvenile color Cyprinus carpio koi and later morphologically identi ed as a poxvirus. The virus causes severe damage to gill lamellae, leading to hypoxia and lethargy, which man-i ests as sleepy behavior, and mortality can reach 80-100% (Ono et al., 1986; Lewisch and Gorgoglione et al., 2015).

The disease is known to be widespread across Japan where koi are cultured and, as a conse-quence, the disease has been managed rather than a empting to eradicate the virus in koi populations. International trade in koi has un-doubtedly led to global spread o CEV/KSD but the occurrence o disease outbreaks has o ten not been recognized and consequently, rarely reported. In the USA, CEV has been detected in association with disease outbreaks in koi at import sites and in hobby ponds since 1996 (Hesami et al., 2015). In Europe, outbreaks o KSD and PCR detections o CEV-like virus were reported rom 2009. At a CEV workshop in Copenhagen in January 2015 (meeting report available rom h p://www.eurl- sh.eu/reports), delegates rom Austria, Czech Republic, France, Germany, Italy, The Netherlands and the UK reported multiple KSD outbreaks in imported koi and also outbreaks in common carp. Follow-ing the workshop in ormation was received on KSD/CEV cases in Poland. A CEV alert paper giving the emergence o CEV in Europe and its signi cance to European aquaculture is cur-rently in preparation (Keith Way, pers.comm.).

A eature o CEV/KSD outbreaks in common carp in Europe, and particularly in the UK, is that many have occurred during periods o low water temperatures (6-9°C) in winter and early spring. Outbreaks o KSD in koi ponds are generally seen at higher temperatures (15-25°C) in late spring or early summer. In the UK a link with Spring Carp Mortality Syndrome (SCMS) was suspected (Way and Stone, 2013). When archived DNA and preserved gill tissue samples, including para n-embedded ormalin

xed gill tissue, were analyzed by PCR, CEV was detected in samples rom SCMS cases rom 2004 and 1998/99.

CEV/KSD is the latest example o a virus disease o cyprinids that has emerged, largely unno-ticed, in Europe and where the true extent o the spread o the virus is not known. The cir-cumstances o the emergence and spread o CEV/KSD are similar to those o KHVD almost 20 years ago.

Possible reasons or why this virus has only rarely been identi ed in carp mortality inves-tigations include: 1) the virus is, at present, non-culturable and not isolated during routine screening; 2) the disease may not always cause high mortalities (extensive outbreaks) in common carp, and 3) CEV/KSD is possibly misdiagnosed as KHVD without con rmation o the true cause. Also, until recently, ew ade-quate diagnostic tools have been available. In the USA in 1996 the virus was identi ed in koi by transmission electron microscopy (TEM) (Hedrick et al., 1997).

The knowledge gaps that, it is suggested, need to be addressed to assess the true impact o the disease on European carp populations include:


1) the mechanisms o virus survival outside the host ; 2) are there susceptible aquatic species other than carp (carriers) ; 3) does the virus exist as a low-level persistent in ection and are there aquatic vectors ; 4) what is the prevalence in carp populations ; 5) what are the important environmental actors responsible or triggering CEV/KSD outbreaks

More tools are needed to provide early warning o emerging pathogenic viruses o aquaculture and resolve aquatic/ sh diseases o unknown aetiology. Possible improvements include: 1) a be er characterization o known viruses and more use o next generation sequencing, partic-ularly to allow e ective design o generic PCR primers that detect a range o DNA viruses; 2) development o qPCR assays or sample screening; 3) be er mechanisms in Europe (with support rom the EU) or collaboration and networking to tackle serious or potentially serious epizootics.

Preliminary investigations on transmission of and susceptibility to Carp Edema Virus CEV / Koi Sleepy Disease KSDB Gorgoglione, E Lewisch, O Schachner and M El-Matbouli

Koi carp are traded worldwide as valuable or-namental sh, particularly appreciated as pets in European garden ponds, which are highly popular in Austria. Common carp Cyprinus carpio is o ten armed in poly-cultural systems, representing the main reshwater sh produc-tion in the world, especially relevant in Asian and Central-East European countries (FAO, 2014). The spreading o CEV/KSD may have

negative trade e ects, threatening the high-val-ued ornamental sh industry and carp arming.

In Austria, the occurrence o KSD was demon-strated or the rst time during the spring o 2014, rom two unrelated cases rom common carp and koi carp (Lewisch and Gorgoglione et al., 2015). In 2015, the rst Austrian case arrived in late February rom a spontaneous KSD out-break a ecting koi rom a large garden pond (> 250 m3), which allowed preliminary trans-mission and susceptibility investigations to be carried out. A marked di erential susceptibility was seen between various individuals rom the same pond, providing evidence o clinically asymptomatic carriers. An adult koi (20 cm total body length) was hospitalized at 17 °C showing a severe in ection with the ectopar-asites Trichodina sp. and Gyrodactylus sp., and multiple skin in ections with opportunistic bac-teria (eg Shewanella putrefaciens). Surprisingly, 6 hours cohabitation was enough to transmit the CEV in ection to other, na ve koi and SPF common carp, showing typical clinical pa erns and mortality by day 7 post-in ection (d.p.i.), (Figure 1). CEV viremia was already visible and detectable at 1 d.p.i., associated with a sleepy behavior, and possibly showing highest DNA levels at 7 d.p.i. (see the thicker band at the gel electrophoresis: Figure 2).

CEV was detected by PCR (using the nested PCR protocol rom CEFAS, Stone Way, pers.comm. 2013), and was only detectable in DNA rom speci cally skin mucus, gills laments

and blood. There ore, we suggest these tissues are the best to be sampled or KSD diagnostics. DNA rom internal organs (including rom parts o the kidney (pronephros and mesonephros), spleen and swim bladder) gave aint positivity,


Figure 1. KSD a ected common carp, in ected with CEV. The carp died at 7 d.p.i. ollowing cohabitation with a CEV-in ected koi carp at 17°C. Clinical changes included emaciation, sunken eyes, mucus aggregations on the body and on the pale gills (le t operculum removed).

Figure 2. CEV detection in DNA samples rom a common carp (Figure 1), which died at 7 d.p.i. ollowing cohabitation with a KSD a ected koi carp. Heparinised blood was sampled during sh hospitalization, while urther tissues were aseptically sampled at necropsy. Gel electrophoresis o amplicons ollowing the second step o a nested PCR (using CEFAS, Stone Way 2013 nested PCR protocol), with 478 bp band indicating positivity.


usually at the second step o the nested PCR, and exclusively rom sh in terminal condition (Figure 2). CEV was also detected by PCR rom mucus o asymptomatic koi up to 4 months ol-lowing co-habitation. KHV detection by PCR (Bercovier et al., 2005) gave negative results.Even a ter e ective anti-ectoparasitic treatments, using FMC (a mixture o 1 L ormalin, 3.5 g mala-chite green and 3.5 g methylene blue; used at the dosage o 1 mL in 100 L) against a severe Ich-

in ection, the skin mucus stayed CEV positive, both tested straight a ter the treatment, and in the longer term (4 months p.i.). Virus cultivation was also a empted by inoculating tissue homogenates (sampled and pooled according to KHV sampling procedures (OIE, 2012)) on Common Carp Brain (CCB) cells. No cytopathic e ect was evident a ter several passages o 7 days each, respectively at 15°C and 20°C, similarly to previous negative results achieved with EPC, BF-2, RTG-2, and CHSE cells (Lewisch and Gorgoglione et al., 2015; Schachner, personal communication).

Our preliminary observations rom clinical cases, although still to be con rmed, provid-ed some intriguing clues about virus detec-tion, transmission and host tissue tropism. The current main aim is to achieve soon the in vitro virus cultivation and isolation. The ability to culture the virus in vitro will allow proper virus characterization, strain di erentiation and in ection challenges to assess the CEV-induced immune response in susceptible and resistant hosts. KSD prevalence is indeed clearly under-estimated; above all when considering the low pathogenicity or adult carp and co-in ections with other pathogens. Thus, assessing the role o asymptomatic carriers should be addressed in uture research e orts.

DiscussionT. Vesel was surprised that already a ter 6 h p.i. clinical signs were observed. N. Adamek and M. Reichert asked about the di erence between samples labelled as mucus and gills in the gel images shown, because gills are also covered by mucus. B. Gorgoglione clari ed to be re erring to mucus when taken rom the skin o the body side, and gills or gill

laments biopsied or sampled at the necropsy. N. Adamek also asked whether KSD should be considered a primary or a background disease B. Gorgoglione replied that rom what is cur-

rently known it is more likely to be a strong background disease, at least or adult sh. E. Lewisch reported about the clinical observa-tion o an in ected garden pond in which a documented outbreak o CEV occurred in koi carp more than one year ago, with some mortal-ity. Fish showed typical clinical signs o KSD and were con rmed CEV positive (KHV and SVCV negative with no other apparent causes o death). Some sh recovered rom the in ec-tion, while some others did not show any sign o disease. A restocking was operated one year later resulting in the death o exclusively all newly stocked sh, and all these sh were con-

rmed as CEV positive (E. Lewisch, personal communication).

Properties of and preventative measures for HVHN in gold sh caused by Cyprinid herpesvirus 2 (CyHV-2)T Ito

Cyprinid herpesvirus 2 (CyHV-2) is the causative agent o herpesviral haematopoietic necrosis (HVHN) in gold sh (Carassius auratus auratus)


and the occurrence o the virus has been re-ported rom several countries (Chang et al., 1999; Dan k et al., 2012; Goodwin et al., 2006; Gro et al., 1998; Je ery et al., 2007; Stephens et al., 2004; Wang et al., 2012). Due to the great di -erence in economic value o gold sh, ranging rom use as live bait sh to high quality orna-

mental sh, it is di cult to ormulate general preventive measures or HVHN. Moreover, recently the virus has also been detected rom Prussian carp (C. gibelio) and crucian carp (C. carassius) rom European and Asian countries. Since the reports suggest that this virus has been spreading, and also CyHV-2 has the potential to in ect other species o the genus Carassius as a host, understanding the properties and the preventative measures o this disease has become important.

Studies on the prevalence o CyHV-2 in organs o gold sh exposed to the virus were described: Kidney is a suitable organ or PCR-based diag-nosis o HVHN in gold sh, since CyHV-2 DNA was detected rom the kidney o all surviving

sh at 31 days post-exposure in experimental in ection (Ito et al., 2013). Results o experimen-tal in ections o indigenous Cyprininae species in Japan were also reported: Susceptibility o indigenous Cyprininae sh species to CyHV-2 is much lower compared to gold sh (Ito and Maeno, 2014a).

Preventative measures against HVHN in gold-sh have also been a empted by control o water

temperature and vaccination. Ryukin gold sh were in ected with CyHV-2 by intraperitoneal injection and maintained at 4 di erent water temperatures. Cumulative mortalities o the 15, 20, 25 and 30°C groups were 10, 90, 90 and 60%, respectively. There ore, the temperature

range o 20 25°C is considered highly permis-sive or HVHN. Moreover most sh in ected with CyHV-2 at 13-15°C acquired resistance to HVHN. This phenomenon might be an ap-plicable preventative measure at low cost. In addition, a simple ormalin-inactivated vaccine was e ective against HVHN o gold sh. This is a use ul preventative measure or high quality ornamental gold sh (Ito and Maeno, 2014b; Ito and Maeno, 2015; Ito and Ototake, 2013).

DiscussionS. Bergmann asked, when a sh is resistant to disease is it also resistant to in ection Answer T. Ito: this was not known, but possibly there is latency.

Cyprinid herpesvirus 2 (CyHV-2) infection in wild gibel carp, Carassius auratus gibelio in the Netherlands O Haenen, M Engelsma and T Ito

Gibel carp, Carassius auratus gibelio is thought to be an ancestral species o gold sh originating rom China, which was introduced into the

Netherlands in the 17th century. In May o 2015, an epizootic with a high mortality occurred in adult wild gibel carp in reshwater lakes in Western-Netherlands. The gibel carp were suspected or in ection with CyHV-2 (Dan k et al., 2012; Wang et al., 2012). Some sh showed a whitish slime layer over their eyes and an erythema o their skin, sometimes with hae-morrhagic scales. The water temperature was 20-25°C during the outbreak. CyHV-2 was de-tected in high amounts (low Ct value) by qPCR (Goodwin et al., 2006).

When testing archived samples o wild gibel


carp rom high mortalities with signs (severe skin and n haemorrhaging) rom May 2011 (2,500 sh dead), and August 2011 (high mor-tality with additionally haemorrhagic eyes, thick slime, loosening skin, and gill in amma-tion with hypertrophy) respectively, rom the same province, these two o three gibel carp groups were also strongly positive by qPCR or CyHV-2. However, a third group with clinical signs, sent in March 2011 was negative in the qPCR or CyHV-2. In 2011, virus isolations on EPC and FHM cells at 15 and 20°C o the three gibel carp groups did not give any cytopathic e ect (CPE) in two passages. In Japan, at the laboratory o T. Ito the our gibel carp organ pools are urther tested, in virus isolation on gold sh cells (Standard Ryukin Taka umi cells (SRTF), and Gold sh Fin cells (GFF) (Ito et al., 2013)), and or molecular characterization, aiming a joint publication.

We report the rst detections since 2011 o CyHV-2 in wild gibel carp in the Netherlands, like in the Czech Republic, which had out-breaks o CyHV-2 disease in gibel carp in Lake

eha ka, close to the Elbe River in May-June o 2011 (Dan k et al., 2012). As gibel carp is a non-native species which seems to be very success ul in our country, the CyHV-2 outbreak is believed to have had a low impact so ar. It is not clear i CyHV-2 is emerging in Europe, as no monitoring has been done. Networking on this new viral cyprinid disease is important, and unding is needed or urther studies.

DiscussionK. Way con rmed that stress is a trigger or CyHV-2 clinical disease. E. Lewisch mentioned, CyHV-2 is also ound in gibel carp in Austria. Similarly T. Wahli and N. Adamek reported

CyHV-2 had also been ound in gold sh rom a closed acility, in Swi erland and in Germany, respectively.

Characterisation of an unknown myxo-like virus from carpR Paley, C Sharp, C Joiner, I Cano-Cejas, R Van Aerle, K Bateman, D Stone and P Dixon

Diagnostic testing o UK and imported cyprin-ids has o ten resulted in the isolation o viruses showing a characteristic syncytial CPE in cell culture that is not caused by the known and expected viruses o carp (22 isolations since 1996), (Figure 3A). For our o these isolates, analysis by electron microscopy showed crystal-line arrays o spherical virions o approximately 90 nm in size in the cytoplasm. A chloro orm test or lipid envelope (absent) and analysis o viral genomic segments by polyacrylamide gel electrophoresis, showing 11 segments, indicated they are aquareoviruses. Subsequent PCR and sequencing showed 94% and 90% similarity to grass carp reovirus RNA genomic segments 2 and 10 respectively.

For the remaining isolates however initial char-acterisation by electron microscopy indicated large (200 nm) pleomorphic, spherical, envel-oped virions that are released rom cells by budding (Figure 3B and 3C). The isolates rep-licate slowly (average o 17 days to develop ull CPE) on carp cell lines producing low titres (3.4 to 5.7 log TCID50/mL). Bromodeoxyuridine and chloro orm treatment indicated RNA genome and presence o a lipid envelope respectively. Together these results indicate a possible myxo-like virus, but the isolates were not able to hae-magglutinate or haemadsorb carp or salmon


erythrocytes. Furthermore, no ampli cation was seen using generic primer sets designed to ampli y orthomyxovirus and paramyxovirus polymerase genes (Tong et al., 2008; W. Ba s, USGS, Western Fisheries Research Centre, per-sonal communication, and this study).

To date, genome segmentation has not been accurately determined due to the inability to obtain su cient puri ed virus or these isolates. Viral nucleic acid has been subjected to Next Generation Sequencing (NGS) by Roche 454 and Illumina HiSeq plat orms, with de novo assem-bly using CLC genomics, Blast (Altschul et al., 1990) comparison against sequence databases and assessment o assignments using Megan (Huson et al., 2007). Despite recovering over 1 million and 10 million sequence reads or isolates in each sequencing plat orm respec-tively no signi cant similarities had yet been identi ed. Isolations rom carp in Germany, o viruses with similar CPE, morphology under electron microscopy and biochemical properties have been previously described (Body et al., 2000; Granzow et al., 2014; Neukirch and Kunz, 2001) and were provisionally ascribed to the amily Paramyxoviridae but without success ul ull characterisation or molecular assignment.

A brie overview o the work undertaken thus ar to characterise our isolations was presented

at the meeting. In conclusion, 4 o the isolates were determined to be grass carp reovirus and a urther 18 isolations were still unknown. These

unknown viruses were deemed either not myxo-like viruses or are very distantly related, or possibly belong to a virus amily with similar morphology e.g. Arenaviridae or Bunyaviridae.

DiscussionAt what water temperature was the myxo-like virus in carp ound/tested R. Paley answered that 20°C is the pre erred growth temperature in vitro. Disease investigations are still under study. T. Vendel Klinge asked whether iso-lates were rom surveillance R. Paley replied samples were rom a mixture o surveillance, import checks and reported disease outbreaks, sometimes but not always associated with pa-thology.

Post meeting note on Paley et al.Recently the HiSeq generated raw sequence reads rom one o the virus isolates were as-sembled again using a lately published new assembly program, IVA (Iterative Virus As-

Figure 3. Typical syncytial cytopathic e ect (A) in EPC cells and enveloped spherical budding virions (B) and capsid ormation (C) or the putative carp paramyxovirus.


sembler), (Hunt et al., 2015). Surprisingly a 16 kb putative viral sequence was generated and subsequently annotated using a selection o bioin ormatics tools including FGENESV0 (h p://linux1.so tberry.com), Prokka (Seemann, 2014) and BLAST. Based on the single segment genome architecture encompassing at least eight putative genes and the average nucleotide iden-tity over putative nucleoprotein, usion and polymerase genes o 21%, the virus was clas-si ed as a putative Paramyxovirus. Failure to group with any o the known paramyxoviruses (including Atlantic Salmon paramyxovirus) in phylogenetic analyses indicates this isolate represents a new species and quite likely a new genus within the amily Paramyxoviridae. Further characterisation is ongoing or this isolate and or the 17 remaining related historical isola-

tions and will be submi ed or publication at a uture date.

VNTR as molecular markers to genotype cyprinivirusesL Bigarré, M Baud, S Labrut, M Jamin and P-M Boitard

The large size o the genome o the Cyprini-viruses is a limiting actor or the rapid geno-typing o eld isolates. The use o speci c and polymorphic molecular markers is there ore o outstanding importance to acilitate molecular epidemiology studies. Mini- and micro-satellites composed o Variable Numbers o Tandem Repeats (VNTR) are use ul targets to rapidly compare isolates rom di erent origins. Tandem repeats are abundant in the known Cyprinivirus genomes. However, their individual mutation rate and mode o evolution (indels combined or not with substitutions) are impossible to predict.

There ore, each candidate VNTR must be evalu-ated by testing samples o various origins, se-quencing the alleles in order to characterize their structure and evaluate its potential as a geographic marker.

For CyHV-3, various markers have been se-lected and success ully used to di erentiate isolates and in er genetic relationships between sequences (Avarre et al., 2012; Sunarto et al., 2010). Un ortunately, tandem repeats do not appear to be conserved between cypriniviruses. There ore, similar, yet distinct, tools are still to be created or other alloherpesviruses, such as CyHV-2, an emerging virus o gold sh (Caras-sius auratus) and Prussian carp (C. gibelio). For this virus, the search or suitable VNTR has been recently initiated. Numerous, at least 280, VNTRs have been ound in this genome, but candidates use ul or genotyping are still to be identi ed. A rst marker di erentiating two isolates rom di erent geographic origins was recently identi ed (Boitard et al., 2015).

The continuous worldwide spread o cyprini-viruses justi es the intensi cation o genetic studies and the use o VNTR to elucidate the geographic movements o these economically important viruses. There is now a need o an open database collating the allelic markers already ound, with epidemiological in orma-tion. A convenient classi cation to distinguish the genotypes is still to be invented. Another issue is the absence o data linking VNTR alleles to virulence, when it is recognised or some eukaryotes that such markers have potential e ects either on the primary structures o the proteins or on non-coding regulatory sequences, with impacts on their unction.


Genomic characterization of novel DNA viruses of cyprinid shes T Wal e , K Subramaniam, P Viadanna, P Thompson, D Stone and K Way

A variety o DNA viruses have been isolated rom cyprinid shes. While some viruses such

as koi herpesvirus have negatively impacted koi and common carp aquaculture, the taxonomy and signi cance o other viruses isolated rom cyprinids remains to be determined. Although known since the 1970’s in Japan, a poxvirus-like agent known as carp edema virus (CEV) has only recently been recognized as a threat to the international koi industry and perhaps the common carp shery. The identity and signi -icance o a novel iridovirus-like agent isolated rom cultured common carp su ering rom

gill disease in Eastern Europe remains unclear. Similarly, an iridovirus-like agent isolated rom moribund cultured European chub is yet to be characterized. Although iridoviruses are well known pathogens o n sh, poxviruses are not. In this investigation, we genetically character-ized the rst poxvirus and iridoviruses isolated rom cyprinid shes using the latest techniques

in Next Generation Sequencing (NGS).

Common carp and koi are the only known sus-ceptible species to CEV. Similar poxvirus-like agents have been partially characterized in ayu (Plecoglossus altivelis), Atlantic salmon (Salmo salar), and Cape seahorses (Hippocampus capen-sis) (reviewed in Hesami et al. 2015). Currently, li le is known about the genetic relationships o CEV and other sh poxviruses. Thus, we sought to elucidate the phylogenetic position o CEV by sequencing its genome. In ected tissues rom a well characterized CEV epizootic in koi rom the USA were subjected to a virion

enrichment strategy prior to DNA extraction and sequencing on an Illumina MiSeq NGS plat orm. De novo assembly o the NGS data recovered the ull CEV genome. Phylogenomic analyses con rmed CEV and the salmon gill poxvirus are sister species and together orm the deepest branch o the Chordopoxvirinae within the amily Poxviridae.

A disease in cultured European common carp resulting in gill necrosis has been known since the 1950s (reviewed in Wol 1998). Early inves-tigations suggested water quality or a virus as the etiology. Popkova and Shchelkunov (1978) isolated an irido-like virus rom the gills and kidneys o diseased Russian common carp. The virus was isolated on FHM and EPC cells at 22-28 °C. Transmission electron microscopy o cell cultures revealed cytoplasmic icosahe-dral shaped nucleocapsids (diameter ~ 200 nm) consistent with an iridovirus. Ultimately, the common carp iridovirus (CCIV) was inconsist-ently isolated rom clinical cases and experi-mental challenge studies ailed to prove it as the sole cause o carp gill necrosis (CGN). The CCIV isolate, archived at CEFAS, was expanded in cell culture and the resulting supernatant was subjected to a virion enrichment strategy prior to DNA extraction and sequencing on an Illu-mina MiSeq NGS plat orm. De novo assembly o the NGS data recovered portions o CCIV genome. Phylogenetic analyses con rmed CCIV as the deepest branch o the genus Ranavirus within the amily Iridovirdae.

In 2005, an aquaculture acility in central England reported elevated mortality in juvenile European chub (Squalius cephalus). The CEFAS received moribund sh and a replicating agent was isolated on a variety o cell lines (BF-2, EPC,


CHSE-214, KF-1, CCB) at 20 °C. Transmission electron microscopy o cell cultures revealed abundant cytoplasmic icosahedral shaped nu-cleocapsids consistent with an iridovirus. The virus was expanded in cell culture and the re-sulting supernatant was subjected to a virion enrichment strategy prior to DNA extraction and sequencing on an Illumina MiSeq NGS plat orm. De novo assembly o the NGS data recovered the ull ECIV genome. Phylogenomic analyses con rmed ECIV and the recently dis-covered Scale Drop Disease Virus are sister species and together orm the deepest branch o the genus Megalocytivirus within the amily Iridovirdae.

A lack o knowledge o sh viruses (including viruses o cyprinids) is the result o a lack o interest/resources and a technology gap. The NGS revolution has aided in the discovery o many viral amilies rom sh hosts including the rst discoveries o iridoviruses and pox-viruses in cyprinids as presented here. These and uture discoveries are expected to greatly expand our understanding o the relationships o sh viruses and will likely radically alter current taxonomic schemes. Given the ease o discovering novel sh viruses using NGS approaches, uture challenges will be proving their signi cance in disease (ie ul lling Koch’s Postulates). Future studies are planned to un-derstand the clinical signi cance o CEV, CCIV, and ECIV.

DiscussionU. Fischer asked, i you sequence, you will always nd viruses. What to do T. Wal ek answered: Proving the signi cance o a virus in disease is best approached by per orming chal-lenge studies and ul lling Koch’s Postulates.

Workshop overall conclusionsIt was concluded, that this workshop was very use ul, to address novel viral cyprinid diseases which should be strongly considered in the near uture, with particular regard to the spread o CEV and KSD. An international CEV network has been active since autumn 2013 and met in January and September 2015, internal reports are available (h p://www.eurl- sh.eu/Reports). The network is currently consider-ing new unding strategies or CEV/KSD joint research (please contact O. Haenen or urther in ormation).

AcknowledgmentsWe thank the organizers o the 17th EAFP International Con erence, particularly Ivona Mladineo, the EAFP meeting secretary, and José Garcia, the EAFP president, or providing this important opportunity to address this work-shop to a large international audience. We also thank all co-authors and colleagues collaborat-ing to any presented work, and all participants or contributing to this success ul event.

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