Detection Of Koi Herpesvirus Research Articles

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.

Optimum Koi Herpesvirus DNA Extraction from the Aquaculture Water

Koi Herpesvirus (KHV) is one of the viral fish disease in the common carp aquaculture. At present, KHV diagnoses is performed using Polymerase Chain Reaction (PCR) method. However, the effort to extract DNA viruses from environmental samples depends on two fundamental problems: inhibition of the extraction and amplification processes and a very small number of viruses. The small amount of virus load in the water requires a concentration process before further molecular analysis. In this study, the sensitivity of three methods of extraction and purification of viral genetic material from the water, namely deposition with PEG, filtration-elution, and flocculation with antibodies for the detection of KHV from the water with the PCR method was performed. Water samples were collected from Cirata reservoir, West Java which is a site location for carp aquaculture. The organic flocculation cannot be carried out in the present study because anti-KHV antibodies cannot be produced. This is because the amount of virus in the sample used by the vaccine is low. Meanwhile, the precipitation method with PEG cannot produce DNAextract’s although quantity and purity are good enough to be amplified. For the filtration-elution method, both with and without the reconcentration process, a clear band of DNA can be produced after amplification. In conclusion, the filtration-elution method is considered as the most suitable method in obtaining KHV DNA’s taken from the water samples.

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.

KHV Detection Based on Thermal Lysis - Nested PCR (Polymerase Chain Reaction)

In the case of common carp (Cyprinus carpio) and goldfish (Carrasius auratus) that were lightly infected with Koi Herpes Virus (KHV), the use of the one-step PCR method was insensitive to detect the presence of KHV DNA. The nested PCR method has been developed for a more sensitive detection system for KHV infection. This study aims to test the sensitivity of one-step and nested PCR methods in detecting the KHV virus in lightly infected common carp to prevent early disease attacks. DNA extraction of the test samples used thermal lysis and PBS (Phosphate Buffered Saline). DNA isolate as a KHV template was used to amplify KHV sequences using KHV-TKf: 5'-GGGTTACCTGTACGAG-3 'and KHV-TKr: 5'-CACCCAGTAGATTATGC-3' primers in the one-step PCR method. The nested PCR method uses two pairs of primers, namely the KHV-TKf: 5'-GACACCACATCTGCAAGGAG-3' and KHV-TKr: 5'-GACACATGTTACAATGGTGGC-3') and the CEFAS primer pair (5'-CGTCGTGAGGAATACGACG-3' and 5'- ACCGTACAGCTCGTACTGG-3') for amplification of KHV sequences. The one-step PCR method was insensitive for detection of KHV in common carp and goldfish infected with viruses without clinical symptoms, and was sensitive enough to detect viruses with mild symptoms with a DNA fragment size of 409 bp using TK primers. The nested PCR method was more sensitive in detecting KHV DNA in fish without clinical symptoms with a DNA fragment size of 348 bp using CEFAS primers.

The First Detection of Koi Herpesvirus (CyHV3) in Migratory Wild Ducks in North America.

A viromics study on the intestinal contents of migratory wild ducks of the genera Anas, Mareca, Spatula, and Oxyura during their winter stay in Mexico showed the presence of the virus family Alloherpesviridae. The genus Cyprinivirus is part of this family and includes cyprinid herpesvirus 3 (CyHV3). This is the etiological agent of the lethal disease known as koi herpesvirosis, which affects different strains of Common Carp Cyrprinus carpio. In this study, samples of the contents of 87 wild duck intestines were analyzed by endpoint PCR, of which 7 samples were positive for the amplification of the TK gene fragment corresponding to CyHV3. These results contribute to the knowledge about the spread of this virus to other species of aquatic animals in areas where fish and ducks coexist.

First detection of koi herpesvirus and carp oedema virus in Iraq associated with a mass mortality in common carp (Cyprinus carpio).

At the end of October 2018, a mass fish mortality occurred in Iraq, involving thousands of tons of cultured and wild common carp (Cyprinus carpio) along Euphrates and Tigris rivers. Fish were found dead or moribund along rivers coasts, showing lethargy, dyspnoea and flared gills. At necropsy, discoloration patches were noticed on the gills. Wet preparations showed rare metacercariae and Dactylogyrus spp. Samples were subjected to bacteriological tests and virological investigation through real-time PCR and nested PCR. Both were positive for koi herpesvirus (KHV) and carp oedema virus. Results obtained were confirmed by the OIE reference laboratory of KHV disease (KHVD) at Cefas (UK) and by sequence analysis. This is the first report on the detection of both viruses in Iraq.

Preparation of monoclonal antibodies against KHV and establishment of an antigen sandwich ELISA for KHV detection

Infections with koi herpesvirus (KHV) in carp are still a severe problem worldwide. Detection and elimination of infected fish are necessary for control of the Koi herpesvirus disease (KHVD). Serum is an excellent specimen for KHV testing because of high survivability of KHV in serum and ease of collection, storage, and handling. The direct detection of fish viruses based on the sandwich ELISA has emerged as a practical and reliable means of diagnosis. Thus, it is important to create monoclonal antibodies (MAbs) against purified KHV. By using hybridoma-monoclonal antibody technology, two hybridoma cell lines secreting MAbs against the KHV were established. By Western blot and IFAT analysis, the secreted MAbs from cell line IB7IB4 and cell line 7C72F7 recognized proteins of KHV. The result demonstrated that the MAbs were highly specific and sensitive to the KHV, and can be used for monitoring the virus quantification of carp, for example, the direct KHV diagnosis by sandwich enzyme-linked immunosorbent assay(ELISA). An antigen sandwich ELISA applying the biotin-avidin system was established using the biotinylated MAb IB7IB4 and 7C72F7 to detect virus in koi sera. These MAbs did not react with any of the tested other viruses by ELISA except KHV. The detection limit of the test was 3.923ng/ml KHV. Thus, this antigen sandwich ELISA is suitable for recognition of KHV.

Detection of koi herpesvirus in healthy common carps, Cyprinus carpio L.

Koi herpesvirus (KHV), a member of Hervesviridae, has been frequently reported to cause mass mortality (80-100%) in common carps (Cyprinus carpio L.). A unique feature of Herverviridae members is latent infection, maintaining their genetic information for an extended period in the absence of productive infection, and reactivate when environmental conditions are favorable for their growth. To prevent this occurs, a monitoring program should be done for early detection. This study aimed at detecting the presence of KHV in healthy common carps reared in West-Nusa Tenggara Province, Indonesia. A total of 80 healthy fish was collected randomly from eight fish farms (Lingsar, Batu Kumbung, Narmada, Tanjung, Lenek, Aik Mel, Brang Rea and Rhee) across West-Nusa Tenggara Province, Indonesia. The presence of KHV genome was detected using a PCR with a commercial kit, IQ 2000TM. The result showed that common carps collected from four farms (Aik Mel, Lenek, Rhee and Brang Rea) were positive KHV. The size of an amplified gene was ~ 550bp which was the same as positive KHV control. The obtained result suggests that KHV as other member of Hervesviridae shows a latent infection in common carps, and should be anticipated for their reactivation. Based on this result it is highly recommended that common carps cultured in this region should be vaccinated. In addition, transporting common carp out from Lombok and Sumbawa Islands should be carefully regulated to prevent the spread of the disease to other areas.

An integrated microfluidic loop-mediated isothermal amplification platform for koi herpesvirus detection

An integrated microfluidic loop-mediated isothermal amplification (LAMP) platform consisting of a microfluidic chip and a portable operating system is proposed for koi herpesvirus (KHV) detection applications. The microfluidic chip is fabricated using a CO2 laser system and comprises a sample chamber, two reagent chambers, a mixing chamber, a serpentine channel, a reaction chamber, and three flow rectifiers based on PDMS membranes. The main components of the operating system include a power source, a time relay, a microfluidic chip holder, four gas pressure valves, a microheater, and a temperature controller. In the proposed detection method, the KHV sample and LAMP reagent are loaded into the sample and reagent chambers, respectively, and are mixed under the effects of an external gas pressure driving force. The mixed solution is then heated at a temperature of approximately 63 °C for 60 min in order to replicate the KHV sample. Finally, the replicated sample is mixed with SYBR Green I dye in order to facilitate fluorescence-based KHV detection. The feasibility of the proposed microfluidic platform is confirmed by comparing the detection results obtained for a sample containing 10 ng/μL KHV with those obtained using a gel electrophoresis PCR-based method. The experimental results shows that the proposed LAMP platform has a KHV detection limit of around 3 × 10−2 ng/μL. Overall, the results presented in this study show that the proposed microfluidic platform provides a compact, reliable and portable tool for KHV detection purposes.

Validation of a KHV antibody enzyme-linked immunosorbent assay (ELISA).

Koi herpesvirus (KHV) causes KHV disease (KHVD). The virus is highly contagious in carp or koi and can induce a high mortality. Latency and, in some cases, a lack of signs presents a challenge for virus detection. Appropriate immunological detection methods for anti-KHV antibodies have not yet been fully validated for KHV. Therefore, it was developed and validated an enzyme-linked immunosorbent assay (ELISA) to detect KHV antibodies. The assay was optimized with respect to plates, buffers, antigens and assay conditions. It demonstrated high diagnostic and analytical sensitivity and specificity and was particularly useful at the pond or farm levels. Considering the scale of the carp and koi industry worldwide, this assay represents an important practical tool for the indirect detection of KHV, also in the absence of clinical signs.

Detection of koi herpesvirus (KHV) using a monoclonal antibody against Cyprinus carpio IgM.

Koi herpesvirus disease (KHVD) is associated with high mortality in both common carp and koi carp (Cyprinus carpio L.) worldwide. The indirect detection of fish viruses based on the identification of antibodies has emerged as a practical and reliable means of diagnosis. Thus, it is important to create monoclonal antibodies (MAbs) against carp IgM. By using hybridoma-monoclonal antibody technology, one hybridoma cell line secreting MAbs against IgM from carp was established. In western blot analysis, the secreted MAb from cell line A5-E10 recognized the heavy chain of IgM from common carp or koi but did not react with immunoglobulins from three different fish species: grass carp (Ctenopharyngodon idella), tilapia (Oreochromis mossambicus) and Mandarin fish (Siniperca chuatsi). These results demonstrated that this MAb is highly specific for the IgM of carp and suggested that it can be used for monitoring the immunity level of carp, for example for indirect KHV diagnosis by antibody ELISA. We therefore established an indirect ELISA, which was tested using 200 serum samples from koi from three farms. The final results showed that 147 (73.5%) samples were confirmed to be KHV antibody negative and 53 (26.5%) were definitely positive, containing antibodies against KHV.

First detection of koi herpesvirus from koi, Cyprinus carpioL. experiencing mass mortalities in Iran: clinical, histopathological and molecular study.

Koi herpesvirus (KHV) is the aetiological agent of an emerging disease (KHVD) associated with mass mortalities in koi and common carp and reported from at least 30 countries. We report the first detection of KHV from koi in Iran using clinical, histopathological and molecular studies. KHV-infected fish showed reduced swimming activity, sunken eyes and increased mucus production on skin and fins. On post-mortem examination, gill necrosis was observed in the majority of fish. Histopathologically, the gill showed diffuse necrosis of the branchial epithelial cells. Margination of chromatin was detected in gills, kidney, heart, spleen, intestine and brain. In addition, sequence analyses of the TK gene, ORF 136 and marker I and II, demonstrates that Iranian KHV isolates were identical and classified as variant A1 of TUSMT1 (J strain) and displayed the I(++) II(+) allele of this Asian genotype.

Detection of koi herpesvirus (KHV) in healthy cyprinid seed stock.

Koi herpesvirus (KHV) disease is a lethal disease in common carp, an important food fish in Asian countries, the seed of which is used in restocking programs for freshwater fishery management. We inspected apparently healthy seed stock of common carp Cyprinus carpio L. and Siberian crucian carp Carassius auratus for the presence of KHV using PCR-based diagnostic tests as a part of a stock enhancement program from 2009 to 2010 in Korea. Consequently, KHV was detected from 24 of 232 inspections with yearly detection percentages of 5.2% in 2009 and 15.5% in 2010 using PCR primer sets for TK or SphI-5 as recommended by the OIE Manual of Diagnostic Tests for Aquatic Animals. Results indicate that the SphI-5 primer set was slightly more sensitive than the TK primer set, as shown by a higher detection rate. To determine the genotype of the KHV strains detected in this study, ORF40-specific PCR amplification was conducted, and the PCR products from 6 samples showed 100% nucleotide sequence identity with a Japanese strain (GenBank accession number AP008984) but not with US (DG657948) and Israeli strains (DG177346). This report conclusively demonstrated the presence of KHV in externally healthy seed of common carp and Siberian crucian carp, indicating a possible risk that subclinically infected seed stock can be released with a potential threat to wild populations.

Development and characterization of a cell line from the snout of koi (Cyprinus carpio L.) for detection of koi herpesvirus

A permanent growing cell line, designated as koi snout (KS), was established from snout tissues of koi utilizing the trypsin method. The KS cell line was maintained in Leibovitz's L-15 medium supplemented with 10–20% fetal bovine serum (FBS) within incubation temperatures ranging from 22 to 27 °C. Maximum cell proliferation rates occurred at 27 °C in Leibovitz's L-15 medium containing 10% FBS. The KS cell line was subcultured more than 115 times, and karyotyping analysis indicated the modal chromosome number was 2n = 100. The results of virus isolation demonstrated that KS cells were susceptible to koi herpesvirus (KHV), which was demonstrated by the presence of obvious cytopathic effects and abundant virus particles with high virus titer of 106.98 TCID50/mL. Immunofluorescence and Western blot assays provided confirmation that KS could replicate KHV. The newly established KS cell line will provide a useful tool for KHV detection.

Sensitivity of seven PCRs for early detection of koi herpesvirus in experimentally infected carp, Cyprinus carpio L., by lethal and non‐lethal sampling methods

Koi herpesvirus (KHV) causes an economically important, highly infectious disease in common carp and koi, Cyprinus carpio L. Since the occurrence of mass mortalities worldwide, highly specific and sensitive molecular diagnostic methods have been developed for KHV detection. The sensitivity and reliability of these assays have essentially focused at the detection of low viral DNA copy numbers during latent or persistent infections. However, the efficacy of these assays has not been investigated with regard to low-level viraemia during acute infection stages. This study was conducted to compare the sensitivity of seven different polymerase chain reaction (PCR) assays to detect KHV during the first hours and days post-infection (hpi; dpi), using lethal and non-lethal sampling methods. The results highlight the limitations of the assays for detecting virus during the first 4dpi despite rapid mortality in experimentally infected carp. False-negative results were associated with time post-infection and the tissue sampled. Non-lethal sampling appears effective for KHV screening, with efficient detection in mucus samples obtained from external swabs during this early infection period (<5dpi), while biopsies from gills and kidney were negative using the same PCR assays. Non-lethal sampling may improve the reliability of KHV detection in subclinical, acutely infected carp.

Production of monoclonal antibody against ORF72 of koi herpesvirus isolated in Taiwan

A monoclonal antibody (MAb) was generated against the capsid protein (ORF 72) of koi herpesvirus (KHV) isolated from diseased koi Cyprinus carpio in Taiwan. The clone of MAb-B2 was obtained by immunizing mice with whole virus particles and further identified using indirect enzyme-linked immunosorbent assay and Western blot assay. In addition, it detected KHV in KHV-infected cells but not in those of mock-infected cells as demonstrated by indirect immunofluorescence assay. The neutralization test showed that MAb-B2 neutralized KHV. Furthermore, we uncovered that MAb-B2 recognizes the ORF72 of KHV as revealed by liquid chromatography-tandem mass spectrometry and Western blot assays. Additionally, MAb-B2 has been used as a diagnostic tool for detection of KHV in clinical samples by immunohistochemistry. Collectively, our results indicated that MAb-B2 could be used in the development of a diagnostic kit for diagnosis of KHV infections and ORF72 protein of KHV might be a candidate for future vaccine development.

Detection of Koi herpesvirus: impact of extraction method, primer set and DNA polymerase on the sensitivity of polymerase chain reaction examinations

Since virus isolation is seldom successful, KHV infection is commonly detected by PCR examination. A number of different PCR assays have been described in recent years. However, at present no commonly accepted PCR method is used amongst different laboratories. The aim of this study was to check if the examination of infected fish by different PCR methods yielded comparable results. We used tissue samples of three KHV-infected koi, one KHVinfected common carp, one KHV-infected goldfish and one non-infected common carp. DNA was extracted with DNAzol Reagent, High Pure PCR Template DNA Preparation Kit and QIAamp DNA Mini Kit. The DNA was tested by PCR with different combinations of published primer sets –KHV-F and -R, KHV-Gray-2F and -2R and KHV-TKf and -TKr – plus different DNA polymerases – a standard Taq DNA polymerase, a Platinum (hotstart) Taq DNA polymerase and a Platinum (hotstart) Pfx DNA polymerase with proofreading activity. The different extraction methods produced DNA solutions with different yields of DNA and different degrees of homogeneity. Also, the sensitivity of the PCR depended on the choice of the primer set and polymerase. Not all infected fish could be identified with all methods; there were large differences in the sensitivity between methods.

Detection of koi herpesvirus (KHV) from koi (Cyprinus carpio koi) broodstock in South Korea

The presence of koi herpesvirus (KHV) was confirmed in three ornamental koi broodstocks for breeding purposes. The broodstocks showed poor appetite, lethargy, abnormal swimming behavior with gasping movements in shallow water and increased mucus secretion. Histopathologically, the gill showed diffuse necrosis of the branchial epithelial cells and diffuse lymphocytic–monocytic interstitial nephritis with necrosis of the tubular epithelial cells predominantly found in these three broodstocks. The presence of viral intranuclear inclusion bodies were also observed. KHV was not isolated using KF-1 cell line but PCR positive results at 484bp DNA product was confirmed in all organs of the fish and quatification of KHV genomic DNA using real-time Taqman PCR in all organs ranged from 1.32×10−9 to 1.39×10−9 copy numbers. The analysis of sequencing data of 484bp fragments of KHV DNA of three Korean isolates revealed 100% identities with each other and closely related (98 to 99%) to the known CyHV-3 type of KHV. This report confirmed the presence of KHV infection in one koi commercial farm in South Korea using pathological and molecular methods.

Koi Herpes Virus: Do Acipenserid Restitution Programs Pose a Threat to Carp Farms in the Disease-Free Zones?

Background. Sturgeons have long been extinct in Polish inland waters. A substantial effort has recently been put into their restitution, covering the drainage areas of two major Polish rivers, the Oder and the Vistula. The stocked fishes are clinically healthy, but very little is known about their potential to transmit viral diseases including koi herpes virus (KHV) to healthy fishes of other species, which may pose a threat to the disease-free zones. This study was intended to determine if sturgeons could be asymptomatic carriers of KHV. Materials and methods. A total of 29 sturgeons (two species; length 8&ndash;37 cm) originating from fish farms in northern Poland with a known KHV history in common carp or koi in the area were examined: 15 Russian sturgeons, Acipenser gueldenstaedtii, with clinical signs of a disease and 14 asymptomatic Atlantic sturgeons, A. oxyrinchus. The former were sent to the laboratory alive while the latter were sent fixed in ethanol. As it is required for detection of a latent KHV infection in acipenserids, two independent procedures were applied. The preliminary results were obtained using PCR. Those findings were subsequently confirmation by nested PCR. The latter procedure consists of sequence analysis of PCR products and direct detection of KHV infected cells in tissue materials by in-situ hybridization on nucleic acid level or indirect immunofluorescence on KHV protein level. Results. KHV genome parts were found in nine Russian sturgeons and four Atlantic sturgeons. Comparison of PCR results obtained from three primer pairs used for KHV diagnostic in sturgeon showed that those designed by Bercovier et al. were most sensitive and robust for this purpose. In order to confirm the presence of viral particles the most useful method was in-situ hybridization (ISH), allowing the detection of KHV in gill samples obtained from live sturgeons. Conclusion. This preliminary study shows that sturgeons can be carriers of KHV. Therefore a viral diagnostics is highly recommended not only for sturgeons obtained from the environment but also for fertilized eggs, fry, and fish intended for re-stocking measurements of inland waters.

Detection of koi herpes virus (CyHV‐3) in goldfish, Carassius auratus (L.), exposed to infected koi

Detection of koi herpes virus (<i>CyHV‐3</i>) in goldfish, <i>Carassius auratus</i> (L.), exposed to infected koi