Koi Herpesvirus Genome Research Articles

The susceptibility of silver crucian carp (Carassius auratus langsdorfii) to infection with koi herpesvirus (KHV).

Koi herpesvirus (KHV) infections cause high mortality in carp (Cyprinus carpio). This study compared the susceptibility of silver crucian carp (Carassius auratus langsdorfii), also called ginbuna, and koi carp to KHV infection. Silver crucian carp and koi carp were challenged with KHV by both intraperitoneal injection and immersion, respectively, and kept in tanks at 22°C. All KHV-exposed koi carp died within 14days post-infection (dpi), whereas no clinics nor mortality was observed in the KHV-exposed silver crucian carp. KHV DNA was detected in both koi and silver crucian carp shortly after infection. At 7dpi, the copy numbers of KHV genome were increased in koi carp but decreased in silver crucian carp. Using reverse transcriptase PCR, KHV mRNA was detected in koi carp but not in silver crucian carp. Cell cultivation on common carp brain (CCB) cell samples from koi carp caused KHV-associated cytopathic effects in CCB cells. Therefore, we concluded that KHV replicated in koi carp but not in silver crucian carp and that silver crucian carp is not susceptible to infection with 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.

Analysis of stress factors associated with KHV reactivation and pathological effects from KHV reactivation

Koi herpesvirus (KHV) is a highly pathogenic virus of common carp and koi. KHV becomes latent in recovered koi or exposed koi without symptoms, and the latent infection can reactivate under stress conditions. KHV reactivation from latency often occurs when water temperature rapidly rises above 17°C. Dissolved O2 is lower at ≥17°C than at non-stress temperatures ≤15°C. To determine whether reduced dissolved O2 level has a role in KHV reactivation during temperature stress, KHV reactivation was investigated in KHV latently infected koi (KHV+ koi) under stress temperatures by maintaining dissolved O2 consistent with the O2 level at 15°C. There was no significant difference in the amount of reactivated virus between KHV+ koi maintained with and without O2 supplementation during temperature stress. Both handling and sampling were found to be stressful to koi and can contribute to KHV reactivation from latency. There was an increase in KHV genome within white blood cells (WBC) during KHV reactivation, which is about 3–4 fold higher than the amount of KHV genome detectable in WBC during the latency stage. At day 15 post-temperature stress (PTS), inflammation and necrosis were observed in multiple tissues, especially in the gills, eye, intestine, skin and kidney. KHV DNA was also detectable in multiple tissues on days 6, 9 and 15 PTS. Following day 3 PTS, the plasma cortisol levels were higher than that observed in koi before temperature stress, suggesting that KHV reactivation is associated with physiological stress in KHV+ koi.

Detection and application of circular (concatemeric) DNA as an indicator of koi herpesvirus infection.

Herpesviruses form a long continuous DNA molecule, or head-to-tail concatemer, as a replicating intermediate in the host. In this study, we developed a DNA-specific PCR assay for detecting the infection stage of koi herpesvirus (KHV) based on the presence of this 'endless' DNA. The 295 kbp double-stranded DNA KHV genome consists of a 251 kbp unique long region and two 22 kbp direct repeats (DRL and DRR) at each genome terminus. We designed a new primer set (DR primer set) based on the DR region spanning the presumed circular or concatemeric junction. Using the DR primer set, a PCR product was obtained from KHV-infected common carp brain (CCB) cells, but not from the virus-infected cell culture supernatant, implying that the PCR assay could detect intracellular virus in the host. The synthesis of a presumptive circular or concatemeric genome in virus-infected CCB cells was examined in a time-course experiment together with viral mRNA of the terminase gene, copy numbers of the viral genome, and infectious viral titer. The mRNA was first detected in the cells at 6 h post-inoculation (hpi), and the copy number of viral genome in the cells started to increase at 12 hpi. Subsequently, circular or concatemeric DNA was detected in the cells at 18 hpi, and progeny virus was detected in the cell culture supernatant at 24 hpi. These findings suggest that detection of the circular or concatemeric KHV genome with the developed PCR method can be used to determine the stage of KHV infection.

Inactivation of koi‐herpesvirus in water using bacteria isolated from carp intestines and carp habitats

Since its first outbreak in Japan in 2003, koi-herpesvirus (KHV) remains a challenge to the carp Cyprinus carpio L. breeding industry. In this study, inactivation of KHV in water from carp habitats (carp habitat water) was investigated with the aim of developing a model for rapidly inactivating the pathogen in aquaculture effluent. Experiments with live fish showed that, in carp habitat water, KHV lost its infectivity within 3days. Indications were that inactivation of KHV was caused by the antagonistic activity of bacteria (anti-KHV bacteria) in the water from carp habitats. Carp habitat water and the intestinal contents of carp were therefore screened for anti-KHV bacteria. Of 581 bacterial isolates, 23 showed anti-KHV activity. An effluent treatment model for the disinfection of KHV in aquaculture effluent water using anti-KHV bacteria was developed and evaluated. The model showed a decrease in cumulative mortality and in the number of KHV genome copies in kidney tissue of fish injected with treated effluent compared with a positive control. It is thought that anti-KHV bacteria isolated from the intestinal contents of carp and from carp habitat water can be used to control KHV outbreaks.

Do wild fish species contribute to the transmission of koi herpesvirus to carp in hatchery ponds?

The koi herpesvirus (KHV) has spread worldwide since its discovery in 1998 and causes disease and mortality in koi and common carp populations with a high impact on the carp production industry. Many investigations have been conducted to examine ways of distribution and to identify possible transmission vectors. The answers, however, raise many new questions. In the present study, different wild fish species taken from carp ponds with a history of KHV infection were examined for their susceptibility to the virus. In the tissue of these fish, the virus load was determined and it was tested whether a release of the virus could be induced by stress and the virus then could be transferred to naive carp. Wild fish were gathered from carp ponds during acute outbreaks of virus-induced mortality in summer and from ponds stocked with carp carrying a latent KHV infection. From these ponds, wild fish were collected during the harvesting process in autumn or spring when the ponds were drained. We found that regardless of season, temperature variation, age and infection status of the carp stock, wild fish from carp ponds and its outlets could be tested positive for the KHV genome using real-time PCR with a low prevalence and virus load. Furthermore, virus transfer to naive carp was observed after a period of cohabitation. Cyprinid and non-cyprinid wild fish can therefore be considered as an epidemiological risk for pond carp farms.

Characterization of Austrian koi herpesvirus samples based on the ORF40 region

Using a PCR that amplifies a region of the thymidine kinase (TK) gene, an epidemic spread of koi herpesvirus (KHV) was determined in koi carps in Austria in 2007. A total of 15 virus samples from different locations in Austria were analyzed to determine their genetic relatedness following PCR and nucleic acid sequencing of the open reading frame 40 (ORF40) region of the KHV genome. ORF40-specific PCR amplification products that were obtained from tissue samples shared 100% nucleotide sequence identity with the published sequence of the Japanese strain of KHV. The ORF40 sequence of one isolate from the UK that was included in the present study was 100% identical with the published sequence of an Israeli strain of KHV. This is the first study that used a larger number of samples and a PCR method, which allowed distinguishing all 3 strains of KHV. The present investigation provides information on the epidemiology of KHV infections in Europe and describes a useful molecular tool for epidemiological studies.

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 8–37 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.

Evaluation of Quenching Probe (QProbe)-PCR Assay for Quantification of the Koi Herpes Virus (KHV)

Koi herpes virus (KHV) has been recognized as a worldwide cause of mortality among populations of koi Cyprinus carpio koi and common carp. KHV disease can be diagnosed by the isolation of the virus, by anatomical assessment, and by the polymerase chain reaction (PCR) assay with agarose gel electrophoresis. Among these methods, the PCR assay has advantages over other methods in accuracy, but it retains a disadvantage with respect to quantification of the KHV genome. The TaqMan real-time PCR assay (TaqMan PCR assay) using the TaqMan-Probe has recently become more commonly used for the quantification of the KHV genome. However, it remains difficult to determine whether or not an amplified sequence originated from the KHV genome, because the TaqMan-Probe is degraded after PCR amplification. To overcome this problem, we utilized a novel quenching probe (QProbe) for quantitative PCR. This probe can be used to perform a melting curve analysis, thus eliminating the likelihood of obtaining false positives for the amplified product after PCR amplification. Two QProbes were designed to target SphI-5 and thymidine kinase, and these probes successfully detected the KHV genome; the amplified sequences could thus be evaluated by melting curve analysis.

Detection and sequence analysis of DNA polymerase and major envelope protein genes in koi herpesviruses derived from Cyprinus carpio in Gunma prefecture, Japan

Koi herpesvirus (KHV), which is believed to be an emerging virus, causes fatal diseases in carps. Since the 1990s, the presence of KHV has been confirmed in several countries. In Japan, from 2003 to 2004, large outbreaks of KHV infection in farmed carps resulted in the death of numerous fishes. From April to May 2004, we collected 43 dead or dying carps exhibiting typical symptoms of KHV infection in Gunma prefecture. To conduct a molecular epidemiologic study of KHV in our prefecture, we amplified DNA polymerase and the major envelope protein genes of KHV derived from carp gills using newly designed primers. We also performed sequence analysis of both genes of KHV. Sensitivity of our PCR method for amplification of DNA polymerase and the major envelope protein genes of KHV was 3 x 10(2) (100 fg) and 3 x 10(3) (1000 fg) copies of KHV genome, respectively. We detected both DNA polymerase and major envelope protein genes in 37 of 43 carps (86%). No mutation was found in both the genes sequenced from 11 strains, which included two foreign strains and one domestic strain. The results suggested that KHV strains derived from carps in our prefecture were closely related genetically to the other KHV strains.

Concentrations of a Koi herpesvirus (KHV) in tissues of experimentally-infected Cyprinus carpio koi as assessed by real-time TaqMan PCR

The Koi herpesvirus (KHV) is a herpes-like virus now recognized as a worldwide cause of mortality among populations of koi Cyprinus carpio koi and common carp Cyprinus carpio carpio. Temperature is a key factor influencing virus replication both in cell culture and in the tissues of experimentally infected fish. Genomic DNA sequences were used to optimize a rapid real-time TaqMan PCR assay to detect and quantify KHV DNA as found in the tissues of virus-exposed fish. The assay allowed analytical enumeration of target KHV genome copies ranging from 10(1) to 10(7) molecules as present in infected cell lines or fish tissues. The new assay was specific for KHV and did not detect DNA from 3 related herpes-like viruses found in fish, the Cyprinid herpesvirus 1 (CyHV-1), Cyprinid herpesvirus 2 (CyHV-2), Ictalurid herpesvirus 1 (IcHV-1) or the KF-1 cell line used for virus growth. Concentrations of KHV DNA were evaluated in 7 different tissues of replicate groups of virus-exposed koi held at water temperatures of 13, 18, 23 and 28 degrees C. Viral DNA was detected among virus-exposed koi at all 4 water temperatures but mortality was only observed among fish at 18, 23, and 28 degrees C. Time and temperature and the interactions between them affected concentrations of viral DNA detected in tissues of koi exposed to KHV. Although there were no recognized patterns to viral DNA concentrations as found in different tissues over time, KHV genome copies for all tissues increased with time post virus exposure and with water temperature. The remarkably rapid and systemic spread of the virus was demonstrated by the presence of viral DNA in multiple tissues 1 d post virus exposure. The greatest DNA concentrations found were in the gill, kidney and spleen, with virus genome equivalents consistently from 10(8) to 10(9) per 10(6) host cells. High levels of KHV DNA were also found in the mucus, liver, gut, and brain. Koi surviving infection at 62 to 64 d post virus exposure contained lower KHV genome copies (up to 1.99 x 10(2) per 10(6) host cells) as present in gill, kidney or brain tissues.