Bull. Eur. Ass. Fish Pathol., 36(1) 2016, 11
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, ¢ȱȱ¢ȱǰȱ§ĵȱŗǰȱŗŘŗŖȱǰȱDzȱ4 Tamaki Laboratory, Aquatic Animal Health Division, National Research Institute of Aquaculture (NRIA), Fisheries Research ¢ǰȱȱśŗşȬŖŚŘřȱ Dzȱ5ȱǰȱ£·ǰȱDzȱ6 College of Veterinary Medicine, Department ȱ ȱȱȱ¢ǰȱ¢ȱȱǰȱ ǰȱȱřŘŜŗŗȬŖŞŞŖǰȱ
* Workshop co-organizer
Introduction In 2007 koi herpesvirus disease (KHVD), caused an open workshop was organized at the 17th by the alloherpesvirus Cyprinid herpesvirus 3 EAFP International Con erence on Diseases o (CyHV-3), was listed by the World Organiza- Fish and Shell sh. Seven short lectures were ol- tion or Animal Health (OIE) and was quickly lowed by a discussion, involving an audience o ollowed by listing as a non-exotic disease in the approximately 60 international experts. New eld European Union (EU), related to the Directive observations and preliminary research on novel 2006/88/EC (EC, 2006). Up until the listing o cyprinid viral in ections were presented, aiming KHVD, Spring Viraemia o Carp (SVC) was to illustrate their impact in various geographic the only viral disease o cyprinids listed by the regions. The main topics presented included OIE (OIE, 2012). issues related to the current emergence and detec- tion o Cyprinid iridoviruses, Carp Edema Virus Apart rom CyHV-3 and SVCV, in the last ve (CEV), Cyprinid Herpesvirus 2 (CyHV-2), and a years, several novel non-noti able cyprinid novel myxo-like virus. Additionally, the promo- viruses have been detected in Europe and their tion o a collaborative approach to be undertaken relation to clinical disease and mortality out- or the genetic characterization o novel cyprinid breaks is a cause or concern. viruses was highlighted. The general aim o the workshop was to identi y potential collaborative To gain a comprehensive overview o current approaches to carry out multidisciplinary studies potential new viral threats or cyprinid sh, and aiming to de ne risks, diagnostic methods and to provide an update on their characterization, suggest adequate prophylactic measures.
* Corresponding author and workshop leading organizer’s email: [email protected] 12, Bull. Eur. Ass. Fish Pathol., 36(1) 2016
Overview of Carp Edema Virus in A eature o CEV/KSD outbreaks in common Europe carp in Europe, and particularly in the UK, is K Way and DM Stone that many have occurred during periods o low water temperatures (6-9°C) in winter and Carp Edema Virus (CEV) is the etiological agent early spring. Outbreaks o KSD in koi ponds o Koi Sleepy Disease (KSD), originally de- are generally seen at higher temperatures (15- scribed in Japan in the 1970’s as a viral edema 25°C) in late spring or early summer. In the UK o juvenile color Cyprinus carpio koi and later a link with Spring Carp Mortality Syndrome morphologically identi ed as a poxvirus. The (SCMS) was suspected (Way and Stone, 2013). virus causes severe damage to gill lamellae, When archived DNA and preserved gill tissue leading to hypoxia and lethargy, which man- samples, including para n-embedded ormalin i ests as sleepy behavior, and mortality can xed gill tissue, were analyzed by PCR, CEV reach 80-100% (Ono et al., 1986; Lewisch and was detected in samples rom SCMS cases rom Gorgoglione et al., 2015). 2004 and 1998/99.
The disease is known to be widespread across CEV/KSD is the latest example o a virus disease Japan where koi are cultured and, as a conse- o cyprinids that has emerged, largely unno- quence, the disease has been managed rather ticed, in Europe and where the true extent o than a empting to eradicate the virus in koi the spread o the virus is not known. The cir- populations. International trade in koi has un- cumstances o the emergence and spread o doubtedly led to global spread o CEV/KSD but CEV/KSD are similar to those o KHVD almost the occurrence o disease outbreaks has o ten 20 years ago. not been recognized and consequently, rarely reported. In the USA, CEV has been detected Possible reasons or why this virus has only in association with disease outbreaks in koi at rarely been identi ed in carp mortality inves- import sites and in hobby ponds since 1996 tigations include: 1) the virus is, at present, (Hesami et al., 2015). In Europe, outbreaks o non-culturable and not isolated during routine KSD and PCR detections o CEV-like virus screening; 2) the disease may not always were reported rom 2009. At a CEV workshop cause high mortalities (extensive outbreaks) in Copenhagen in January 2015 (meeting report in common carp, and 3) CEV/KSD is possibly available rom h p://www.eurl- sh.eu/reports), misdiagnosed as KHVD without con rmation delegates rom Austria, Czech Republic, France, o the true cause. Also, until recently, ew ade- Germany, Italy, The Netherlands and the UK quate diagnostic tools have been available. In reported multiple KSD outbreaks in imported the USA in 1996 the virus was identi ed in koi koi and also outbreaks in common carp. Follow- by transmission electron microscopy (TEM) ing the workshop in ormation was received on (Hedrick et al., 1997). KSD/CEV cases in Poland. A CEV alert paper giving the emergence o CEV in Europe and The knowledge gaps that, it is suggested, need its signi cance to European aquaculture is cur- to be addressed to assess the true impact o the rently in preparation (Keith Way, pers.comm.). disease on European carp populations include: Bull. Eur. Ass. Fish Pathol., 36(1) 2016, 13
1) the mechanisms o virus survival outside the negative trade e ects, threatening the high-val- host ; 2) are there susceptible aquatic species ued ornamental sh industry and carp arming. other than carp (carriers) ; 3) does the virus exist as a low-level persistent in ection and are there In Austria, the occurrence o KSD was demon- aquatic vectors ; 4) what is the prevalence in strated or the rst time during the spring o carp populations ; 5) what are the important 2014, rom two unrelated cases rom common environmental actors responsible or triggering carp and koi carp (Lewisch and Gorgoglione et CEV/KSD outbreaks al., 2015). In 2015, the rst Austrian case arrived in late February rom a spontaneous KSD out- More tools are needed to provide early warning break a ecting koi rom a large garden pond o emerging pathogenic viruses o aquaculture (> 250 m3), which allowed preliminary trans- and resolve aquatic/ sh diseases o unknown mission and susceptibility investigations to be aetiology. Possible improvements include: 1) carried out. A marked di erential susceptibility a be er characterization o known viruses and was seen between various individuals rom the more use o next generation sequencing, partic- same pond, providing evidence o clinically ularly to allow e ective design o generic PCR asymptomatic carriers. An adult koi (20 cm primers that detect a range o DNA viruses; total body length) was hospitalized at 17 °C 2) development o qPCR assays or sample showing a severe in ection with the ectopar- screening; 3) be er mechanisms in Europe asites Trichodina sp. and Gyrodactylus sp., and (with support rom the EU) or collaboration multiple skin in ections with opportunistic bac- and networking to tackle serious or potentially teria (eg Shewanella putrefaciens). Surprisingly, serious epizootics. 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 Preliminary investigations on and mortality by day 7 post-in ection (d.p.i.), transmission of and susceptibility to (Figure 1). CEV viremia was already visible and Carp Edema Virus CEV / Koi Sleepy detectable at 1 d.p.i., associated with a sleepy Disease KSD behavior, and possibly showing highest DNA B Gorgoglione, E Lewisch, O Schachner and levels at 7 d.p.i. (see the thicker band at the gel M El-Matbouli electrophoresis: Figure 2).
Koi carp are traded worldwide as valuable or- CEV was detected by PCR (using the nested namental sh, particularly appreciated as pets PCR protocol rom CEFAS, Stone Way, pers. in European garden ponds, which are highly comm. 2013), and was only detectable in DNA popular in Austria. Common carp Cyprinus rom speci cally skin mucus, gills laments carpio is o ten armed in poly-cultural systems, and blood. There ore, we suggest these tissues representing the main reshwater sh produc- are the best to be sampled or KSD diagnostics. tion in the world, especially relevant in Asian DNA rom internal organs (including rom parts and Central-East European countries (FAO, o the kidney (pronephros and mesonephros), 2014). The spreading o CEV/KSD may have spleen and swim bladder) gave aint positivity, 14, Bull. Eur. Ass. Fish Pathol., 36(1) 2016
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. Bull. Eur. Ass. Fish Pathol., 36(1) 2016, 15 usually at the second step o the nested PCR, Discussion and exclusively rom sh in terminal condition T. Vesel was surprised that already a ter 6 h p.i. (Figure 2). CEV was also detected by PCR rom clinical signs were observed. N. Adamek and mucus o asymptomatic koi up to 4 months ol- M. Reichert asked about the di erence between lowing co-habitation. KHV detection by PCR samples labelled as mucus and gills in (Bercovier et al., 2005) gave negative results. the gel images shown, because gills are also Even a ter e ective anti-ectoparasitic treatments, covered by mucus. B. Gorgoglione clari ed using FMC (a mixture o 1 L ormalin, 3.5 g mala- to be re erring to mucus when taken rom chite green and 3.5 g methylene blue; used at the skin o the body side, and gills or gill the dosage o 1 mL in 100 L) against a severe Ich- laments biopsied or sampled at the necropsy. ¢ȱę in ection, the skin mucus N. Adamek also asked whether KSD should be stayed CEV positive, both tested straight a ter considered a primary or a background disease the treatment, and in the longer term (4 months B. Gorgoglione replied that rom what is cur- p.i.). Virus cultivation was also a empted by rently known it is more likely to be a strong inoculating tissue homogenates (sampled and background disease, at least or adult sh. E. pooled according to KHV sampling procedures Lewisch reported about the clinical observa- (OIE, 2012)) on Common Carp Brain (CCB) tion o an in ected garden pond in which a cells. No cytopathic e ect was evident a ter documented outbreak o CEV occurred in koi several passages o 7 days each, respectively at carp more than one year ago, with some mortal- 15°C and 20°C, similarly to previous negative ity. Fish showed typical clinical signs o KSD results achieved with EPC, BF-2, RTG-2, and and were con rmed CEV positive (KHV and CHSE cells (Lewisch and Gorgoglione et al., SVCV negative with no other apparent causes 2015; Schachner, personal communication). o death). Some sh recovered rom the in ec- tion, while some others did not show any sign Our preliminary observations rom clinical o disease. A restocking was operated one year cases, although still to be con rmed, provid- later resulting in the death o exclusively all ed some intriguing clues about virus detec- newly stocked sh, and all these sh were con- tion, transmission and host tissue tropism. rmed as CEV positive (E. Lewisch, personal The current main aim is to achieve soon the in communication). vitro virus cultivation and isolation. The ability to culture the virus in vitro will allow proper virus characterization, strain di erentiation and Properties of and preventative in ection challenges to assess the CEV-induced measures for HVHN in goldęsh caused immune response in susceptible and resistant by Cyprinid herpesvirus 2 hosts. KSD prevalence is indeed clearly under- (CyHV-2) estimated; above all when considering the low T Ito pathogenicity or adult carp and co-in ections with other pathogens. Thus, assessing the role Cyprinid herpesvirus 2 (CyHV-2) is the causative o asymptomatic carriers should be addressed agent o herpesviral haematopoietic necrosis in uture research e orts. (HVHN) in gold sh (Carassius auratus auratus) 16, Bull. Eur. Ass. Fish Pathol., 36(1) 2016 and the occurrence o the virus has been re- range o 20 25°C is considered highly permis- ported rom several countries (Chang et al., sive or HVHN. Moreover most sh in ected 1999; Dan k et al., 2012; Goodwin et al., 2006; with CyHV-2 at 13-15°C acquired resistance Gro et al., 1998; Je ery et al., 2007; Stephens et to HVHN. This phenomenon might be an ap- al., 2004; Wang et al., 2012). Due to the great di - plicable preventative measure at low cost. In erence in economic value o gold sh, ranging addition, a simple ormalin-inactivated vaccine rom use as live bait sh to high quality orna- was e ective against HVHN o gold sh. This is mental sh, it is di cult to ormulate general a use ul preventative measure or high quality preventive measures or HVHN. Moreover, ornamental gold sh (Ito and Maeno, 2014b; Ito recently the virus has also been detected rom and Maeno, 2015; Ito and Ototake, 2013). Prussian carp (C. gibelio) and crucian carp (C. carassius) rom European and Asian countries. Discussion Since the reports suggest that this virus has been S. Bergmann asked, when a sh is resistant to spreading, and also CyHV-2 has the potential disease is it also resistant to in ection Answer to in ect other species o the genus Carassius T. Ito: this was not known, but possibly there as a host, understanding the properties and is latency. the preventative measures o this disease has become important. Cyprinid herpesvirus 2 (CyHV-2) Studies on the prevalence o CyHV-2 in organs infection in wild gibel carp, Carassius o gold sh exposed to the virus were described: auratus gibelio in the Netherlands Kidney is a suitable organ or PCR-based diag- O Haenen, M Engelsma and T Ito nosis o HVHN in gold sh, since CyHV-2 DNA was detected rom the kidney o all surviving Gibel carp, Carassius auratus gibelio is thought to sh at 31 days post-exposure in experimental be an ancestral species o gold sh originating in ection (Ito et al., 2013). Results o experimen- rom China, which was introduced into the tal in ections o indigenous Cyprininae species Netherlands in the 17th century. In May o 2015, in Japan were also reported: Susceptibility o an epizootic with a high mortality occurred indigenous Cyprininae sh species to CyHV-2 in adult wild gibel carp in reshwater lakes is much lower compared to gold sh (Ito and in Western-Netherlands. The gibel carp were Maeno, 2014a). suspected or in ection with CyHV-2 (Dan k et al., 2012; Wang et al., 2012). Some sh showed Preventative measures against HVHN in gold- a whitish slime layer over their eyes and an sh have also been a empted by control o water erythema o their skin, sometimes with hae- temperature and vaccination. Ryukin gold sh morrhagic scales. The water temperature was were in ected with CyHV-2 by intraperitoneal 20-25°C during the outbreak. CyHV-2 was de- injection and maintained at 4 di erent water tected in high amounts (low Ct value) by qPCR temperatures. Cumulative mortalities o the (Goodwin et al., 2006). 15, 20, 25 and 30°C groups were 10, 90, 90 and 60%, respectively. There ore, the temperature When testing archived samples o wild gibel Bull. Eur. Ass. Fish Pathol., 36(1) 2016, 17 carp rom high mortalities with signs (severe CyHV-2 had also been ound in gold sh rom a skin and n haemorrhaging) rom May 2011 closed acility, in Swi erland and in Germany, (2,500 sh dead), and August 2011 (high mor- respectively. tality with additionally haemorrhagic eyes, thick slime, loosening skin, and gill in amma- tion with hypertrophy) respectively, rom the Characterisation of an unknown myxo- same province, these two o three gibel carp like virus from carp groups were also strongly positive by qPCR or R Paley, C Sharp, C Joiner, I Cano-Cejas, R CyHV-2. However, a third group with clinical Van Aerle, K Bateman, D Stone and P Dixon signs, sent in March 2011 was negative in the qPCR or CyHV-2. In 2011, virus isolations on Diagnostic testing o UK and imported cyprin- EPC and FHM cells at 15 and 20°C o the three ids has o ten resulted in the isolation o viruses gibel carp groups did not give any cytopathic showing a characteristic syncytial CPE in cell e ect (CPE) in two passages. In Japan, at the culture that is not caused by the known and laboratory o T. Ito the our gibel carp organ expected viruses o carp (22 isolations since pools are urther tested, in virus isolation on 1996), (Figure 3A). For our o these isolates, gold sh cells (Standard Ryukin Taka umi cells analysis by electron microscopy showed crystal- (SRTF), and Gold sh Fin cells (GFF) (Ito et line arrays o spherical virions o approximately al., 2013)), and or molecular characterization, 90 nm in size in the cytoplasm. A chloro orm aiming a joint publication. test or lipid envelope (absent) and analysis o viral genomic segments by polyacrylamide gel We report the rst detections since 2011 o electrophoresis, showing 11 segments, indicated CyHV-2 in wild gibel carp in the Netherlands, they are aquareoviruses. Subsequent PCR and like in the Czech Republic, which had out- sequencing showed 94% and 90% similarity to breaks o CyHV-2 disease in gibel carp in Lake grass carp reovirus RNA genomic segments 2 eha ka, close to the Elbe River in May-June and 10 respectively. o 2011 (Dan k et al., 2012). As gibel carp is a non-native species which seems to be very For the remaining isolates however initial char- success ul in our country, the CyHV-2 outbreak acterisation by electron microscopy indicated is believed to have had a low impact so ar. It is large (200 nm) pleomorphic, spherical, envel- not clear i CyHV-2 is emerging in Europe, as oped virions that are released rom cells by no monitoring has been done. Networking on budding (Figure 3B and 3C). The isolates rep- this new viral cyprinid disease is important, and licate slowly (average o 17 days to develop ull unding is needed or urther studies. CPE) on carp cell lines producing low titres (3.4
to 5.7 log TCID50/mL). Bromodeoxyuridine and Discussion chloro orm treatment indicated RNA genome K. Way con rmed that stress is a trigger or and presence o a lipid envelope respectively. CyHV-2 clinical disease. E. Lewisch mentioned, Together these results indicate a possible myxo- CyHV-2 is also ound in gibel carp in Austria. like virus, but the isolates were not able to hae- Similarly T. Wahli and N. Adamek reported magglutinate or haemadsorb carp or salmon 18, Bull. Eur. Ass. Fish Pathol., 36(1) 2016 erythrocytes. Furthermore, no ampli cation A brie overview o the work undertaken thus was seen using generic primer sets designed ar to characterise our isolations was presented to ampli y orthomyxovirus and paramyxovirus at the meeting. In conclusion, 4 o the isolates polymerase genes (Tong et al., 2008; W. Ba s, were determined to be grass carp reovirus and a USGS, Western Fisheries Research Centre, per- urther 18 isolations were still unknown. These sonal communication, and this study). unknown viruses were deemed either not myxo- like viruses or are very distantly related, or To date, genome segmentation has not been possibly belong to a virus amily with similar accurately determined due to the inability to morphology e.g. Arenaviridae or Bunyaviridae. obtain su cient puri ed virus or these isolates. Viral nucleic acid has been subjected to Next Generation Sequencing (NGS) by Roche 454 and Discussion Illumina HiSeq plat orms, with de novo assem- At what water temperature was the myxo-like bly using CLC genomics, Blast (Altschul et al., virus in carp ound/tested R. Paley answered 1990) comparison against sequence databases that 20°C is the pre erred growth temperature and assessment o assignments using Megan in vitro. Disease investigations are still under (Huson et al., 2007). Despite recovering over study. T. Vendel Klinge asked whether iso- 1 million and 10 million sequence reads or lates were rom surveillance R. Paley replied isolates in each sequencing plat orm respec- samples were rom a mixture o surveillance, tively no signi cant similarities had yet been import checks and reported disease outbreaks, identi ed. Isolations rom carp in Germany, o sometimes but not always associated with pa- viruses with similar CPE, morphology under thology. electron microscopy and biochemical properties have been previously described (Body et al., Post meeting note on Paley et al. 2000; Granzow et al., 2014; Neukirch and Kunz, Recently the HiSeq generated raw sequence 2001) and were provisionally ascribed to the reads rom one o the virus isolates were as- amily Paramyxoviridae but without success ul sembled again using a lately published new ull characterisation or molecular assignment. 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. Bull. Eur. Ass. Fish Pathol., 36(1) 2016, 19 sembler), (Hunt et al., 2015). Surprisingly a There ore, each candidate VNTR must be evalu- 16 kb putative viral sequence was generated ated by testing samples o various origins, se- and subsequently annotated using a selection quencing the alleles in order to characterize o bioin ormatics tools including FGENESV0 their structure and evaluate its potential as a (h p://linux1.so tberry.com), Prokka (Seemann, geographic marker. 2014) and BLAST. Based on the single segment genome architecture encompassing at least eight For CyHV-3, various markers have been se- putative genes and the average nucleotide iden- lected and success ully used to di erentiate tity over putative nucleoprotein, usion and isolates and in er genetic relationships between polymerase genes o 21%, the virus was clas- sequences (Avarre et al., 2012; Sunarto et al., si ed as a putative Paramyxovirus. Failure to 2010). Un ortunately, tandem repeats do not group with any o the known paramyxoviruses appear to be conserved between cypriniviruses. (including Atlantic Salmon paramyxovirus) There ore, similar, yet distinct, tools are still to in phylogenetic analyses indicates this isolate be created or other alloherpesviruses, such as represents a new species and quite likely a new CyHV-2, an emerging virus o gold sh (Caras- genus within the amily Paramyxoviridae. Further sius auratus) and Prussian carp (C. gibelio). For characterisation is ongoing or this isolate and this virus, the search or suitable VNTR has or the 17 remaining related historical isola- been recently initiated. Numerous, at least 280, tions and will be submi ed or publication at VNTRs have been ound in this genome, but a uture date. candidates use ul or genotyping are still to be identi ed. A rst marker di erentiating two isolates rom di erent geographic origins was VNTR as molecular markers to recently identi ed (Boitard et al., 2015). genotype cypriniviruses L Bigarré, M Baud, S Labrut, M Jamin and The continuous worldwide spread o cyprini- P-M Boitard viruses justi es the intensi cation o genetic studies and the use o VNTR to elucidate the The large size o the genome o the Cyprini- geographic movements o these economically viruses is a limiting actor or the rapid geno- important viruses. There is now a need o an typing o eld isolates. The use o speci c and open database collating the allelic markers polymorphic molecular markers is there ore o already ound, with epidemiological in orma- outstanding importance to acilitate molecular tion. A convenient classi cation to distinguish epidemiology studies. Mini- and micro-satellites the genotypes is still to be invented. Another composed o Variable Numbers o Tandem issue is the absence o data linking VNTR alleles Repeats (VNTR) are use ul targets to rapidly to virulence, when it is recognised or some compare isolates rom di erent origins. Tandem eukaryotes that such markers have potential repeats are abundant in the known Cyprinivirus e ects either on the primary structures o the genomes. However, their individual mutation proteins or on non-coding regulatory sequences, rate and mode o evolution (indels combined or with impacts on their unction. not with substitutions) are impossible to predict. 20, Bull. Eur. Ass. Fish Pathol., 36(1) 2016
Genomic characterization of novel enrichment strategy prior to DNA extraction DNA viruses of cyprinid ęshes and sequencing on an Illumina MiSeq NGS T Walĵe, K Subramaniam, P Viadanna, P plat orm. De novo assembly o the NGS data Thompson, D Stone and K Way recovered the ull CEV genome. Phylogenomic analyses con rmed CEV and the salmon gill A variety o DNA viruses have been isolated poxvirus are sister species and together orm the rom cyprinid shes. While some viruses such deepest branch o the Chordopoxvirinae within as koi herpesvirus have negatively impacted koi the amily Poxviridae. and common carp aquaculture, the taxonomy and signi cance o other viruses isolated rom A disease in cultured European common carp cyprinids remains to be determined. Although resulting in gill necrosis has been known since known since the 1970’s in Japan, a poxvirus-like the 1950s (reviewed in Wol 1998). Early inves- agent known as carp edema virus (CEV) has tigations suggested water quality or a virus as only recently been recognized as a threat to the etiology. Popkova and Shchelkunov (1978) the international koi industry and perhaps the isolated an irido-like virus rom the gills and common carp shery. The identity and signi - kidneys o diseased Russian common carp. icance o a novel iridovirus-like agent isolated The virus was isolated on FHM and EPC cells rom cultured common carp su ering rom at 22-28 °C. Transmission electron microscopy gill disease in Eastern Europe remains unclear. o cell cultures revealed cytoplasmic icosahe- Similarly, an iridovirus-like agent isolated rom dral shaped nucleocapsids (diameter ~ 200 nm) moribund cultured European chub is yet to be consistent with an iridovirus. Ultimately, the characterized. Although iridoviruses are well common carp iridovirus (CCIV) was inconsist- known pathogens o n sh, poxviruses are not. ently isolated rom clinical cases and experi- In this investigation, we genetically character- mental challenge studies ailed to prove it as ized the rst poxvirus and iridoviruses isolated the sole cause o carp gill necrosis (CGN). The rom cyprinid shes using the latest techniques CCIV isolate, archived at CEFAS, was expanded in Next Generation Sequencing (NGS). in cell culture and the resulting supernatant was subjected to a virion enrichment strategy prior Common carp and koi are the only known sus- to DNA extraction and sequencing on an Illu- ceptible species to CEV. Similar poxvirus-like mina MiSeq NGS plat orm. De novo assembly agents have been partially characterized in ayu o the NGS data recovered portions o CCIV (Plecoglossus altivelis), Atlantic salmon (Salmo genome. Phylogenetic analyses con rmed CCIV salar), and Cape seahorses (Hippocampus capen- as the deepest branch o the genus Ranavirus sis) (reviewed in Hesami et al. 2015). Currently, within the amily Iridovirdae. li le is known about the genetic relationships o CEV and other sh poxviruses. Thus, we In 2005, an aquaculture acility in central sought to elucidate the phylogenetic position England reported elevated mortality in juvenile o CEV by sequencing its genome. In ected European chub (Squalius cephalus). The CEFAS tissues rom a well characterized CEV epizootic received moribund sh and a replicating agent in koi rom the USA were subjected to a virion was isolated on a variety o cell lines (BF-2, EPC, Bull. Eur. Ass. Fish Pathol., 36(1) 2016, 21
CHSE-214, KF-1, CCB) at 20 °C. Transmission Workshop overall conclusions electron microscopy o cell cultures revealed It was concluded, that this workshop was abundant cytoplasmic icosahedral shaped nu- very use ul, to address novel viral cyprinid cleocapsids consistent with an iridovirus. The diseases which should be strongly considered virus was expanded in cell culture and the re- in the near uture, with particular regard to the sulting supernatant was subjected to a virion spread o CEV and KSD. An international CEV enrichment strategy prior to DNA extraction network has been active since autumn 2013 and and sequencing on an Illumina MiSeq NGS met in January and September 2015, internal plat orm. De novo assembly o the NGS data reports are available (h p://www.eurl- sh.eu/ recovered the ull ECIV genome. Phylogenomic Reports). The network is currently consider- analyses con rmed ECIV and the recently dis- ing new unding strategies or CEV/KSD joint covered Scale Drop Disease Virus are sister research (please contact O. Haenen or urther species and together orm the deepest branch in ormation). o the genus Megalocytivirus within the amily Iridovirdae. Acknowledgments We thank the organizers o the 17th EAFP A lack o knowledge o sh viruses (including International Con erence, particularly Ivona viruses o cyprinids) is the result o a lack o Mladineo, the EAFP meeting secretary, and José interest/resources and a technology gap. The Garcia, the EAFP president, or providing this NGS revolution has aided in the discovery o important opportunity to address this work- many viral amilies rom sh hosts including shop to a large international audience. We also the rst discoveries o iridoviruses and pox- thank all co-authors and colleagues collaborat- viruses in cyprinids as presented here. These ing to any presented work, and all participants and uture discoveries are expected to greatly or contributing to this success ul event. expand our understanding o the relationships o sh viruses and will likely radically alter References current taxonomic schemes. Given the ease Altschul SF, Gish W, Miller W, Myers EW and Lipman DJ (1990). Basic local alignment o discovering novel sh viruses using NGS search tool. Journal of Molecular Biology 215, approaches, uture challenges will be proving 403-410. their signi cance in disease (ie ul lling Koch’s Avarre JC, Santika A, Bentenni A, Zainun Postulates). Future studies are planned to un- Z, Madeira JP, Maskur M, Bigarré L and derstand the clinical signi cance o CEV, CCIV, Caruso D (2012). Spatio-temporal analysis and ECIV. o cyprinid herpesvirus 3 genetic diversity at a local scale. Journal of Fish Diseases 35, 767-774. Discussion Bercovier H, Fishman Y, Nahary R, Sinai S, U. Fischer asked, i you sequence, you will Zlotkin A, Eyngor M, Gilad O, Eldar A always nd viruses. What to do T. Wal ek and Hedrick RP (2005). Cloning o the answered: Proving the signi cance o a virus in koi herpesvirus (KHV) gene encoding disease is best approached by per orming chal- thymidine kinase and its use or a highly sensitive PCR based diagnosis. BMC lenge studies and ul lling Koch’s Postulates. 22, Bull. Eur. Ass. Fish Pathol., 36(1) 2016
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