Initial characteristics of koi herpesvirus and development of a polymerase chain reaction assay to detect the virus in koi,Cyprinus carpio koi

Since 1998, episodes of mass mortality have occurred in populations of common carp Cyprinus carpio carpio in Israel and in populations of koi Cyprinus carpio koi in Israel and the USA. A herpesvirus isolated from infected fish has been shown in experimental studies to induce disease and mortality similar to those observed in outbreaks at infected farms. Initial characteristics of the virus show that it is clearly different from Herpesvirus cyprini (CHV), the most commonly known herpesvirus from cyprinid fish. The koi herpesvirus (KHV) has 31 virion polypeptides. Twelve of the virion polypeptides of KHV have similar molecular weights to those of CHV and 10 are similar to those of channel catfish virus (CCV). Both virion polypeptide and restriction fragment length polymorphism analyses of genomic DNA showed that the first KHV isolates from Israel and the USA were identical. In contrast, the genomic DNA restriction fragments clearly distinguish KHV from CHV and CCV. A polymerase chain reaction (PCR) assay to detect the virus in koi tissues was developed with sequences obtained from 1 restriction fragment of KHV DNA. The PCR assay effectively detected a 484 base pair sequence from KHV but did not amplify genomic DNA from either CHV or CCV. The PCR assay detected as little as 1 pg of KHV DNA mixed with 100 ng of host DNA. Viral sequences were amplified from koi obtained from field collections and from koi that were experimentally exposed to 10(2) TCID50 ml(-1) of KHV via the waterborne route. All KHV exposed fish dying of infection between 8 and 10 d post exposure or surviving to 14 d post exposure were found to be positive by PCR, while unexposed control koi were all negative. The assay also showed the presence of KHV DNA in tissues of koi obtained from farms in Israel. The PCR assay should assist virus isolation procedures and histologic and electron microscopic analyses now commonly used to detect KHV infection. Current studies are examining the possibility of using the PCR to detect KHV DNA in live fish and the relative sensitivity and specificity of the KHV PCR assay compared with other diagnostic tests.     

An unusual koi herpesvirus associated with a mortality event of common carp Cyprinus carpio in New York State, USA

Koi herpesvirus (KHV), a highly contagious and lethal virus that affects both koi (Cyprinus carpio koi) and common carp (Cyprinus carpio), was isolated in 1998 from two outbreaks of koi suffering mass mortality in New York State, USA, and in Israel. The disease had been described as early as 1996 in Europe. In July 2004, this virus was found associated with a mass mortality event in wild common carp in the Chadakoin River, New York, USA (42 degrees 07' N, 79 degrees W). Affected fish typically showed marked hyperplasia of gill tissues, abdominal adhesions, and severe multifocal to diffuse external hemorrhages. The virus isolated in this outbreak was somewhat unusual in that it initially replicated well in fathead minnow cell cultures, which is typical of spring viremia of carp virus. Testing at the National Veterinary Services Laboratories, Ames, Iowa, USA, confirmed the virus's identity to be KHV. Koi herpesvirus is not currently on the OIE (World Organisation for Animal Health) list of notifiable diseases; however, it is capable of causing mass mortality in susceptible fish at permissive temperatures.     

Cyprinid herpes virus-3 (CyHV-3) bears genes of genetically distant large DNA viruses

A large DNA virus, designated koi herpes virus (KHV), carp interstitial nephritis gill necrosis virus (CNGV) and Cyprinid herpes virus-3 (CyHV-3), causes massive mortality of carp. Morphologically, the virus resembles herpes viruses, but it contains a genome of ca 295 kbp, larger than that of any Herpesviridae member. Interestingly, three CyHV-3 genes, thymidylate monophosphate kinase (TmpK), ribonucleotide reductase and thymidine kinase, which are involved in deoxynucleotide tri-phosphate synthesis, resemble those of pox viruses. In addition to the TmpK gene, which is nonexistent in the genome of herpes viruses, CyHV-3 contains a B22R-like gene, exclusively expressed by pox viruses. These results raise questions on the phylogenic origin of CyHV-3.     

Reactivation of koi herpesvirus infections in common carp Cyprinus carpio

Two co-habitation studies with common carp were conducted to determine whether latent infections of koi herpesvirus (KHV) exist. Fish were exposed to KHV using 2 different temperature profiles, which induced low and high initial mortality. Subsequently, certain groups of fish were co-habited with naive fish while others were not. Koi herpesvirus was reactivated in fish from 3 of the 5 experimental tanks. Reactivation of the virus occurred regardless of the initial mortality associated with the virus or whether fish were co-habited with naive fish. The reactivation of the virus in our experiments occurred several months after the initial exposure to KHV and appeared to be temperature dependent.     

Cloning of the koi herpesvirus (KHV) gene encoding thymidine kinase and its use for a highly sensitive PCR based diagnosis

Background  

Outbreaks with mass mortality among common carp Cyprinus carpio carpio and koi Cyprinus carpio koi have occurred worldwide since 1998. The herpes-like virus isolated from diseased fish is different from Herpesvirus cyprini and channel catfish virus and was accordingly designated koi herpesvirus (KHV). Diagnosis of KHV infection based on viral isolation and current PCR assays has a limited sensitivity and therefore new tools for the diagnosis of KHV infections are necessary.     

Detection of the herpesviral hematopoietic necrosis disease agent (Cyprinid herpesvirus 2) in moribund and healthy goldfish: validation of a quantitative PCR diagnostic method

Cyprinid herpesvirus 2 (CyHV-2) is a pathogen of goldfish Carassius auratus auratus L. that causes herpesviral hematopoietic necrosis (HVHN) disease. The disease is associated with necrosis of hematopoietic tissues and anemia with high mortality. We have developed a real time 5'-nuclease PCR method (Taqman) that quantitatively detects CyHV-2 with a linear response over 8 logs of target concentration. The coefficient of variability on replicate samples tested on different days was 13% and the calculated sensitivity approached 1 target molecule per reaction. The assay does not cross-react with other similar fish herpesviruses, including CyHV-1 (carp pox) and CyHV-3 (koi herpesvirus), but reliably detects known CyHV-2 positive fish. The assay detects CyHV-2 not just in clinical cases of HVHN but also in apparently healthy 1 yr old goldfish fingerlings and even in 3 to 5 yr old broodfish.     

Genome sequences of three koi herpesvirus isolates representing the expanding distribution of an emerging disease threatening koi and common carp worldwide

Since the mid-1990s, lethal infections of koi herpesvirus (KHV) have been spreading, threatening the worldwide production of common carp and koi (both Cyprinus carpio). The complete genome sequences of three KHV strains from Japan, the United States, and Israel revealed a 295-kbp genome containing a 22-kbp terminal direct repeat. The finding that 15 KHV genes have clear homologs in the distantly related channel catfish virus (ictalurid herpesvirus 1) confirms the proposed place of KHV in the family Herpesviridae, specifically in the branch with fish and amphibian hosts. KHV thus has the largest genome reported to date for this family. The three strains were interpreted as having arisen from a wild-type parent encoding 156 unique protein-coding genes, 8 of which are duplicated in the terminal repeat. In each strain, four to seven genes from among a set of nine are fragmented by frameshifts likely to render the encoded proteins nonfunctional. Six of the affected genes encode predicted membrane glycoproteins. Frameshifts or other mutations close to the 3' ends of coding sequences were identified in a further six genes. The conclusion that at least some of these mutations occurred in vivo prompts the hypothesis that loss of gene functions might be associated with emergence of the disease and provides a basis for further investigations into the molecular epidemiology of the virus.     

Mass mortality associated with koi herpesvirus in wild common carp in Canada

Koi herpesvirus (KHV) was identified as being associated with more than one mortality event affecting common carp in Canada. The first was an extensive mortality event that occurred in 2007 in the Kawartha Lakes region, Ontario, affecting Lakes Scugog and Pigeon. Fish had branchial necrosis and hepatic vasculitis with an equivocal interstitial nephritis. Several fish also had branchial columnaris. Subsequent mortality events occurred in 2008 in additional bodies of water in south-central Ontario, such as Lake Katchewanooka and outside of Ontario in Lake Manitoba, Manitoba. Koi herpesvirus was detected in fish submitted for examination from all of these lakes by polymerase chain reaction (PCR), and sequence of the PCR product revealed 100% homology to KHV strains U and I. Real-time PCR analysis of KHV-infected wild carp revealed viral loads ranging from 6.02×10(1) to 2.4×10(6) copies μg(-1) host DNA. This is the first report of KHV in Canada.     

The alloherpesviral counterparts of interleukin 10 in European eel and common carp

Viral interleukin 10 (IL-10) like open reading frames have been identified in several pox- and herpesviruses, including the fish herpesviruses Anguillid herpesvirus 1 (AngHV-1) and Cyprinid herpesvirus 3 (CyHV-3). European eel (Anguilla anguilla) IL-10 was sequenced, in order to compare European eel and common carp (Cyprinus carpio) IL-10 with their alloherpesviral counterparts. Homology between the virus and host IL-10 amino acid sequences is low, which is confirmed by phylogenetic analysis. However, the three dimensional structures of the fish and alloherpesviral IL-10 proteins as predicted by modeling are highly similar to human IL-10. Closely related AngHV-1 and CyHV-3 are expected to have obtained their viral IL-10 genes independently in the course of coexistence with their respective hosts. The presence and structural conservation of these alloherpesviral IL-10 genes suggest that they might play an important role in the evolution of pathogenesis.     

Resistance of common carp (Cyprinus carpio L.) to Cyprinid herpesvirus-3 is influenced by major histocompatibility (MH) class II B gene polymorphism

The role of MH class II B (Cyca-DAB1-like) genes in resistance of common carp (Cyprinus carpio L.) to Cyprinid herpesvirus-3 (CyHV-3), also known as koi herpesvirus (KHV) was analysed. The material consisted of 934 fish from six carp crosses. Fish were challenged with CyHV-3 at an age of 7 and 10 months. During challenge experiments the peak of mortality caused by CyHV-3 was observed at days 8–12 p.i. and the overall cumulative mortality reached 79.9%. Among six Cyca-DAB1-like genotypes, revealed by PCR-RF-SSCP analysis, one genotype (E) was found associated with higher resistance to CyHV-3. Three other genotypes (B, H and J) could be linked to higher susceptibility to CyHV-3. Analysis of the alleles that compose the Cyca-DAB1-like genotypes linked one particular allele (Cyca-DAB1*05) to significantly increased, and two alleles (Cyca-DAB1*02 and Cyca-DAB1*06) to significantly decreased resistance to CyHV-3. Our data indicate that MH class II B genes could be used as potential genetic markers in breeding of common carp for resistance to this virus.     

重组酶聚合酶扩增技术结合侧向流动试纸条快速检测锦鲤疱疹病毒

为建立一种快速、灵敏并适用于临床检测Ⅲ型鲤疱疹病毒(Cy HV-3, KHV)的方法,实验根据KHV SphI-5基因的保守序列片段设计引物及探针,采用重组酶聚合酶扩增技术结合侧流层析试纸条(RPA-LFD)检测KHV。重组酶聚合酶扩增技术(RPA)具恒温扩增及高灵敏度特点,简化了设备要求的同时又能做到高效检测病毒,再结合侧向流动试纸条(LFD)将RPA结果快速地可视化,提高了该疾病的检测效率。结果表明,在38℃的最适反应温度下,采用RPA-AGE技术仅需10min便可检测出病原的目标片段,结合LFD方法仅需5min便可将RPA结果通过试纸条可视化呈现。研究研发的KHV RPA-LFD检测方法简单、快捷,可为实验条件有限的养殖场快速诊断需求提供技术支撑。     

Environment�CKHV�Ccarp�Chuman linkage as a model for environmental diseases

To predict outbreaks of infectious disease and to prevent epidemics, it is essential not only to conduct pathological studies but also to understand the interactions between the environment, pathogen, host and humans that cause and spread infectious diseases. Outbreaks of mass mortality in carp caused by Cyprinid herpesvirus 3 (CyHV-3), formerly known as koi herpesvirus (KHV), disease have occurred worldwide since the late 1990s. We proposed an environment?CKHV?Ccarp?Chuman linkage as a conceptual model for ??environmental diseases?? and specify research subjects that might be necessary to construct and shape this linkage.     

Seasonal Distribution of Cyprinid Herpesvirus 3 in Lake Biwa,Japan

The seasonal distribution of the cyprinid herpesvirus 3 (CyHV-3) in Lake Biwa, Japan, was investigated. CyHV-3 was distributed all over the lake 5 years after the first outbreak. The mean concentration of CyHV-3 in water showed annual oscillation, with a peak in the summer and a trough in winter. Our results suggested that CyHV-3 is present at high density in reductive environments, such as reed zones and turbid or eutrophic water.A novel fatal disease of fish caused by cyprinid herpesvirus 3 (CyHV-3), also known as koi herpesvirus or carp interstitial nephritis and gill necrosis virus, which infects the common carp (Cyprinus carpio carpio) and ornamental koi (Cyprinus carpio koi) was reported at the end of the 1990s, and it has spread rapidly worldwide (13). In 2003, the first mass mortality in Japan was observed in Lake Kasumigaura, Ibaraki Prefecture (17), and the disease immediately spread all over the country.Lake Biwa is the largest and oldest freshwater lake in Japan. It occupies 670 km², and the total length of the coastline is 241 km. The first and relatively small-scale mortality caused by CyHV-3 was observed in the autumn of 2003 in Lake Biwa, and mass mortality occurred in the following spring, in which more than 100,000 carp died (10). Thus, the disease is a great threat not only to the cultivation industry and koi collectors but also to the natural carp population. Carp is regarded as an ecological engineer that has an impact on freshwater ecosystems, and therefore, mass mortality will affect the entire lake ecosystem (10).The pathogenesis and diagnosis of the disease have been studied intensively, but the dynamics of CyHV-3 has not been clarified. Recently, the major portal of CyHV-3 entry was reported to be fish skin (2), which means that infection via water is possible. Thus, to determine the method by which the virus spreads and to evaluate the infectious risk in the environment, one must clarify the CyHV-3 dynamics in the natural environment. In the present study, we surveyed the seasonal distribution pattern of CyHV-3 in Lake Biwa, Japan, using a quantitative method.     

Detection of carp interstitial nephritis and gill necrosis virus in fish droppings

Carp interstitial nephritis and gill necrosis virus (CNGV) is an unclassified large DNA virus that morphologically resembles members of the Herpesviridae but contains a large (ca. approximately 280-kbp) linear double-stranded DNA. This virus has also been named koi herpesvirus, koi herpes-like virus, and cyprinid herpesvirus 3. CNGV is the cause of a lethal disease that afflicts common carp and koi. By using immunohistochemistry, molecular analysis, and electron microscopy we previously demonstrated that this virus is present mainly in the intestine and kidney of infected fish. Based on these observations, we postulated that viruses and/or viral components may appear in droppings of infected carp. Here we report that (i) by using PCR we demonstrated that fish droppings contain viral DNA, (ii) fish droppings contain viral antigens which are useful for CNGV diagnosis, and (iii) fish droppings contain active virus which can infect cultured common carp brain cells and induce the disease in na?ve fish following inoculation. Thus, our findings show that CNGV can be identified by using droppings without taking biopsies or killing fish and that infectious CNGV is present in the stools of sick fish. The possibility that fish droppings preserve viable CNGV during the nonpermissive seasons is discussed.     

Koi herpesvirus disease

Koi herpesvirus (KHV) disease emerged at the late 1990s, and has rapidly spread to the world. In Japan, KHV disease first occurred at October 2003. The disease resulted in mass mortality of wild carp as well as cultured carp. Until now, KHV-infected carp were found in 42 out of 47 prefectures in Japan. Only carp Cyprinus carpio is susceptible to KHV, while goldfish, closely-related species to carp, is not. The affected carp swim lethargically. Sunken eyes and gill necrosis are frequently noticed, but no marked internal signs are observed. Optimal water temperature for the disease is 18-23 degrees C. Under 13 degrees C or over 28 degrees C, no death occurs. Keep at over 30 degrees C cures KHV disease, but can make the fish latent carriers. Because the fish do not get acquired immunity against KHV disease under low water temperature, the disease recurs with increase of water temperature. Isolation of KHV is difficult. KHV disease is diagnosed through epidemiological investigation, disease signs and PCR detection of KHV DNA. Vaccine development is ongoing for restart of culturing carp at KHV-contaminated places.     

Cloning of the koi herpesvirus genome as an infectious bacterial artificial chromosome demonstrates that disruption of the thymidine kinase locus induces partial attenuation in Cyprinus carpio koi

Koi herpesvirus (KHV) is the causative agent of a lethal disease in koi and common carp. In the present study, we describe the cloning of the KHV genome as a stable and infectious bacterial artificial chromosome (BAC) clone that can be used to produce KHV recombinant strains. This goal was achieved by the insertion of a loxP-flanked BAC cassette into the thymidine kinase (TK) locus. This insertion led to a BAC plasmid that was stably maintained in bacteria and was able to regenerate virions when permissive cells were transfected with the plasmid. Reconstituted virions free of the BAC cassette but carrying a disrupted TK locus (the FL BAC-excised strain) were produced by the transfection of Cre recombinase-expressing cells with the BAC. Similarly, virions with a wild-type revertant TK sequence (the FL BAC revertant strain) were produced by the cotransfection of cells with the BAC and a DNA fragment encoding the wild-type TK sequence. Reconstituted recombinant viruses were compared to the wild-type parental virus in vitro and in vivo. The FL BAC revertant strain and the FL BAC-excised strain replicated comparably to the parental FL strain. The FL BAC revertant strain induced KHV infection in koi carp that was indistinguishable from that induced by the parental strain, while the FL BAC-excised strain exhibited a partially attenuated phenotype. Finally, the usefulness of the KHV BAC for recombination studies was demonstrated by the production of an ORF16-deleted strain by using prokaryotic recombination technology. The availability of the KHV BAC is an important advance that will allow the study of viral genes involved in KHV pathogenesis, as well as the production of attenuated recombinant candidate vaccines.