Assessment of commercial test kits for identification of spring viraemia of carp virus

Two test kits for the identification of spring viraemia of carp virus (SVCV), one an enzyme-linked immunosorbent assay (ELISA) using a rabbit polyclonal antiserum, and the other an indirect fluorescent antibody test (IFAT) using a mouse monoclonal antibody, were assessed for specificity using a range of virus isolates. The test viruses were selected from 4 recently described genogroups of piscine rhabdoviruses: Genogroup I (SVCV), Genogroup II (grass carp rhabdovirus), Genogroup III (pike fry rhabdovirus) and Genogroup IV ('tench rhabdovirus'). The test viruses included SVCV isolates from all 4 subgroups of Genogroup I. The ELISA was non-specific for these viruses and did not distinguish between SVCV and isolates from the other 3 Genogroups. However, the IFAT was too specific and detected SVCV isolates from only 1 of the 4 SVCV subgroups. Reliance on these test kits alone could result in misidentification of this OIE notifiable disease.     

Nucleotide sequence analysis of the glycoprotein gene of putative spring viraemia of carp virus and pike fry rhabdovirus isolates reveals four genogroups

RT-PCR methods have been applied to the detection and sequencing of the glycoprotein gene of putative spring viraemia of carp viruses (SVCV) and pike fry rhabdoviruses (PFRV), including isolates from tench, grass carp, roach, bream and false harlequin, sheatfish and orfe. Phylogenetic analysis of a 550 nucleotide (nt) region of the glycoprotein gene identified 4 groups, I to IV. Significantly, the majority of viruses previously identified as PFRV formed a distinct cluster (Genogroup IV) which shared <80% nucleotide identity with the PFRV reference strain (Genogroup III). The similarity between another PFRV-like virus isolated from grass carp and representatives of Genogroups III and IV was also <80%, indicating that this virus belonged to a third group (Genogroup II). All of the putative SVC viruses were assigned to a 4th group (Genogroup I), sharing <61% nucleotide identity with viruses in Genogroups II to IV.     

Spring viremia of carp (SVC)

Spring viremia of carp (SVC) is an important disease affecting cyprinids, mainly common carp Cyprinus carpio. The disease is widespread in European carp culture, where it causes significant morbidity and mortality. Designated a notifiable disease by the Office International des Epizooties, SVC is caused by a rhabdovirus, spring viremia of carp virus (SVCV). Affected fish show destruction of tissues in the kidney, spleen and liver, leading to hemorrhage, loss of water-salt balance and impairment of immune response. High mortality occurs at water temperatures of 10 to 17 degrees C, typically in spring. At higher temperatures, infected carp develop humoral antibodies that can neutralize the spread of virus and such carp are protected against re-infection by solid immunity. The virus is shed mostly with the feces and urine of clinically infected fish and by carriers. Waterborne transmission is believed to be the primary route of infection, but bloodsucking parasites like leeches and the carp louse may serve as mechanical vectors of SVCV. The genome of SVCV is composed of a single molecule of linear, negative-sense, single-stranded RNA containing 5 genes in the order 3'-NPMGL-5' coding for the viral nucleoprotein, phosphoprotein, matrix protein, glycoprotein, and polymerase, respectively. Polyacrylamide gel electrophoresis of the viral proteins, and sequence homologies between the genes and gene junctions of SVCV and vesicular stomatitis viruses, have led to the placement of the virus as a tentative member of the genus Vesiculovirus in the family Rhabdoviridae. These methods also revealed that SVCV is not related to fish rhabdoviruses of the genus Novirhabdovirus. In vitro replication of SVCV takes place in the cytoplasm of cultured cells of fish, bird and mammalian origin at temperatures of 4 to 31 degrees C, with an optimum of about 20 degrees C. Spring viremia of carp can be diagnosed by clinical signs, isolation of virus in cell culture and molecular methods. Antibodies directed against SVCV react with the homologous virus in serum neutralization, immunofluorescence, immunoperoxidase, or enzyme-linked immunosorbent assays, but they cross-react to various degrees with the pike fry rhabdovirus (PFR), suggesting the 2 viruses are closely related. However, SVCV and PFR can be distinguished by certain serological tests and molecular methods such as the ribonuclease protection assay.     

Spring viremia of carp virus RNA and virion-associated transcriptase activity

An RNA-dependent RNA polymerase activity has been demonstrated for spring viremia of carp virus (SVCV). The optimal temperature for in vitro synthesis of RNA was 20 to 25 degrees C. The SVCV enzyme activity was stimulated when the methyl donor S-adenosyl-L-methionine was included in the reaction mixture. S-adenosyl-L-methionine was not particularly effective in stimulating the virion RNA polymerase activity of vesicular stomatitis virus or pike fry rhabdovirus. The 5' nucleotide of the SVCV viral RNA is pppAp.     

mtDNA sequence data supports an Asian ancestry and single introduction of the common carp into the Danube Basin

Common carp Cyprinus carpio ( n =24) from the upper Danube (wild or feral forms, scaleless varieties) as well as Japanese koi shared the identical mtDNA haplotype (565 bp). In contrast, Amur River carp ( n =5) exhibited unique haplotypes, 1–12 bp divergent from the Danubian one. These results support an Asian origin and single introduction or domestication event leading to the present diversity of common carp in Europe, as well as the ornamental Japanese koi.     

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.     

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.     

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.     

Triploidy in hybrids of carp with other representatives of the Cyprinidae family]

As a result of remote hybridization of carp (Cyprinus carpio L.) with the representatives of certain other subfamilies of Cyprinidae a few viable F1 hybrids were obtained for the first time. These hybrids proved to be allotriploids. In the cross Cyprinus carpio L. female X Hemiculter eigenmanni (Jord. et Metz.) male the rate of survival of this year's brood was 0,5% of the fertilized eggs and 3n=128; whereas in the cross Cyprinus carpio L. male X Ctenopharyngodon idella (val.) female the rate of survival was 0,008%, while the chromosome number (3n) varied from 124 to 128. The chromosome number of non-viable E1 hybrids derived from this cross was intermediate (2n=76). The diploidization of the maternal chromosome set was spontaneous, since no stimulating agents were used to induce it. It is assumed by the authors that it is the tetraploid nature of carp that affords the possibility of the initiation of allotriploids.     

Does the stock density of stillwater catch and release fisheries affect the growth performance of introduced cultured barbel?

In comparison to barbel Barbus barbus in their natural, riverine environment, the growth performance of cultured barbel, following introduction into stillwater environments, was depressed when the extant stock density was above 500 kg ha⁻¹ and common carp Cyprinus carpio were present.     

Genetic diversity and population structure inferred from the partially duplicated genome of domesticated carp,Cyprinus carpio L.

Genetic relationships among eight populations of domesticated carp (Cyprinus carpio L.), a species with a partially duplicated genome, were studied using 12 microsatellites and 505 AFLP bands. The populations included three aquacultured carp strains and five ornamental carp (koi) variants. Grass carp (Ctenopharyngodon idella) was used as an outgroup. AFLP-based gene diversity varied from 5% (grass carp) to 32% (koi) and reflected the reasonably well understood histories and breeding practices of the populations. A large fraction of the molecular variance was due to differences between aquacultured and ornamental carps. Further analyses based on microsatellite data, including cluster analysis and neighbor-joining trees, supported the genetic distinctiveness of aquacultured and ornamental carps, despite the recent divergence of the two groups. In contrast to what was observed for AFLP-based diversity, the frequency of heterozygotes based on microsatellites was comparable among all populations. This discrepancy can potentially be explained by duplication of some loci in Cyprinus carpio L., and a model that shows how duplication can increase heterozygosity estimates for microsatellites but not for AFLP loci is discussed. Our analyses in carp can help in understanding the consequences of genotyping duplicated loci and in interpreting discrepancies between dominant and co-dominant markers in species with recent genome duplication.     

Pathogenesis of acute viral disease induced in fish by carp interstitial nephritis and gill necrosis virus

A lethal disease of koi and common carp (species Cyprinus carpio) has afflicted many fish farms worldwide since 1998, causing severe financial losses. Morbidity and mortality are restricted to common carp and koi and appear in spring and autumn, when water temperatures are 18 to 28 degrees C. We have isolated the virus causing the disease from sick fish, propagated it in koi fin cell culture, and shown that virus from a single clone causes lethal disease in carp and koi upon infection. Intraperitoneal virus injection or bathing the fish in virus-containing water kills 85 to 100% of the fish within 7 to 21 days. This virus is similar to the previously reported koi herpesvirus; however, it has characteristics inconsistent with the herpesvirus family, and thus we have called it carp interstitial nephritis and gill necrosis virus. We examined the pathobiology of this disease in carp by using immunohistochemistry and PCR. We found large amounts of the virus in the kidneys of sick fish and smaller amounts in liver and brain. A rapid increase in the viral load in the kidneys was detected by using both immunofluorescence and semiquantitative PCR. Histological analyses of fish at various times after infection revealed signs of interstitial nephritis as early as 2 days postinfection, which increased in severity up to 10 days postinfection. There was severe gill disease evidenced by loss of villi with accompanying inflammation in the gill rakers. Minimal focal inflammation was noted in livers and brains. This report describes the etiology and pathology of a recently described viral agent in fish.     

Study of the haemoglobin content of a single erythrocyte (M index) in various cultured fish species

Investigations of the erythrocyte count, haemoglobin content and M index of 7 fish species were performed under equivalent environmental conditions in the same period of the year. The species were: bighead Hypophthalmichthys nobilis (Richardson), carp Cyprinus carpio L., silver carp Hypophthalmichthys molitrix (Val.), grass carp Ctenopharyngodon idella (Val.), sheat-fish Silurus glanis L. , pike Esox Indus L., pike-perch Lucioperca lucioperca (L.). Bighead and carp are carnivorous and omnivorous, silver carp and grass carp are herbivorous and sheat-fish, pike and pike-perch are predatory. Significant differences were demonstrable between M values of fish living on different type of food, where the M index is: It is concluded that M index values are useful as one parameter differentiating between fish with distinctly different food habits.     

鲤春血症病毒中国分离株糖蛋白基因和氨基酸序列的初步解析

2002～2004年间从国内养殖鲤科鱼类和观赏鱼类中分离出8株鲤春血症病毒(SVCV)。根据SVCV参考株 全序列,设计引物,用逆转录聚合酶链式反应扩增出8株SVCV糖蛋白编码基因片段,并对扩增产物进行了克隆和 序列测定。用生物信息学方法对测得的序列进行分析,结果8个国内分离株的糖蛋白基因序列与参考株的基因序 列相似性均在92％以上,8个国内分离株之间基因序列相似性均在97．7％以上;8个国内分离株之间糖蛋白推导 出的氨基酸序列相似性均在94．5％以上,与参考株氨基酸序列相似性在92．9％-94．9％之间。系统发育树分析结 果表明,SVCV国内分离株与USA株、980451株、980528株和970469株的进化方向一致,与其它SVCV毒株在进 化方向上不同。8个毒株有19个共同的酶切位点,推导出的氨基酸序列中有10个亲水区、10个可能的抗原位点 和10个跨膜蛋白区域,其峰值基本一致。对SVCV国内分离株的糖蛋白6个功能位点(天冬酰胺糖基化位点、精 氨酸-甘氨酸-天冬氨酸序列、酪蛋白激酶Ⅱ磷酸化位点、蛋白激酶C磷酸化位点、酪氨酸磷酸化位点和肉豆蔻酰 基化位点)进行了初步分析。     

Discovery of an ancient lineage of Cyprinus carpio from Lake Biwa, central Japan, based on mtDNA sequence data, with reference to possible multiple origins of koi

MtDNA sequence data ( c . 2100 bp of complete D‐loop + cytochrome b gene regions) showed a principal phylogenetic split within common carp Cyprinus carpio between the Lake Biwa wild form ( n  = 3) and 'Eurasian' wild ( n  = 18) and domesticated ( n  = 35) forms. This supports the idea of the ancient origin of the Lake Biwa wild form, as well as the East Asian origin of wild common carp, previously proposed on the basis of fossil data. In addition, a possible multiple origin of koi carp was indicated by the polyphyletic distribution of five mtDNA haplotypes of koi carp within the 'Eurasian' clade.