Koi Herpesvirus Disease Research Articles

Propagation of koi herpesvirus using embryonated chicken eggs: a potential substitute method for fish vaccine production?

Koi herpesvirus disease (KHVD) is caused by a large DNA virus that commonly infects carp, Cyprinus carpio (koi and common carp). KHVD has spread rapidly across the globe and caused high mortality in all ages of common carp and koi. Until now, no effective treatment has been applied to prevent KHV infection impacting the mass production of koi and common carp . An environmentally friendly alternative strategy for controlling fish disease is vaccination. One of the challenges facing conventional viral vaccine production is the requirement for koi or common carp cell cultures, which must be frequently maintained with expensive materials required for virus propagation. This study aims to obtain KHV that has been propagated in embryonated chicken eggs (ECE) to formulate an affordable KHV vaccine for the aquaculture industry. This research consisted of three stages; the first stage was virus inoculation into various parts of eggs (allantoic fluid, chorioallantoic membrane, amniotic fluid, and egg yolk). The second stage was observing viral growth and collecting ECE fluid and membranes. The third stage involved quantitatively determining the viral genomic copy numbers using the quantitative Polymerase Chain Reaction (qPCR) assay. KHV propagation in various parts of ECE resulted in varying viral genomic copy numbers with a high DNA copy number reported in allantoic fluid on the third day after inoculation. Further work is required to monitor virus titer in later passages and optimize methodology for using ECE as the potential alternative to cultured cells as the medium for virus propagation. In the future the system could be developed to produce promising vaccine candidates with more affordable vaccine prices for the Indonesian fish farming industry.

Immunoinformatics Design of Multi-Epitope Peptide-Based Vaccine Against Cyprinid Herpesvirus-3 (CyHV-3) Targeting Thymidine Kinase Proteins

The common carp Cyprinus carpio is a freshwater teleost and is among the most economically significant fishes in aquaculture throughout the world. Taxonomically, C. carpio are a complex of species including subspecies Cyprinus carpio carpio. C. carpio are now threatened by Cyprinid Herpesvirus-3 (CyHV-3), the causative agent of Koi Herpesvirus Disease (KHVD), which causes severe morbidity and mortality in ornamental koi and common carp and can infect or be transmitted by other species. Despite these devastating circumstances, effective vaccinations or other medications for the control of KHVD are not readily available. For this reason, the aim of the current study was to formulate a multi-epitope vaccine against Cyprinid Herpesvirus-3 (CyHV-3) using an immunoinformatics approach. To assess the immunodominant T- and B-cell epitopes, the CyHV-3 proteomes were employed. Following a thorough evaluation, we constructed a strategy for vaccination employing four possible epitopes selected from among each of the three relevant epitope groups: cytotoxic T-lymphocyte, helper T-lymphocyte and linear B-lymphocyte. Important qualities used in the evaluation of the resultant vaccine are that it will be highly soluble, antigenic, immunogenic and non-allergenic. Among acceptable physicochemical qualities, the anticipated structure of the vaccine bears a close resemblance to that of the original protein. Additional considerations include a robust and sustained predicted binding between the vaccine and the Toll-Like Receptor (TLR9). Simulations of molecular dynamics confirm the likelihood of a strong binding stability and structural tightness. Moreover, the computer-generated immunological simulation revealed that the vaccine, when administered to fish, should induce immune responses comparable to those in real life. Finally, codon optimization based on Escherichia coli K12 produced favorable indications of GC content and acceptably high CAI value, as applicable to the cloning vector pET28+ (a). Overall, these results show that the proposed peptide vaccine is a promising option for CyHV-3 prophylaxis.

First detection of Koi herpesvirus disease (KHVD) in Garmian, Kurdistan region of Iraq: A clinical and molecular study.

Koi herpesvirus disease (KHVD) is attributed to cyprinid herpesvirus-3 (CyHV-3) and predominantly affects common carp and ornamental koi carp (Cyprinus carpio). This viral infection leads to substantial morbidity and mortality among these fish species. This study aimed to confirm the presence of KHVD in the Kurdistan region of Iraq by employing clinical and optimized molecular assays on fish populations experiencing high mortality among common carp in carp farms. The present research was conducted in the Kalar district, situated at the heart of Garmian province in Iraqi Kurdistan. four samples from common carp fish farms were received by our laboratory. These samples specifically displaying clinical signs associated with koi herpesvirus (KHV) infection, were subjected to clinical examinations, and PCR assay in addition to sequence analysis. The results of the current study revealed that the observed clinical signs, particularly gill necrosis, skin lesions, and sunken eyes, closely resembled the clinical signs of KHVD in common carp fish. In addition, PCR, nested PCR, and sequence analysis assay detected appropriate DNA fragments of the CyHV-3 major capsid protein gene confirming the first detection of KHVD in common carp fish in the Kurdistan region of Iraq. In this study, the results confirm the detection of KHVD in the Kurdistan region, Iraq, for the first time. This study revealed that CyHV-3 was responsible for KHVD-related signs and symptoms. Based on these results, it is strongly recommended that comprehensive studies be initiated to investigate the prevalence and distribution of CyHV-3.

The engineered ORF56–57 deletion mutant leads to attenuation of an Asian koi herpesvirus strain

Koi herpesvirus (KHV) poses a significant threat to the koi and common carp aquaculture industry due to its high mortality rate among infected fish. In this study, we successfully isolated an Asian KHV strain designated as SS09 and investigated the potential attenuation of the SS09 through the deletion of open reading frames 56 and 57 (ORF56–57). By utilizing homologous recombination technology, we generated a mutant strain with a deletion of ORF56–57, known as SS09 Δ56–57. The mutant strain exhibited slightly reduced viral replication efficiency in common carp brain (CCB) cells. Furthermore, challenge trials conducted on healthy koi populations revealed that fish infected with the SS09 Δ56–57 did not show mortality, whereas the mortality rate of fish infected with the wild strain was nearly 100%. This suggests that the deletion of ORF56–57 can lead to the attenuation of the Asian KHV strain. To further validate the feasibility of SS09 Δ56–57 as novel vaccine candidates or therapeutic interventions against KHV, we conducted immersion immunization of koi fish with three different doses of SS09 Δ56–57. Serum neutralizing antibody titers were determined, and challenge tests were performed three weeks after immunization. The results showed that SS09 Δ56–57 at different doses exhibited a relative percentage survival (RPS) ranging from 70.0% to 96.6%. The neutralization titer of anti-serum in fish following 3 weeks of inoculation also displayed a dose-dependent response. In summary, owing to its outstanding immunogenicity, safety, and efficacy, SS09 Δ56–57 can be regarded as a promising candidate vaccine strain for KHV disease.

Prevalence of Cyprinid herpesvirus 3 and ORF150 genomic variations in carp populations of Indonesia

Cyprinid herpesvirus-3 (CyHV-3) is the etiological agent of koi herpesvirus disease (KHVD) in common and koi carp farming. This highly contagious pathogen has a 295-kb genome that harbours 156 open reading frames. Recent in vitro experimental evolution studies highlighted strong dynamics of genomic structural variations (SVs), in particular in the region of ORF150, an ORF potentially involved in virus multiplication and host inflammatory response. Among these SVs, a 1363-bp deletion could be associated with a loss of virulence. The present study aimed at investigating the genomic variations in the ORF150 region, and especially the deletion, in viruses isolated from carp populations of Indonesia. A screening of 236 fish from 43 different farms revealed a high prevalence of CyHV-3 (nearly 70%), both in symptomatic and asymptomatic common carp. However, in contrast with the results obtained in vitro, long read sequencing of the ORF150 region revealed a low level of genetic variations and the absence of the 1363-pb deletion. The complex interactions between the virus, the environment and the host, particularly the immune system, probably play an important role in this reduced variability.

Purification and concentration of infectious koi herpesvirus using steric exclusion chromatography.

Koi herpesvirus (KHV) is the causative agent of a koi herpesvirus disease (KHVD) inducing high mortality rates in common carp and koi (Cyprinus carpio). No widespread effective vaccination strategy has been implemented yet, which is partly due to side effects of the immunized fish. In this study, we present an evaluation of the purification of infectious KHV from host cell protein and DNA, using the steric exclusion chromatography. The method is related to conventional polyethylene glycol (PEG) precipitation implemented in a chromatographic set-up and has been applied for infectious virus particle purification with high recoveries and impurity removal. Here, we achieved a yield of up to 55% of infectious KHV by using 12% PEG (molecular weight of 6 kDa) at pH 7.0. The recoveries were higher when using chromatographic cellulose membranes with 3-5 μm pores in diameter instead of 1 μm. The losses were assumed to originate from dense KHV precipitates retained on the membranes. Additionally, the use of >0.6 M NaCl was shown to inactivate infectious KHV. In summary, we propose a first step towards a purification procedure for infectious KHV with a possible implementation in fish vaccine manufacturing.

Genetic parameters and genomic prediction of resistance to koi herpesvirus disease using a low-density SNP panel on two Amur mirror carp populations

Koi herpesvirus disease (KHVD), caused by Cyprinid herpesvirus-3 (CyHV-3), is one of the most serious threats to carp farming. In the present study, we investigated the efficiency of a low-density (LD) SNP panel for estimating genetic parameters and breeding values to KHVD resistance in the Amur mirror carp (AMC). Two populations (Pop 1 and Pop 2) of AMC generated from unrelated parents were created using a partial factorial design. One-year old fish (Pop 1 = 1500 individuals.; Pop 2 = 1200 individuals) were challenged with CyHV-3 and phenotyped to KHVD resistance. 218 SNPs originating from a medium genotyping platform previously applied to Pop 1 (15615 SNPs; denoted as MD panel) with the highest association to KHVD resistance were used as a LD panel to genotype individuals of Pop 2. Genetic parameters and estimated pedigree-based BLUP (EBV) and genomic-based GBLUP (GEBV_MD and GEBV_LD) breeding values were calculated and obtained for Pop 1 using either pedigree, MD or LD panel and for Pop 2 using either pedigree or the LD panel. The heritability estimates of KHVD resistance were very high for both populations ranging from 0.42 to 0.96. Selection for KHVD resistance in Pop 2 using the LD panel would have led to a relative increase of ∼7% in prediction accuracy compared to the pedigree-based selection. Pearson correlation coefficients between pedigree-based and genomic-based estimated breeding values (EBV vs. GEBV_MD; EBV vs. GEBV_LD; GEBV_MD vs. GEBV_LD) showed a strong association for both populations (0.79 – 0.91). In addition, the concordance rate of individuals selected by pedigree-based (EBV) and genomic-based breeding values (GEBV_MD and GEBV_LD) within selection pressures of 5%, 10% and 20% were not statistically different in most cases. In conclusion, the low-density SNP panel could be useful for a selection program focused on the genetic improvement of KHVD resistance.

Development and Application of a Triplex PCR Assay for Detection of Koi Herpes Virus (KHV) Gene Infected Common Carp (Cyprinus carpio L.)

Koi herpesvirus (KHV) has been responsible for worldwide KHV disease (KHVD) outbreaks, resulting in high mortality rates, and has been reported in Cyprinus carpio cultivation. A Triplex Polymerase Chain Reaction (T-PCR) was developed for the detection of KHV gene to improve the diagnosis of KHV infection. Three sets of primers were designed targeting specific sequences of KHV gene which were frequently used in PCR assay for detecting KHV (modifications primer by Yuasa,290 bp; Gilad 484 bp, and TK, 409 bp) in once PCR running. The validity test was conducted by verified using DNA sample of Aeromonas hydrophila. Sensitivity test of singleplex and Triplex conducted by dilution. The result showed that T-PCR method using these pairs of primers is at 55 annealing temperature with consistency of T-PCR method has been repeated in triplicates, and with the same results. The validity test found that no band detected with A. hydrophila. This Triplex PCR-based assay may have useful for the clinical diagnosis tool of KHV disease.

The analysis of a cluster of koi herpesvirus disease outbreaks in intensive carp aquaculture using molecular and conventional epidemiology.

The analysis of a cluster of koi herpesvirus disease outbreaks in intensive carp aquaculture using molecular and conventional epidemiology.

The Babylon River's common carp (Cyprinus carpio) gills were used for the histopathological examination and PCR detection of Koi herpesvirus disease (KHVD)

The first outbreak of koi herpesvirus in Iraq occurred in Babylon Province in the floating cages in the Euphrates River from the Musayyib thermal electric power station to the Al-Hindiya Dam. Fish were suffering from gills rot that did not respond to treatment and lesions and ulcers on the fish's body. The fatalities reached 80%. was water temperature 24°C, pH 6.85, Salinity 760 ppt. So the present study aims to highlight the pathological changes in gills after the Koi herpes virus infection in Babylon Province. The study started after the detection of (KHV) by polymerase chain reaction ( PCR) from the suspected samples (30 ) for the detection of virus nucleic acid. Also, study the water characterization during the periods of outbreaks. The gills specimen was collected from the positive cases for gross and histopathological examination. The result showed that ten samples were positive for (KHV) infection. The gross and histopathological examination results showed severe congestion with necrotic foci and sloughing of secondary lamella with increased mucus secretion. In addition, necrosis in the primary lamellae, Edam and inflammatory cells infiltration with hyperplasia of the secondary lamellae with the presence of intranuclear inclusion body in all exanimated slides. Keywords: Koi Herpes; Cyprinus Carpio; Euphrates River; KHV; secondary lamellae; floating cages

The application of exopolysaccharides (EPS) can prevent viral disease of fish

In the frame of investigations on the use of exopolysaccharides (EPS) from Arthrospira platensis in carp and koi cultures, two animal experiments were carried out to assess their efficiency as prophylactic and metaphylactic (therapeutic) measures against koi herpesvirus disease (KHVD). In experiment 1, carp were treated with algae biomass (BM) and EPS before and after infection with European lineage koi herpesvirus (KHV-E). In experiment 2, carp were treated with EPS prior and after the infection with Taiwan isolate of KHV (KHV-T), only prior to infection with higher concentration of EPS or only after the infection with KHV-T. No conclusive protection against KHV was observed in experiment 1 in carp treated with BM. In groups where EPS was applied, carp were protected to a certain extent. In experiment 2, carp were protected significantly against a severe KHVD outbreak. In the prophylactic group, which received a double EPS concentration for six weeks, and in the metaphylactic group, KHVD was stopped. Fish developed antibodies against EPS as well as against KHV at day 30 post infection.

Development of an attenuated vaccine against Koi Herpesvirus Disease (KHVD) suitable for oral administration and immersion

Since the end of the1990ies, Cyprinid herpesvirus 3 (also known as koi herpesvirus, KHV) has caused mass mortality events of koi and common carp all over the globe. This induced a high economic impact, since the KHV disease cannot be cured up to now, but only prevented by vaccination. Unfortunately, there is only one commercial vaccine available which is not approved in most countries. Therefore, there is an urgent need for new, safe and available vaccines. In this study, a live attenuated vaccine virus was generated by cell culture passages of virulent KHV, and shown to protect carp or koi after immersion or oral application against wild type challenge. An advantage of boost immunization was demonstrated, especially after oral application. Vaccination induced no or mild clinical signs and protecting antibodies have been measured. Additionally, the vaccine virus allowed differentiation of infected from vaccinated animals (DIVA) by PCR. The attenuation of the newly generated vaccine was tracked down to a partial deletion of open reading frame 150. This was confirmed by the generation of engineered ORF150 deletion mutants of wild-type KHV which exhibited a similar attenuation in vivo.

Cytokines Studied in Carp (Cyprinus carpio L.) in Response to Important Diseases

Cytokines belong to the most widely studied group of intracellular molecules involved in the function of the immune system. Their secretion is induced by various infectious stimuli. Cytokine release by host cells has been extensively used as a powerful tool for studying immune reactions in the early stages of viral and bacterial infections. Recently, research attention has shifted to the investigation of cytokine responses using mRNA expression, an essential mechanism related to pathogenic and nonpathogenic-immune stimulants in fish. This review represents the current knowledge of cytokine responses to infectious diseases in the common carp (Cyprinus carpio L.). Given the paucity of literature on cytokine responses to many infections in carp, only select viral diseases, such as koi herpesvirus disease (KHVD), spring viremia of carp (SVC), and carp edema virus disease (CEVD), are discussed. Aeromonas hydrophila is one of the most studied bacterial pathogens associated with cytokine responses in common carp. Therefore, the cytokine-based immunoreactivity raised by this specific bacterial pathogen is also highlighted in this review.

Investigation of Cyprinid Herpesvirus 3 (CyHV-3) Disease Periods and Factors Influencing CyHV-3 Transmission in A Low Stocking Density Infection Trial.

Cyprinid herpesvirus 3 (CyHV-3) is the etiological agent of koi herpesvirus disease (KHVD) and important pathogen of aquaculture and wild populations of common carp worldwide. Understanding the relative contributions of direct and indirect transmission of CyHV-3 as well as the factors that drive CyHV-3 transmission can clarify the importance of environmental disease vectors and is valuable for informing disease modeling efforts. To study the mechanisms and factors driving CyHV-3 transmission we conducted infection trials that determined the kinetics of KHVD and the contributions of direct and indirect forms of CyHV-3 transmission, as well as the contributions of contact rate, viral load, pathogenicity and contact type. The incubation period of KHVD was 5.88 + 1.75 days and the symptomatic period was 5.31 + 0.87 days. Direct transmission was determined to be the primary mechanism of CyHV-3 transmission (OR = 25.08, 95%CI = 10.73-99.99, p = 4.29 × 10-18) and transmission primarily occurred during the incubation period of KHVD. Direct transmission decreased in the symptomatic period of disease. Transmissibility of CyHV-3 and indirect transmission increased during the symptomatic period of disease, correlating with increased viral loads. Additionally, potential virulence-transmission tradeoffs and disease avoidance behaviors relevant to CyHV-3 transmission were identified.

Health status and microbial quality of common carp reared in a pond fed with treated wastewater from a slaughterhouse

Wastewater from slaughterhouses in many countries is still discharged into rivers, without having been adequately treated. Such wastewater contains plenty of organic matter which is an ideal source of nutrients for fish, but also for the development of microorganisms. Thus, usage of wastewater in aquaculture could become a health risk for humans, fish due to the introduction of microorganisms into the aquatic environment. In the available literature, there is insufficient data on health and meat safety regarding common carp reared in purified wastewater. The aim of this study was to assess the health and meat safety of common carp cultivated in a fishpond supplemented with slaughterhouse wastewater that was subjected to tertiary treatment. The number of parasites was not significant and not a single parasitic disease was found in this study, but the number of parasite species detected was as expected and typical for carp production. No spring viraemia of carp or koi herpesvirus disease was found. The carp cultivated were in good health and completely safe for human consumption in terms of the presence of microbial contaminants. The safe use of wastewater for fish rearing should be encouraged, but proper treatment of wastewater must be applied before its use.

Serological analysis of historical field samples reveals major inconsistency between PCR and antibody ELISA for establishing KHV infection status of groups and individual koi

Koi herpesvirus (KHV) is the causative agent of a highly infectious and notifiable disease of Cyprinus carpio L. Serology has the potential to identify koi or carp that have been previously exposed to KHV and which may be possible carriers of the virus. In the present study, sera (n = 162) from groups of farmed koi carp, previously screened for KHV using a variety of molecular methods as part of a surveillance program in Asia from 2008 to 2010, were subsequently tested here individually by ELISA using plates coated with purified virus (American isolate KHV-I, H361). Only 31% of koi from PCR-positive KHV fish groups or populations associated with KHV disease (n = 59/162) were seropositive when screened in the ELISA at a serum dilution of 1/200, in contrast to 52.9% of seropositive koi that were KHV-negative by PCR (n = 103/162). Furthermore >34% of those seropositive/PCR negative fish had titres of >1/400 (moderate-strong responders). This field data highlights the concerns related to carp populations that have been screened for KHV using molecular methods alone and supports the need for serology to accompany molecular testing in carp for this notifiable virus.

Serological responses to koi herpesvirus (KHV) in a non-cyprinid reservoir host.

Koi herpesvirus (KHV) is a highly contagious virus that causes KHV disease (KHVD) inducing high mortality in carp and koi (Cyprinus carpio L.). In the late stage, latency occurs with very low, often non-detectable virus concentrations, which represents a challenge for virus detection. After validation according to OIE recommendations, an antibody ELISA was established to recognize antibodies of C.carpio against KHV infection. In this study, the ELISA was modified to detect anti-KHV antibodies from a non-cyprinid fish. Experimentally infected rainbow trout (Oncorhynchus mykiss) were able to transmit KHV to naïve carp at two different temperatures, demonstrating their potential as a reservoir host. At 20°C, KHVD was induced in carp but not at 15°C. Unexpectedly, rainbow trout developed humoral response against KHV at both temperatures. In contrast to carp, at 15°C trout produced neutralizing antibodies but not at 20°C. While antibodies obtained from infected carp sera reacted in a similar way against all KHV, antibodies from rainbow trout sera reacted differently to the same isolates by ELISA. The data show that even when non-cyprinid fish species are infected with KHV, they can produce antibodies that differ from those observed in carp.

The role of energy reserves in common carp performance inferred from phenotypic and genetic parameters

In temperate zones, energy reserves of fish are closely related to survival during the first winter of their life. In this study, the genetic and phenotypic background of the accumulation, mobilization and utilization of energy reserves was investigated in Amur mirror carp. To achieve this, the role of traits related to energy reserves on fish performance during the first winter and further periods of rearing was investigated. The experimental stock was established by four full-factorial matings of 5 dams and 10 sires to generate up to 200 full-sibling families. The offspring were sampled before and after the first winter rearing period. Seasonal variation in direct and indirect measures of energy status was examined using Fulton's condition factor (FC), hepato-somatic index (HSI), visceral index (VSI_NO), glycogen, fat and protein in hepatopancreas (HP) and muscle fat content. Other performance traits were also recorded (weight, resistance to koi herpesvirus disease). All traits related to energy reserves, except HP protein, were significantly lower after the first winter. Overall, HP glycogen and fat from muscle, HP and viscera were mobilized during winter. However, genetic correlations between same traits recorded in autumn and spring were lower than 0.8 for most of the traits, implying that not all families responded to overwintering in a similar manner. Heritability also differed before and after the first winter. Before the first winter, all traits had low to medium heritability (0.05–0.35), but after the winter the same traits were moderately or highly heritable (0.22–0.58). Interestingly, HP glycogen traits, unlike HP fat and HP protein, and HSI recorded in yearlings were positively genetically correlated with survival during the third growing season (rg = 0.49–0.72). This study provides the first evidence of a genetically based strategy for energy mobilization related to overwintering of common carp. Measuring of FC and HSI could be used to monitor the energy status of common carp and to provide a supplementary tool for management of carp stocks.

Development of a double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) for the detection of KHV.

Koi herpesvirus disease (KHVD) caused by the koi herpesvirus (KHV) is difficult to diagnose in live fish, presenting a challenge to the koi industry. The enzyme-linked immunosorbent assay (ELISA) method cannot be widely used to detect KHV because few commercial anti-KHV antibody exists. Here, we developed an anti-ORF132 polyclonal antibody and confirmed its reactivity via indirect immunofluorescence assay and Western blotting. A double-antibody sandwich ELISA (DAS-ELISA) was established to detect KHV, monoclonal antibody 1B71B4 against ORF92 was used as the capture antibody, and the detection antibody was the polyclonal antibody against the truncated ORF132. The lowest limit was 1.56ng/ml KHV. Furthermore, the DAS-ELISA reacted with KHV isolates, while no cross-reactions occurred with carp oedema virus, spring viraemia of carp virus, frog virus 3 and grass carp reovirus. Two hundred koi serum samples from Guangdong, China, were used in the DAS-ELISA test, and the positive rate of the koi sera was 13%. The clinical sensitivity and specificity of the DAS-ELISA relative to the traditional PCR method were 66.7% and 97.6%, respectively. Our findings may be useful for diagnosing and preventing KHVD in koi and common carp.

A Seasonal Study of Koi Herpesvirus and Koi Sleepy Disease Outbreaks in the United Kingdom in 2018 Using a Pond-Side Test.

Simple SummaryCyprinid herpesvirus (CyHV)-3 and carp edema virus (CEV), the causative agents of koi herpesvirus disease and koi sleepy disease, respectively, are emerging DNA viruses infecting koi and common carp. Similarities in their clinical presentation present difficulties for its on-site identification based on gross pathology. Fluorescence real-time loop-mediated isothermal amplification (LAMP) assays for detecting CyHV-3 and CEV DNA were designed to use border inspection posts and local testing by national authorities for outbreak control. The limit of these tests’ detection (102 and 103 viral copies for CyHV-3 and CEV, respectively) allows for the amplification of viral DNA in clinical samples in less than 20 min. The assays’ field performance was tested with 63 common carp mucus swabs taken during disease investigations in 2018, and the results validated with the reference laboratory analysis. Overall, the good performance, ease of use, and cost-effectiveness of these tests make them good candidates for a point of care test. However, further work is required to incorporate reliable internal controls and improve the sensitivity of these tests’ asymptomatic testing.Fluorescence real-time LAMP assays were designed for the orf43 gene of CyHV-3 European genotype and the p4a gene of the CEV genogroup I. A third LAMP assay to detect the ef1a gene of the host common carp was designed as an internal control. The limit of detection was 102 and 103 viral copies under 25 min for CyHV-3 and CEV, respectively. The specificity of the CyHV-3 LAMP assay was 95.6% of 72 fish herpesviruses tested. Sixty-three non-lethal common carp mucus swabs were collected across 16 sites during disease investigations. DNA extractions were performed in under 10 min using the QuickExtract™ digestion buffer. The LAMP amplification of CyHV-3 DNA in mucus swabs from clinical cases was detected from 4 to 13 min in 13 sites, while a co-infection of CyHV-3 and CEV was confirmed by LAMP in a single site. The LAMP results agreed with the results of the reference laboratory. The common carp ef1a was amplified only in 61% of the mucus swabs collected, preventing its use as a robust internal control to distinguish false negatives from invalid tests. After further optimization, these tests could be implemented for border inspection posts surveillance and decentralizing testing during disease outbreaks.