J Fish Dis. 2019;42:1609–1621. wileyonlinelibrary.com/journal/jfd  | 1609© 2019 John Wiley & Sons Ltd

1  | INTRODUC TION

Common carp (Cyprinus carpio), hereafter referred to as carp, (and its selectively bred ornamental variety, koi), is a species that has signif‐icant economic, recreational and cultural value in many parts of the world (FAO, 2015; Rahman, 2015; Vilizzi, 2012). Due to its popular‐ity and hardiness, carp are one of the oldest and most widespread cultured species worldwide and have unfortunately proven to be a destructive invasive species in many shallow lake regions (Bajer et al., 2016; Kloskowski, 2011; Weber & Brown, 2009). Intensive culture of carp and international trade has also led to the global spread of viral

pathogens of carp (Gotesman, Kattlun, Bergmann, & El‐Matbouli, 2013; Haenen et al., 2016) including cyprinid herpesvirus‐1 (CyHV‐1), koi herpesvirus (KHV; cyprinid herpesvirus‐3), carp oedema virus (CEV) and spring viremia of carp virus (SVCV). These viral patho‐gens are major threats to farm‐raised and wild carp populations in areas where carp are valued (Haenen et al., 2016; Walker & Winton, 2010). The intentional introduction of species‐specific viruses (e.g. KHV) has also been suggested as a strategy for biological control in areas where carp have invaded (McColl, Sunarto, & Neave, 2018; Saunders, Cooke, McColl, Shine, & Peacock, 2010); however, this strategy remains controversial (Boutier et al., 2019; Kopf et al., 2019).

Received:28June2019  |  Revised:6August2019  |  Accepted:6August2019DOI: 10.1111/jfd.13082

O R I G I N A L A R T I C L E

Koi herpesvirus and carp oedema virus: Infections and coinfections during mortality events of wild common carp in the United States

Soumesh K. Padhi1,2 | Isaiah Tolo1,2 | Margaret McEachran1,2 | Alexander Primus1,3 | Sunil K. Mor1,3 | Nicholas B. D. Phelps1,2

Padhi and Tolo equally contributed to this work.

1Minnesota Aquatic Invasive Species Research Center, University of Minnesota, St. Paul, MN, USA2College of Food, Agriculture and Natural Resource Sciences, Department of Fisheries, Wildlife and Conservation Biology, University of Minnesota, St. Paul, MN, USA3College of Veterinary Medicine, Department of Veterinary Population Medicine and Veterinary Diagnostic Laboratory, University of Minnesota, St. Paul, MN, USA

CorrespondenceNicholas B. D. Phelps, Minnesota Aquatic Invasive Species Research Center and the Department of Fisheries Wildlife and Conservation Biology, University of Minnesota, St. Paul, MN, USA.Email: phelp083@umn.edu

Funding informationMinnesota Aquatic Invasive Species Research Center; Minnesota Environment and Natural Resources Trust Fund

AbstractKoi herpesvirus (KHV; cyprinid herpesvirus‐3) and carp oedema virus (CEV) are im‐portant viruses of common and koi carp (Cyprinus carpio); however, the distribution of these viruses in wild common carp in North America is largely unknown. During the summers of 2017 and 2018, 27 mass mortalities of common carp were reported from four states in the USA (Minnesota, Iowa, Pennsylvania and Wisconsin), the major‐ity of which were distributed across eight major watersheds in southern Minnesota. Samples from 22 of these mortality events and from five clinically healthy nearby carp populations were screened for KHV, CEV and SVCV using real‐time polymer‐ase chain reaction (qPCR). KHV was confirmed in 13 mortality events, CEV in two mortality events and coinfections of KHV/CEV in four mortality events. Nucleotide sequence analysis revealed that the KHV and CEV detected here are closely related to European lineages of these viruses. While molecular detection alone cannot con‐clusively link either virus with disease, the cases described here expand the known range of two important viruses. This is also the first reported detection of KHV and CEV coinfections in wild carp populations.

K E Y W O R D S

carp oedema virus, coinfection, emerging disease, koi herpesvirus

1610  |     PADHI et Al.

Koi herpesvirus and carp oedema virus are both DNA viruses with a narrow host specificity. Though KHV and CEV both infect and cause disease in carp, and its ornamental variety, koi (OIE, 2018; Way et al., 2017), KHV also infects goldfish and can cause disease in goldfish × carp hybrids (Bergmann et al., 2010; Hedrick, Waltzek, & McDowell, 2006). Mortality during epizootic events caused by these viruses can reach 100% (Haenen et al., 2016; Jung‐Schroers et al., 2015; Way et al., 2017), though disease outcomes are influenced by water temperature and quality, population density, fish age and condition, as well as the breed of carp and viral genotype (Gao et al., 2018; Shapira et al., 2005; Thresher, Allman, & Stremick‐Thompson, 2018; Uchii, Okuda, Minamoto, & Kawabata, 2013).

The clinical presentation of KHV and CEV can be similar and may include discoloured skin, haemorrhages, scale and skin loss, over or underproduction of mucous, enophthalmia, discoloration and necro‐sis of gills and fins, or be absent altogether (OIE, 2018; Way et al., 2017). For both KHV and CEV, highly sensitive molecular assays are the most reliable diagnostic methods and several detection proto‐cols have been developed (Adamek et al., 2016; Gilad et al., 2004; Matras et al., 2017). Importantly, molecular methods can be useful for detecting pathogens even when fresh samples are not available, which is often the case with outbreaks of disease in wild fish (La & Cooke, 2011; Phelps et al., 2019).

Koi herpesvirus was first characterized from two outbreaks which occurred in the late 1990s and has been associated with disease outbreaks of wild and farm‐raised carp populations in at least 28 countries in Europe, Asia, South Africa, Canada and North America (reviewed in OIE, 2018). In North America, 17 mass mor‐tality events of wild carp have been reported and confirmed to be associated with KHV in the USA and Canada, beginning in New York in 2004 (Thresher et al., 2018). Surveys of healthy wild carp popu‐lations have resulted in the detection of KHV at low prevalence in 2011 at sampling sites in four lakes (Lakes Michigan, Huron, St. Clair and Ontario) along the USA/Canada border (Cornwell et al., 2012) and in 2010 from two asymptotic carp populations in Oregon (Xu et al., 2013).

Carp oedema virus was first characterized in the mid‐1970s in cultured koi and was largely restricted to Japan until 2013, but has since been confirmed in wild and farm‐raised carp populations in Europe (Way et al., 2017) and cultured stocks of koi in Brazil (Viadanna, Pilarski, Hesami, & Waltzek, 2015), South Korea (Kim et al., 2018) and North America (Stevens et al., 2018). Interestingly, a short report from Hedrick, Antonio, and Munn (1997) of a poxvirus‐like agent identified in koi with clinical signs consistent with CEV and high mortality suggests that CEV was present outside of Japan as early as 1996. To date, there has been only one report of CEV in wild populations of carp in North America (Lovy, Friend, Al‐Hussinee, & Waltzek, 2018).

Recently, a coinfection of KHV and CEV was reported in a dis‐ease outbreak in cultured ornamental koi in China (Ouyang et al., 2018) suggesting that coinfections of these viruses could occur in wild or cultivated environments where the viruses coexist. Indeed, herein we report the first detection of KHV and CEV coinfection in

wild carp populations. In addition, we report the first detection of KHV in wild carp in Minnesota and Pennsylvania, and the first de‐tection of CEV in Iowa, Minnesota and Wisconsin.

2  | MATERIAL S AND METHODS

2.1 | Sample collection

Dead and moribund carp were collected from natural mortality events in the Upper Midwest region of the United States. This was an opportunistic survey, whereby the project team relied on re‐ports from agency biologists, fish health laboratories and the public. Efforts were made to standardize fish kill investigations and labora‐tory procedures; however, since fish kills are unpredictable events often initially reported by the public (i.e. http://z.umn.edu/fishkill), the number of fish collected, extent of decomposition of samples, reliability of field observations and response time are often variable (Phelps et al., 2019). Attempts were made to collect at least five carp with clinical signs representative of the mortality event and to re‐cord field observations within 24 hr of the initial report. Carp were collected by agency biologists or field technicians and sent to the University of Minnesota Veterinary Diagnostic Laboratory (UMN VDL) same day or overnight on ice for necropsy and analysis. In some cases, the necropsy was performed at another fish health laboratory and samples submitted on ice to the UMN VDL for analysis. Due to the poor post‐mortem condition of most samples collected during mortality events, tissues were not suitable for bacterial isolation, his‐topathology or parasitology. However, most carp were subjected to cell culture and all carp were tested by pathogen‐specific PCR for KHV, CEV and SVCV according to the methods below.

To determine the disease status of apparently healthy popula‐tions of carp near mortality events, carp from five Minnesota lakes were screened for KHV, CEV and SVCV. Approximately 60 fish from each location were collected by box netting as part of commercial fishing activities and delivered on ice the same day to the UMN VDL for necropsy. Fish were either necropsied same day or kept in a walk‐in refrigerator for up to 48 hr prior to necropsy and analysis. All sam‐ples were tested by pathogen‐specific PCR for KHV, CEV and SVCV according to the methods below.

2.2 | Virology

A necropsy was performed on all carp mortality events to remove the kidney, spleen and heart. If tissues were of sufficient quality (i.e. recognizable), they were collected and pooled in groups of up to five fish. Cell culture methods were performed according to the US Fish and Wildlife Service and American Fisheries Society—Fish Health Section Blue Book (USFWS and AFS‐FHS 2016). Briefly, tissues were homogenized in a 1:10 (weight:volume) suspension of Hank's Balanced Salt Solution (HBSS; Cellgro, VA, USA). The homogenized samples were centrifuged at 2,360 g for 15 min. The supernatant was inoculated in the epithelioma papulosum cyprini (EPC; Fijan et al., 1983) and fathead minnow (FHM; Gravell & Malsberger, 1965)

     |  1611PADHI et Al.

cell lines and incubated at 15 and 25°C for 14 days. A blind passage was performed for an additional 14 days if no cytopathic effects were observed on the first passage. The common carp brain (CCB) and koi fin (KF) cell lines were not available at the time of this study.

2.3 | Detection of KHV, CEV and SVCV by qPCR

During the necropsy of all fish, additional samples were collected for molecular detection of viruses. If tissues were of sufficient quality, brain, gill, kidney and spleen were collected from each fish and were pooled in groups of up to five fish per tissue type (e.g. gills from five fish in one pool). Pools of tissue were homogenized in PBS in a 1:10 ratio (weight:volume) and centrifuged at 2,360 g for 25 min at 4°C. Thesupernatantswerestoredat−80°Cuntilextraction.Nucleicacidpurification from tissue supernatants was done using a MagMAX CORE Nucleic Acid Purification Kit using a benchtop KingFisher Flex platform (Applied Biosystems) for all samples. Due to the low quality of tissues collected from mortality events, a second method of nucleic acid extraction was performed on all tissue supernatants originating from mortality events using the Qiagen DNeasy blood & tissue kit (QIAGEN GmbH), following the manufacturer's standard protocol.

A duplex Taqman probe‐based qPCR for KHV and CEV detection was developed in our laboratory using published primers targeting the ORF89 and p4a genes, for KHV and CEV, respectively (Table 1) (Gilad et al., 2004; Matras et al., 2017). The qPCR for SVCV was op‐timized in our laboratory using published primers/probe targeting the glycoprotein (G) gene (Yue et al., 2008) using Path‐ID™ Multiplex One‐Step RT‐PCR Kit (Applied Biosystems) (Table 1).

In order to generate the standard curves necessary for quanti‐fication of viruses, a 998‐bp laboratory‐synthesized DNA fragment containing a concatenated sequence of the ORF89 (KHV), P4a (CEV) and glycoprotein (SVCV) gene fragments was designed (gBlocks®, Integrated DNA Technologies). The total copy number of ORF 89, P4a and G protein gene fragments were calculated as follows: number of copies = (1,000 ng of DNA × 6.022 × 1,023)/ (length of fragment [i.e., 988 bp] × 1 × 109 × 650). This formula assumes that the average weight of a base pair is 650 Da. Using this formula, the total copy number of three gene fragments was calculated to be 9.38 × 1,011. The dried gene fragment was suspended in 100 μl of sterile TE buffer, resulting in a stock concentration of 9.38 × 109 cop‐ies/μl. From this stock solution, eight 10‐fold dilutions were made (108 to 10 copies) and used to construct standard curves. The results for virus load are presented as the number of virus copies per 250 ng of total DNA/RNA.

Total nucleic acids preparations from mortality events and clin‐ically healthy populations were screened in triplicate for all three viruses. The 70 μl extracted total nucleic acid was used for KHV‐CEV duplex qPCR using Path‐ID™ qPCR Master Mix (Ambion) including 150 nM (for KHV) and 200 nM (for CEV) probes and 400 nM of each primer. The reaction mix was subjected to an initial denaturation at 95°C for 10 min, followed by 40 cycles of denaturation at 95°C for 30 s and annealing at 60°C for 30 s using QuantStudio Real‐Time PCR (Applied Biosystems). A threshold cycle of 38 was used as a cut‐off for both KHV and CEV.

The reaction mix for SVCV contained 120 nM probe and 400 nM each primer. The amplification programme included a one‐step re‐verse transcription step for 10 min at 48°C and denaturation for

TA B L E 1   List of oligonucleotides used during this study

Primer name Target Primer sequence (5’−3’) ReferencesTarget gene (Amplicon bp‐length)

Enlarged TK Fwd: KHV AAC‐GCG‐GGC‐CAG‐CTG‐AAC‐AT Kurita et al. (2011) TK (1,001)

Englarded TK Rev: TGT‐GTG‐TAT‐CCC‐AAT‐AAA‐CG

TK‐IT Fwd KHV CTT‐GAC‐GAC‐CAG‐TGT‐CTG‐CT This study TK (1,426)

TK‐IT‐Rev TTG‐GTC‐AGA‐GTG‐GTT‐CAC‐CG

KHV−86f KHV GAC‐GCC‐GGA‐GAC‐CTT‐GTG Gilad et al. (2004) ORF 89 (78)

KHV−163r CGG‐GTT‐GTT‐ATT‐TTT‐GTC‐CTT‐GTT

KHV−109p [Cy5] CTT‐CCT‐CTG‐CTC‐GGC‐GAG‐CAC‐G‐[IBRQ]

CEFAS_F CEV ATG‐GAG‐TAT‐CCA‐AAG‐TAC‐TTA‐G Matras et al. (2017) p4a (528)

CEFAS_R CTC‐TTC‐ACT‐ATT‐GTG‐ACT‐TTG

CEFAS_nF (nested) CEV GTT‐ATC‐AAT‐GAA‐ATT‐TGT‐GTA‐TTG p4a (478)

CEFAS_nR (nested) TAG‐CAA‐AGT‐ACT‐ACC‐TCA‐TCC

CEFAS_qF CEV AGT‐TTT‐GTA‐KAT‐TGT‐AGC‐ATT‐TCC Matras et al. (2017) p4a (76)

CEFAS_qR GAT‐TCC‐TCA‐AGG‐AGT‐TDC‐AGT‐AAA

CEV qProbe [FAM]‐AGA‐GTT‐TGT‐TTC‐TTG‐CCA‐TAC‐AAA‐CT‐[BHQ1]

SVCVF SVCV TGC‐TGT‐GTT‐GCT‐TGC‐ACT‐TAT‐YT Yue et al. (2008) G protein (81)

SVCVR TCA‐AAC‐KAA‐RGA‐CCG‐CAT‐TTC‐G

SVCVP [FAM]‐ATG‐AAG‐ARG –AGT‐AAA‐CKG‐CCT‐GCA‐ACA‐GA‐[3IABkFQ]

1612  |     PADHI et Al.

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     |  1613PADHI et Al.

10 min at 95°C, followed by 40 cycles of 15 s at 95°C and 45 s at 60°C using QuantStudio Real‐Time PCR (Applied Biosystems).

2.4 | Sanger Sequencing for genotyping

In order to determine the potential geographic origin and relation‐ship between KHV and CEV genotypes/variants collected in this study to previously reported strains, we amplified genomic regions of KHV and CEV from infected tissues.

For KHV, a set of primers, TK‐IT forward and TK‐IT reverse, were designed to amplify a 1,426 bp region which encompasses a region of the thymidine kinase gene (TK) amplified by previously published primers to distinguish between genotype variants of KHV by Kurita et al. (2009) (Table 1). The thermocycling conditions were one cycle for 30 s at 94°C; 35 cycles of: 30 s at 94°C, 30 s at 58°C, two min at 72°C and a final extension step of seven min at 72°C. PCR products were visualized by 1% agarose gel electrophoresis and then purified by precipitation with a 20% PEG, 2.5 M NaCl solution. Sanger se‐quencing for the TK gene was done using the TK‐IT primers as well as previously published primers by Bercovier et al. (2005). Resulting sequences from these primer sets were aligned for each sample.

For CEV, a 478 bp fragment of the partial 4a gene was amplified by means of an end point PCR using the nested primers by Matras et al., (2017) (Table 1). The amplification programme included an initial denaturation at 95°C for 5 min, followed by 45 cycles of denatur‐ation at 95°C for 30 s, annealing at 55°C for 30 s and elongation at 72°C for 30 s and a final elongation step for seven min at 72°C. The PCR products were visualized by 1% agarose gel electrophoresis and purified using a Qiaquick PCR purification kit (QIAGEN). Purified PCR products were sequenced in both orientations using the ampli‐fication primers. Sanger sequencing for both KHV and CEV was per‐formed at the University of Minnesota Genomic Center (Minnesota, USA).

2.5 | Sequence analysis

Sequences were assembled using Sequencher 5.1 software (http://genco des.com). For KHV, final good quality sequences of 1,003 bp were used after trimming and assembling for further analysis and for alignment with the enlarged TK gene region (Kurita et al., 2009), which includes the 651 bp open reading frame of the TK gene as well as a 409 bp sequence, which is the target for the KHV detec‐tion primers published by Bercovier et al. (2005). For CEV, final assembled sequences of a 478 bp partial p4a gene were used. Sequences of the TK gene and p4a gene region were further con‐firmed by BLASTn analysis at the National Center of Biotechnology, and best hit reference sequences were selected and used for com‐parison and phylogenetic analysis. Selected reference sequences were aligned with our study sequences using Clustal W in MEGA v7. For KHV, sequence variants were aligned with a TK gene sequence (GenBank accession no. AB375389) using MEGA v7 and analysed using Microsoft Excel v16.24. For CEV, neighbour‐joining trees were

constructed with the nucleotide substitution model GTR (General Time Reversible + G (Gamma distribution with four rate categories) using 1,000 bootstrap replicates.

3  | RESULTS

3.1 | Sample collection

In total, 27 mortality events of wild carp were reported from four states during the 2017–2018 field seasons to the project team; however, carp were only collected from 22 events (Table 2, Figure 1). Twenty‐one of the total events occurred in Minnesota between May – October 2017 (n = 10) and May – August 2018 (n = 11). Notably, there were no repeated mortality events in the same lakes reported during the two‐year study period. Seven of the mortality events occurred in connected and nearby lakes in the Cannon River and Le Sueur River watersheds. Additional mor‐tality events of carp were investigated in Wisconsin that occurred between August and September 2017 in four lakes in the Rock River System (samples were only collected from three lakes), one mortality event in Pennsylvania between August and September 2017 in the Pymatuning Reservoir and one mortality event in Iowa in September 2018 in Pine Lake (Table 2). In addition, two mortal‐ity events involving captive ornamental koi were reported in the summer of 2018 in Minnesota: one involved a recently purchased koi from a breeder out of state, and the other had been recently purchased at a local pet shop. Specifics on these two cases are unavailable due to client confidentiality, and tissue was only ob‐tained from one case for this study.

Carp from mortality events ranged in length from 23 to 71 cm (Table 2). In cases where water quality was recorded, temperatures were 20.0–24.5°C. However, one event in the fall of 2017 occurred at a considerably lower temperature of 9.9°C. Dissolved oxygen at the time of sample collection ranged between 4.9 and 10.3 mg/L. Many samples were collected in advanced states of decay, and clin‐ical signs were not always recorded. In cases where fresh samples were obtained, clinical signs were observed in seven of eleven cases and included petechial haemorrhaging of the skin, fins, necrotic white or grey lesions on the gills and enopthalmia (Figure 2). In most cases, mortalities were estimated between 100 and 1,000, and up to 10,000 fish, but mortality varied between lakes and precise quanti‐fication was not routinely performed by field biologists. There were no other species observed in close proximity to dead or moribund carp during mortality events—the mortality events appeared to be species‐specific.

3.2 | Virology

Samples suitable for cell culture were collected from 15 mortality events. Consistent with expectations of KHV and CEV isolation on cell culture, no growth was observed on EPC or FHM cell lines at 15°C or 20°C from any of the lakes tested.

1614  |     PADHI et Al.

     |  1615PADHI et Al.

3.3 | Detection of KHV, CEV and SVCV by qPCR

From samples collected following reported mortality events of wild carp, we confirmed 13 events associated with KHV alone, two events with CEV alone and four events with coinfections of KHV and CEV (Table 2, Figure 1). Of the 13 events positive for KHV alone, ten occurred in Minnesota, two in Wisconsin and one in Pennsylvania. The two mortality events associated with CEV alone occurred one each in Minnesota and Iowa. Of the four co‐infections of KHV and CEV, three occurred in Minnesota and one in Wisconsin. Finally, KHV was also confirmed in a backyard koi pond in Minnesota (Copy number = 3.65 × 106). In coinfections of KHV and CEV, copy numbers of KHV were generally higher (Table 2). KHV was detected in all tissues tested (brain, gill, kid‐ney and spleen), and CEV was primarily detected in gill tissue but was also detected in some brain and kidney samples (Supporting Information S1). SVCV was not detected in any of the carp mortal‐ity events.

All clinically healthy carp collected from Minnesota lakes in 2017 were negative for KHV, CEV and SVCV. There were no re‐ports of mortality events from these lakes during the study period and no previous reports of fish kills according to the University of Minnesota's online fish kill database (http://z.umn.edu/fishkill; Phelps et al., 2019). These lakes are not directly connected to lakes

with mortality events reported in this study, and only one lake was located in the same watershed as an outbreak lake.

3.4 | Sequencing and phylogenetic analysis

Fourteen sequences of the TK gene of KHV were obtained from three states: Minnesota (10), Wisconsin (2) and Pennsylvania (2) (Table 3) (submitted in GenBank with accession no. MK987085‐MK987098). All 14 sequences were 99.30%–100% identical to each other. The criteria of Aoki et al. (2007) were used to define genotypes of KHV based on SNPs and INDELs. Our study sequences were compared with 32 published sequences available in the GenBank database rep‐resenting variants of the Asian (A) and European (E) genotypes. There were two polymorphisms which were fully discriminatory between A and E genotypes, a deletion at alignment position 817–818 and a substitution at alignment position 782 (A/G) (Table 3). A deletion at alignment position 965–966 also discriminates A from E genotypes with the exception of E5 which has a four‐nucleotide deletion at the 5′endoftheTKgene.Thesequencesfromthisstudycanbedesig‐nated as being from the European lineage based on these variants, which have been previously reported by Kurita et al. (2009). Among the sequences obtained from wild carp in this study, there were only two variant sites: one single nucleotide deletion specific to F111 at alignment position 891 and a substitution at alignment position 957

F I G U R E 1   (a) KHV and CEV detections from mortality events of wild common carp in Iowa, Minnesota and Wisconsin during this study; (b) All KHV and CEV detections from wild common carp during this study; (c) All published KHV and CEV detections from wild common carp in North America from Cornwell et al. (2012), Xu et al. (2013), Thresher et al. (2018), and Lovy et al. (2015). Symbols denote the detection of KHV alone (filled black circles), CEV alone (filled red circles), KHV and CEV (red circle with black bulleseye), and no detection of KHV or CEV (black diamond) from mortality events in this study. Major watersheds (HUC 8) are outlined in light grey. Major rivers and lakes (1 × 106 scale) are denoted in light blue. State and province boundaries are denoted in dark grey [Colour figure can be viewed at wileyonlinelibrary.com]

F I G U R E 2   Gross pathology of freshly dead wild carp from Cottonwood Lake with KHV and CEV coinfection. Loss of skin and scales on the surface of the body (a). Necrosis in the gill filaments (b). Haemorrhages and loss of scales on the surface of the body (c). Enopthalmos (d) [Colour figure can be viewed at wileyonlinelibrary.com]

(a) (b)

(c) (d)

1616  |     PADHI et Al.

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138

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782

817

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890

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965

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     |  1617PADHI et Al.

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1618  |     PADHI et Al.

(A/T) (Table 3). Sequences from samples obtained from wild carp from Minnesota, Pennsylvania and Wisconsin in this study were vir‐tually identical to those of isolate PoB3 (Accession no. KX544842) isolated from carp in Poland and to the European genotype/variant E4 (Accession no. AB375387). Interestingly, the sequence of KHV obtained from the ornamental koi (Koi MN 19880) had Asian geno‐type alleles of the three discriminatory variants and was identical to reference strain TUMST1 (Accession no. AP008984).

For CEV, six sequences of the partial 4a gene (478 nucleo‐tides) were obtained from mortality events in Iowa, Minnesota and Wisconsin (submitted in GenBank with accession no. MK990708‐MK990715). All six sequences from this study clustered together with the genogroup I sequences reported from Poland with 98.33%–100% nucleotide identity (Figure 3). Study sequences showed 94.35%–95.61% identity with genotype II sequences reported from Europe and Asia. Three unique nucleotides substitutions were observed in CEV sequences from Iowa and one sequence from Minnesota.

4  | DISCUSSION

This opportunistic survey of wild carp mortality events indicates a broader distribution of KHV and CEV in North America than previ‐ously reported in the literature (Figure 1). Though prior cases of KHV have been reported in wild carp in the Midwest, Lovy et al.

(2018) only recently detected the first case of CEV in wild carp in the USA in 2017. Our findings indicate that the range of CEV is more extensive than previously known and overlaps with that of KHV. Since both KHV and CEV are hypothesized to have spread worldwide through international trade in ornamental koi (Haenen et al., 2016; Rakus et al., 2013), it is not surprising that KHV and CEV have developed overlapping ranges in this region. Regardless, the detection timeline of KHV along the USA/Canada border be‐ginning in New York in 2004 (Grimmet, Warg, Getchell, Johnson, & Bowser, 2006) and later in Canada in 2007 (Garver et al., 2010) and the Great Lakes and Midwestern States from 2011 to 2017 (Thresher et al., 2018) may indicate a spread westward along this course. Alternatively, this could be the result of sampling and/or reporting bias.

One implication of both individual and concomitant detections of KHV and CEV across a wide geographic range may be that coin‐fections are common in regions where KHV and CEV are present. Recently, the first reported incidence of coinfection of KHV and CEV occurred in a domestic koi pond in Chengdu, China (Ouyang et al., 2018). Though our reports are the first of coinfections of these viruses occurring in wild carp, it is possible that coinfections occur wherever both viruses are endemic. It is likely that coinfections have not been detected until recently since surveys for CEV have not been undertaken in the USA and surveys for KHV are sparse (Thresher et al., 2018).

F I G U R E 3   Phylogeny (neighbour‐joining) of partial 4a sequences of CEV from carp and koi. Genogroups I and II are denoted by red and blue, respectively. Node labels indicate bootstrap support values [Colour figure can be viewed at wileyonlinelibrary.com]

     |  1619PADHI et Al.

If coinfections prove to be more widespread or an emerging trend, it will be important to determine what type of interaction ex‐ists between KHV and CEV in coinfections. Currently, the lack of a cell line for culture of CEV presents a significant obstacle to further research. In general, concomitant infections of viruses in fish result in viral interference of one or both viruses (Kotob, Menanteau‐Ledouble, Kumar, Abdelzaher, & El‐Matbouli, 2016). However, Ouyang et al. (2018) speculated that lower titres of KHV compared with CEV in infected koi may indicate that the coinfections they de‐tected were due to infection with CEV in fish with pre‐existing latent infections of KHV. In contrast, we detected higher copy numbers of KHV compared with CEV in three of four coinfections with the exception of Lake Jonathan where copy numbers of KHV and CEV were comparable. Despite the novelty of coinfection of KHV with another virus, there is existing evidence that CEV has been associ‐ated with other viruses, including archived samples previously found to be positive for SVCV (Lewisch, Gorgoglione, Way, & El‐Matbouli, 2015; Way et al., 2015) and with CyHV‐1 (Stevens et al., 2018). Thus, testing for CEV should be done whenever viral infections of carp are suspected in wild or cultured populations. The duplex qPCR de‐veloped in this study will be useful to detect coinfection in a single reaction cost‐effectively. Further reporting and research are needed to determine the significance of coinfections of KHV and CEV and the potential risks they pose to wild carp and cultured koi.

The impacts of KHV and CEV on wild carp in the Midwest re‐main unclear. Based on previous estimates of carp biomass in lakes in southern Minnesota (Bajer & Sorensen, 2012), it is unlikely that out‐breaks observed in the present study represent a high percentage of the population. However, our estimates of mortality are derived from informal observations of visible dead carp which likely under‐estimate the actual level of mortality. Thresher et al. (2018) reported that mortality events of carp attributed to KHV generally do not ap‐pear to have significant short‐term impacts on populations of adult carp and that conspicuous mortality events of wild carp are consis‐tently one‐off events. Indeed, subsequent mortality events were not reported in 2018 in lakes where mass mortality of carp occurred in 2017. However, it has been shown that certain developmental stages of pre‐adult carp are more susceptible to KHV than adults (Raj et al., 2011), which may result in recruitment declines as juvenile carp are exposed to persistently infected adults over time. Although impos‐sible to retrospectively test, it has been speculated that widespread and continuing population declines of carp in the Upper Mississippi River Basin could be associated with recruitment failure caused by an infectious disease, such as KHV (Gibson‐Reinemer, Chick, VanMiddlesworth, & Casper, 2017).

We determined that isolates of KHV from wild carp in three sepa‐rate states are of the European lineage by molecular characterization of the TK gene. Both the European and Asian genotypes have been iso‐lated from carp and ornamental koi (Dong, Li, Weng, Xie, & He, 2013; Kurita et al., 2009). We found few mutations among strains identified in this study, which is consistent with the low genetic diversity found among even geographically distant strains of KHV (Aoki et al., 2007, Hammoumi et al., 2016). The TK gene open reading frame had enough

polymorphisms to distinguish between the two primary genotypes of KHV; however, full genome sequencing should be pursued to identify subtypes. For CEV, sequencing of the P4a gene (Matras et al., 2017) revealed that the new sequences identified from Minnesota, Iowa and Wisconsin clustered in genogroup I along with the recently reported sequence from New Jersey by Lovy et al. (2018). To date, all CEV se‐quences derived from koi worldwide have clustered with genogroup II, suggesting that CEV has been introduced to multiple states via move‐ment of carp rather than the release of ornamental koi. It will be im‐portant to identify the mechanisms of spread for both KHV and CEV in wild populations of carp if new introductions are to be controlled.

This study demonstrates the value of investigating fish kill events. While it is possible that this study was coincidentally timed with regionally widespread emerging outbreaks of KHV and CEV in wild carp, we believe this is unlikely. It is more likely that the dedi‐cated effort to increase reporting and pursue investigation of mor‐tality events of wild carp revealed the widespread distribution of the viruses. Indeed, during the course of this study, previously undocu‐mented carp kills were acknowledged by agency biologists. These reports, however, were not included in this study due to concerns for recall bias and data limitations. While fish kill samples are often col‐lected in advanced stages of decomposition, we found that the use of molecular diagnostics was useful in screening for viral pathogens. Though this approach is unable to conclusively link viral detection with clinical disease, it has nevertheless provided more information than would have otherwise been possible. We recommend an in‐creased awareness and targeted sampling of carp mortality events (vs. screening apparently healthy populations) to provide a more cost‐effective approach to better understand the distribution and impacts of KHV and CEV in the USA.

ACKNOWLEDG EMENTS

This project would not have been possible without the dedicated efforts of those who collected carp for this study, including the Minnesota Department of Natural Resources pathology laboratory and field biologists, Carp Solutions LLC, Wisconsin Department of Natural Resources, Pennsylvania Fish and Boat Commission and the Bajer Lab at the University of Minnesota Aquatic Invasive Species Research Center. Funding for this project was provided by the Environment and Natural Resources Trust Fund as recommended by the Minnesota Aquatic Invasive Species Research Center.

CONFLIC T OF INTERE S T

The authors have no conflicts of interest to declare.

DATA AVAIL ABILIT Y S TATEMENT

The authors confirm that the data supporting the findings of this study are available within the article and its supplementary materi‐als. Sequence data are available in GenBank (GenBank accession no. MK987085‐MK987098 and MK990708‐MK990715).

1620  |     PADHI et Al.

ORCID

Nicholas B. D. Phelps https://orcid.org/0000‐0003‐3116‐860X

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SUPPORTING INFORMATION

Additional supporting information may be found online in the Supporting Information section at the end of the article.

How to cite this article: Padhi SK, Tolo I, McEachran M, Primus A, Mor SK, Phelps NBD. Koi herpesvirus and carp oedema virus: Infections and coinfections during mortality events of wild common carp in the United States. J Fish Dis. 2019;42:1609–1621. https ://doi.org/10.1111/jfd.13082