A British pharmaceutical company, Henderson Morley plc have announced that they have been working on producing a vaccine against KHV (Koi Herpes Virus) over the past 10 months and from these initial studies are now ready to start field trials. According to the most recent virus taxonomy reported in 2012 by the International Committee on Taxonomy of Viruses (ICTV), DEV (also referred to as Anatid herpesvirus 1) is classified into the genus Mardivirus and the subfamily Alphaherpesvirinae of Herpesviridae. In addition, we summarize the clinical and histopathological manifestations of the disease. Remember to have a suitable filtration system for the size of your pond, Koi can produce a large amount of waste which in turn will cause poor water quality and cause nothing but problems for your koi. Koi Herpesvirus (KHV) is a serious disease causing high mortalities and it creates consequences for Koi that survive it. In each strain, four to seven genes from among a set of nine are fragmented by frameshifts likely to render the encoded proteins nonfunctional. Twenty eight days post vaccination, the fish were challenged by injecting 10-4 ml/fish of KHV.
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. (2008) 82:4955-4964. The KHV-gD protein belongs to the transmembrane glycoprotein, with only a little part inside the cell, and the transmembrane spiral area of this protein is located at No.4-25of N end and No.341-363of C end, the part between which belongs to the envelope region. Culture of common carp provides a key source of protein for human consumption in Asia, Europe, and the Middle East (3, 35). As we concentrate on our Animal Health research programme, his vast experience will be of enormous benefit to the Company and its scientific advisory board”. www.fishupdate.com is published by Special Publications.
has produced and marketed KV-3, a live attenuated vaccine against koi herpes virus disease. The virus does not appear clinically when fish are held at cold water temperatures; however the fish does not mount an effective immune response at these cold temperatures to prevent further clinical appearance of the virus. Considerably more information has since been obtained on the properties of this virus, including its host range, the effects of water temperature on disease outcome, development of detection methods, and novel attempts at control (1, 4, 15-17, 20, 26, 27, 34, 37-39). The virus has now been found associated with mass mortality events on most continents, including countries throughout Europe, the United States, Japan, Indonesia, South Africa, Thailand, Taiwan, China, and Malaysia (22, 29, 42, 45). In the interim period white patches of dead tissue may appear on the gills and the skin. Bowser and J. Therefore no rights can be derived from this article.
As with any other disease, the quicker you act, the better the prognosis for the fish (the rest of the fish). Hedrick, unpublished data). Goldfish of any age may be infected with higher mortalities in juvenile fish. Several sequence deletions were observed in the microsatellite zone in KHV-K. A substantial number of viruses in the mammalian HV clade have been sequenced, but only three in the fish HV clade (ictalurid HV 1, IcHV-1, also known as channel catfish virus, and two ranid HVs [7, 10]) and one in the bivalve HV clade (ostreid HV 1, OsHV-1, also known as oyster HV ). It has been proposed that these clades should be classified as three families encompassed by an order, the Herpesvirales (8, 12). In phylogenetic trees (A-C), sequences from the USA (KHV-U), Israel (KHV-I), and Japan (KHV-J) CyHV-3 were clustered into one group, but amplified sequences from the Korean CyHV-3 (KHV-K) was highly distinguished from this group.
Early observations demonstrated the clear developmental and morphological affinities of KHV to the HVs (24), but subsequent findings prompted others to suggest that the virus should not be considered a member of the Herpesviridae (26, 38, 39). Key factors in this argument included the estimated genome size of KHV (277 kbp), which is greater than that observed previously among HVs (125 to 245 kbp), and the failure to demonstrate convincing genetic relationships between KHV and recognized HVs from limited sequence analyses. The encapsulated molecules will generally have completely different properties (e.g., solubility or circulating half-life) compared to the non-encapsulated ones. KHV is related closely to cyprinid HVs 1 and 2 (CyHV-1 and CyHV-2), the agents associated with carp pox and hematopoietic necrosis in goldfish, respectively (28, 41), and distantly to IcHV-1 and ranid HV 1 (RaHV-1, also known as Lucké tumor herpesvirus of frog) (46). Therefore, KHV has been proposed formally as a member of the Alloherpesviridae under the species name Cyprinid herpesvirus 3. On the other hand, vaccine-induced activation of APCs is limited in the absence of antigen replication for non-replicative vaccines. We confirm that KHV shares significant similarities to fish HVs and belongs as a new species in the family Herpesviridae.
The 295 kb genome of CyHV-3 codes for 156 open reading frames (Aoki et al., 2007) and the relative transcriptional timing for each gene has been annotated (Ilouze et al., 2012). Growth of viruses.KHV strain J was isolated from a dead koi on a farm in Japan. (b) Norris, A., Hutchison, M., Chilcott, K., and Stewart, D. Strain I was isolated in 1998 from adult koi during large mortalities in a producer plant on the coastal plain of Israel. It would be very stupid to risk every thing for one fish. Bulk virus preparations were made (passages 3, 12, and 30 for J, U, and I, respectively) and purified by sucrose gradient ultracentrifugation (16). Genome DNA was purified by phenol extraction and ethanol precipitation.
DNA sequencing.The complete genome sequences were determined commercially (Hitachi Instruments Service Co., Ltd., Tokyo, Japan) by standard shotgun sequencing. Butler, L-M. The KHV DNA inserts were sequenced from both ends of the plasmids by using the M13 forward and reverse primers with a BigDye Terminator v3.1 cycle sequencing kit and ABI 3700 and 3730xl DNA analyzers. DNA sequence analysis.The DNA sequences were assembled by using Phrap (14), employing the quality files and default settings to produce a consensus sequence that was edited by using Consed (18). The final consensus sequence in each case represented an average eightfold redundancy at each base position. Gap closure was achieved by primer walking of gap-spanning clones and sequencing of PCR products. The two ORF encoding the most abundant envelope proteins demonstrated interesting sequence homologies.
The genome termini in U were located precisely by PCR amplification of virion DNA that had been flush ended and ligated to a partially double-stranded adaptor oligonucleotide as described previously (13), using a primer matching part of the adaptor plus 5′-CGCAGTAGGCCTTGACCAGCA-3′ for the left terminus or 5′-AGGGTCTGAACATGGCTTAGG-3′ for the right terminus. The locations of the termini determined from sequencing of 12 plasmid clones of each product were assumed to be identical in J and I. The GCG suite (Accelrys) and other programs (19, 33) were used for standard analyses of the sequences. ATG-initiated open reading frames (ORFs) of >50 codons were evaluated initially for protein coding potential by using GAMBLER, which semiautomates analysis of the output from genome assembler software, assigns ORFs automatically, and carries out homology searching (40). Additional homology searches were carried out by using the FastA (36) and BLAST (2) tools, and alignments were made by using CLUSTAL W (44). The preliminary gene map was refined by using GCG Codonpreference, which measures codon preference and third position codon G+C bias on the basis of statistics generated from standard genes. Since KHV happens to be strongly biased in these measures, the analysis proved useful in revising the preliminary map, as a result of which most ORFs were confirmed as likely to be protein coding and several ambiguities were clarified.
Vaccines against virus infections, including infectious pancreatic necrosis, have also been used in commercial fish farming. Additional criteria routinely used in HV genome analysis were used, in particular the lack of extensive overlap between coding regions and the presence of polyadenylation signals downstream from ORFs or families of similarly oriented ORFs. Splicing is relatively rare in HVs, and predictions for KHV based on the presence of appropriately located splice donor and acceptor signals were kept to a minimum. The availability of an incomplete sequence database for the majority of the related CyHV-1 genome (T. Waltzek and R. Hedrick, unpublished data) enabled most predicted protein-coding regions in KHV (including splice sites) to be confirmed by homology and helped clarify many remaining difficulties. The vaccine may incite a more severe reaction if it is injected into the wrong portion of the fish.
Analysis of mutated regions.Regions containing frameshift mutations in one or another strain were amplified by standard PCR from U at passages 2 and 12 (the former from infected cell DNA and latter from infected cell DNA and purified virion DNA). In addition, data were obtained for a fourth KHV strain (CO5-53-3) at passage 0 (i.e., directly from infected fish tissue) and 2. The CO5-53-3 strain was obtained in 2005 from a moribund wild common carp during a mortality episode in the lower San Joacquin River, California. The core size was in the 96- to 105-nm range, with an average diameter of 103 nm. Genome characteristics.The size of the KHV genome as determined from the sequences is 295 kbp (295,271, 295,146, and 295,138 bp for J, U, and I, respectively). Restriction endonuclease digestion of the DNA preparations with NotI or XbaI yielded identical profiles for the three strains, with fragment sizes as predicted from the sequences (data not shown). The genome has a 22-kbp direct repeat at each terminus (22,437, 22,469, and 22,485 bp for J, U, and I, respectively).
The overall nucleotide composition for each is 59.2% G+C, and the frequency of the CG dinucleotide is as expected from this value. The genomes are highly similar to each other at the sequence level, with U and I more closely related to each other than either is to J. For example, in respect of single nucleotide substitutions (not counting duplicates in the terminal repeat), J differs from U and I at 181 of the 217 loci that are not conserved in all three strains; that is, there is a nucleotide difference every 1.5 kbp on average. Of the remaining 36 nonconserved loci, I differs from J and U at 32 loci, and U differs from J and I at 4 loci. These relationships imply a history in which an ancestral KHV strain gave rise initially to two branches: the J lineage that led eventually to J and the U/I lineage, which subsequently split into the branches leading to U and I. This conclusion is also consistent with the pattern of differences due to insertions and deletions. Genetic content.The close relationship among the three strains resulted in the same predicted gene map for each, with the exception of a number of fragmented ORFs discussed below.
Figure 1 shows the predicted layout of protein-coding genes in an ancestral (wild-type) KHV as envisaged prior to gene fragmentation events. The genetic complement is 156, with 8 duplicated in the terminal repeat, yielding a total of 164 in the genome. Table 1 lists the features of KHV genes for which information was derived from bioinformatic analysis. Gene layout in wild-type KHV. The locations of predicted protein-coding ORFs (based on the strain J coordinates) are shown as defined in the key at the foot, with conserved genes (those with clear IcHV-1 homologs) differentiated from nonconserved genes, which include five gene families. We don’t know for sure where it hides when it’s not causing active disease. Nomenclature is given with the ORF prefix omitted, and the terminal repeat is shown in a thicker format than the rest of the genome.
Other symptoms are; excess of skin slime, loss of the slime coat, skin damage, loss of appetite, uncoordinated swimming and gasping for air. We view the gene layout as substantially accurate, but expect improvements to be made in the future. Alterations are most likely to come in the form of additional small ORFs, further splicing, and removal of present ORFs (e.g., perhaps ORF58 and ORF105, which we consider as the least likely ORFs to encode proteins). Mortality may be up to 100% in some outbreaks. Indeed, despite a substantially greater genome size, KHV is predicted to have fewer genes than the largest human HV, human cytomegalovirus (165 genes in a genome of 236 kbp, or 0.70 per kbp; 13). The KHV genome contains 15 genes (colored red in Fig. 1) that have clear homologs in IcHV-1.
The data for IcHV-1 and other HVs (7-9) indicate that these genes encode proteins involved in capsid morphogenesis (ORF92, ORF72, and ORF78, encoding two structural components of the capsid shell and a candidate protease involved in capsid maturation), nucleotide metabolism (ORF19 and ORF123 encoding deoxynucleoside kinase and deoxyuridine triphosphatase, respectively), DNA replication (ORF79, ORF71, and ORF46, encoding DNA polymerase, helicase, and a candidate primase), and DNA packaging (ORF33, which encodes the putative ATPase subunit of terminase; the three exons that comprise this coding region are shown in Fig. 1 as connected by introns). In addition, the conserved genes include one encoding a large membrane glycoprotein (ORF99) and five encoding proteins with unknown functions (ORF47, ORF61, ORF80, ORF90, and ORF56). During the production process with all these methods impurities are formed, thus requiring an additional purification step . When the ranid HV sequences (10) are taken into account, only 13 genes are convincingly conserved: the 15 genes conserved between KHV and IcHV-1 as described above, less ORF19 and ORF123. These findings indicate that the fish HV clade is considerably more divergent overall than the mammalian HV clade, in which 43 genes have been inherited from a common ancestor (8). Thus, it is difficult to determine the threshold level of IgT able to deter pathogen attachment, colonization, and entry at mucosal surfaces.
It is likely that the number of conserved genes in the fish HV clade will increase as data for other species facilitate more sensitive comparisons, but not to the number observed in the mammalian HV clade. As is the case with distantly related viruses in the mammalian HV clade (8), conserved genes are located centrally in the KHV and IcHV-1 genomes, and their arrangement is not conserved. The remaining 141 KHV genes are in the nonconserved set. These are colored pink in Fig. 1, except for five families of related genes. The RING family encodes four proteins containing a zinc ion-coordinating motif known as the RING finger. This motif is ubiquitous among the HVs and is encoded by representatives of the three clades.
The tumor necrosis factor receptor (TNFR) family encodes two versions of a secreted form of TNFR, which presumably have roles in immune evasion, as demonstrated with the poxviruses (43). The ORF2 and ORF22 families encode two and three proteins, respectively. Haq, K., K.A. The putative products of several KHV genes are convincingly related to enzymes in addition to those described above. These include the large and small subunits of ribonucleotide reductase (ORF141 and ORF23, respectively), thymidine kinase (ORF55), thymidylate kinase (ORF140), uracil-DNA glycosylase (ORF98), serine protease (ORF94), and serine-threonine protein kinase (ORF104). KHV also encodes proteins related to G protein-coupled receptors (ORF16), eukaryotic DUF614 proteins (ORF31), a family of iridovirus proteins (ORF32), nucleoside transporters (ORF64), a cellular protein of unknown function (ORF114), and a poxvirus B22R protein (ORF139) that is likely to be involved in immune evasion (30). Like some members of the mammalian HV clade (6), KHV encodes a protein that is clearly related to interleukin-10 (ORF134), which may modulate host immune responses.
sequences available. It is apparent from the conserved and nonconserved gene sets that KHV evolution has been characterized by gene capture from the cell or other viruses. Strain evolution.As mentioned above, the three KHV strains differ from each other at only a small number of loci. A few of the insertions and deletions represent mutations (usually frameshifts) in one or more strains that disrupt coding regions. It should be noted that the identification of affected ORFs depends upon the accuracy of the predicted gene set and, moreover, that genes mutated in all strains may only be identified in certain circumstances. This is possible when such genes are members of a family or are related to genes in other organisms or where the encoded proteins contain characteristic features, such as those of a membrane protein with a signal sequence and transmembrane region. Despite these analytical limitations, 15 ORFs (10% of the complement) appear to be mutated in one or more strains (Table 1).
Of these, the unmutated forms of 11 ORFs encode proteins with features implying that their assignment as protein-coding regions at least is correct. It is notable that the majority of mutated ORFs encode membrane glycoproteins and that none of the conserved genes is affected. Nine genes (listed as “broken” in Table 1) are probably rendered nonfunctional by frameshifts located centrally in the coding regions, and the wild-type gene is identified readily. Six genes (listed as “frameshifted”) might retain function since the mutations are close (sometimes very close) to the 3′ ends of the ORFs, making it more difficult to identify the wild-type gene (see the assumptions explained in the footnotes to Tables 1 and 2). Nonetheless, even though identification of the precise number of mutated genes in each genome is problematic, all three sequenced strains are evidently multiple mutants derived from a wild-type ancestor. Since the sequenced DNAs were obtained from KHV strains passaged in cell culture, it is possible that some of the mutations occurred after isolation from infected fish. Unfortunately, the tissues from which the strains had been isolated were no longer available, and it was not possible to examine the unpassaged viruses.
This process may take several months or longer, depending upon the situation. The distribution of mutations in a subset of the loci in Table 1 is summarized in Table 2. The combined data indicate that mutations in three genes arose in vivo, since they are present in more than one of the sequenced viruses: that in ORF30 in U and I and those in ORF26 and ORF40 in J, U, and I. Mutations in six genes (ORF16, ORF55, ORF87, ORF94, ORF108, and ORF116) are present in a single strain each and therefore may have arisen in vivo or in vitro. The skin showed a lack of luster, with pale patches and increased mucus secretions (22, 23, 34, 35; Bergmann, unpublished data). We conclude that mutations occurred in vivo in ORF26 and ORF40 prior to divergence of the three strains from a wild-type parent, with a mutation then arising in vivo in ORF30 after the U/I lineage had diverged from the J lineage. In the absence of the original infected tissues, it was not possible to determine whether subsequent mutations occurred in vivo or in vitro.
The sequence comparisons indicate that the three sequenced KHV strains arose via the loss of genetic functions, as evidenced by frameshifted coding regions, with at least some of the cognate mutations having occurred in vivo. Elsewhere among large DNA viruses, fragmented genes have been documented extensively in the Poxviridae, particularly the Orthopoxvirus genus, where up to 16% of genes in a single virus may be fragmented (21). These genes are invariably not required for virus growth in cell culture and are therefore presumed to contribute to some aspect of growth in the host. The simplest interpretation is that some orthopoxviruses have become associated with their hosts relatively recently and have diverged from an ancestral virus by losing certain functions either because these functions are not required or because they reduce fitness. Gene fragmentation was also observed in the bivalve HV, OsHV-1, which had not been passaged in cell culture, suggesting that this might have contributed to the striking pathogenicity (and perhaps increased host range) of this emerging virus in farmed shellfish (12). The apparent loss of KHV gene functions, particular among those encoding membrane glycoproteins that may be associated with host specificity and critical to virulence, presents a provocative parallel. Nonetheless, a role for gene loss in the emergence of KHV is currently speculative.
Origins of KHV.The epidemiologic and pathogenic features of KHV-associated disease are new, and we doubt that they were overlooked previously (23, 46). The earliest known archival evidence indicates the presence of KHV among wild common carp in the United Kingdom as early as 1996 (K. Way, unpublished data), preceding observations of the disease in koi in Germany that were first recorded in 1997 (5). The active and often unregulated movements of large numbers of koi have contributed to a rapid spread of the virus presumably from these origins (22). The first cases of the disease in the eastern United States (strain U) occurred in 1998 following a koi show in New York that involved fish from Israel (24), a finding consistent with the high degree of similarity between U and I. Causal links for the origin of KHV among common carp in Japan are less defined (42). Despite these observations, the origins of KHV remain obscure.
It is possible that it has derived from an innocuous virus of C. In the research center marked carps are bred in a sterile environment so it is a 100% certainty that they do not carry any virus. In this respect, findings with KHV have relevance beyond carp aquaculture, since similar phenomena have been observed with herpesviruses of other intensively cultured animals, including Marek’s disease virus in chickens, where the evolution of increasingly virulent strains is exacerbated by vaccination (32), and perhaps OsHV-1 in oysters (12). The sequence comparisons prompt the hypothesis that intensive culture of common carp and koi, combined with large-scale movements of live koi, may have favored transmission of genetically deficient KHV strains of enhanced virulence. Testing of this hypothesis will include further extensive comparisons of KHV isolates that have not been passaged in cell culture in order to understand the extent and timescale of gene loss and specific mutagenesis studies in order to assess any contribution of gene loss to pathogenicity. Whether or not further adaptation might occur, more active control measures, including listing of the KHV disease by international organizations charged with disease control, are probably needed to reduce future economic and ecologic impacts of this important viral pathogen. This research was supported in part by the Tokyo University of Marine Science and Technology, by the United Kingdom Medical Research Council, and by a grant from the Center for Study of Emerging Diseases (Israel) and BARD (United States-Israel Binational Agricultural Research and Development Fund project no.