1 Department of Medicine and Epidemiology, School of Veterinary Medicine, University of California, Davis, CA 95616, USA
2 The Centre for the Environment, Fisheries and Aquaculture Science, Weymouth Laboratory, Barrack Road, The Nothe, Weymouth, Dorset DT4 8UB, UK
3 Department of Basic Sciences, College of Veterinary Medicine, PO Box 6100 Mississippi State, MS 39762, USA
4 Department of Marine Biosciences, Tokyo University of Marine Science and Technology, Konan 4-5-7, Minato-ku, Tokyo 108-8477, Japan
5 MRC Virology Unit, Institute of Virology, Church Street, Glasgow G11 5JR, UK
Correspondence
Thomas B. Waltzek
tbwaltzek{at}ucdavis.edu
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession numbers for the sequences reported in this paper are KHV helicase (AY939857)/triplex (AY939859), KHV DNA polymerase (AY939862) and KHV major capsid protein (AY939864); corresponding CyHV-1 sequences (AY939858, AY939860, AY939868 and AY939865, respectively); CyHV-2 sequences for parts of helicase (AY939867), triplex (AY939861) and DNA polymerase (AY939863); CyHV-1 sequence homologous to IcHV-1 ORF56 (AY939866).
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MAIN TEXT |
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Current placement of viruses in the family Herpesviridae is based upon the presence of a linear, double-stranded DNA genome packaged within an icosahedral capsid that is surrounded by a proteinaceous tegument layer and finally a host-derived envelope (Minson et al., 2000). Capsid architecture is highly conserved among the herpesviruses and consists of 162 capsomers arranged as a T=16 icosadeltahedral lattice. In herpes simplex virus type 1 (HSV-1), the 150 hexameric and 12 pentameric capsomers consist of the major capsid protein (VP5) plus a small protein (VP26) associated with the exterior of each subunit of the hexamers. These are joined by intercapsomeric triplexes, each consisting of two copies of one protein (VP23) and one copy of another (VP19C) (Zhou et al., 2000
). Production of herpesvirions involves capsid formation, capsid maturation, DNA replication, DNA packaging, addition of tegument, envelopment of capsids and release of mature particles. Each of these processes requires a suite of virally encoded proteins. DNA replication is dependent on seven viral proteins, which include a two-subunit DNA polymerase, a three-subunit helicaseprimase complex, a single-stranded DNA-binding protein and a helicase that recognizes the origins of DNA replication (Roizman & Pellett, 2001
; Wu et al., 1988
). DNA packaging also requires seven proteins, including a terminase that is unique to double-stranded DNA bacteriophages and herpesviruses (Brown et al., 2002
; Serwer et al., 2004
). Subsequent processes in viral morphogenesis and maturation are at least as complex and relatively poorly understood.
The existence of three fundamental clades within the family Herpesviridae is evident from the almost complete lack of sequence similarity between herpesviruses that infect mammals, birds and reptiles, herpesviruses that infect fish and frogs and the single recognized herpesvirus that infects an invertebrate (the oyster) (Bernard & Mercier, 1993; Davison, 1992
, 1998
, 2002
; Davison et al., 1999
, 2005
; McGeoch et al., 2000
). This has led to the proposal that the clades should be established as separate families under the umbrella of an order, Herpesvirales (Davison, 2002
; Davison et al., 2005
). At present, however, herpesviruses of fish and frogs, including two previously classified cyprinid herpesviruses (CyHV-1 and CyHV-2), remain as members of the family Herpesviridae (Minson et al., 2000
).
In order to investigate the genetic relationships between KHV, CyHV-1 and CyHV-2, sequences were determined and compared for two genes presumed to be involved in DNA replication and two genes encoding putative capsid proteins. The sequences were also compared to the most extensively characterized fish herpesvirus, channel catfish virus (Ictalurid herpesvirus 1, IcHV-1). In addition, genetic variation in a region of the DNA polymerase gene was assessed for 12 KHV isolates from koi and common carp in the USA, Israel, Malaysia and Taiwan.
Details of the KHV isolates are listed in Table 1. The CyHV-1 (Sano et al., 1985
) and CyHV-2 isolates were provided by one of the authors (H. Fukuda). The KHV and CyHV-1 isolates were grown in the KF-1 cell line, and the CyHV-2 isolate was grown in a goldfish fin cell line (GF-1) provided by H. Fukuda. Stocks of each virus (1 ml), previously frozen at 80 °C, were thawed and used to inoculate flasks containing monolayers of the appropriate cell line. After CPE was complete, cells and culture media were collected. All centrifugation steps were carried out at 10 °C. Cellular debris was separated from the culture medium by centrifugation at 3500 g for 20 min. The pellet was then Dounce-homogenized with 10 ml minimal essential medium containing 2 % (v/v) fetal bovine serum. The supernatant obtained by centrifugation at 3500 g for 20 min was combined with the culture medium and centrifuged at 95 300 g for 90 min. The virus pellet was resuspended by Dounce homogenizing in 1 ml TNE (50 mM Tris/HCl, 150 mM NaCl, 1 mM EDTA, pH 7·5) and layered onto a 1060 % (w/v) linear sucrose gradient in TNE. After centrifugation at 77 000 g for 18 h, two visible bands near the bottom of the tube were collected together, diluted in fresh TNE and centrifuged at 151 000 g for 1 h. The resulting virus pellet was eluted in 1 ml TNE and stored at 80 °C. Difficulties in growing significant quantities prevented CyHV-2 from being purified by using this protocol. Instead, the infected cell pellet was collected after CPE was complete, eluted into 1 ml TNE and stored at 80 °C. DNA was isolated from each preparation by treatment with proteinase K in lysis buffer [50 mM Tris/HCl, 100 mM NaCl, 100 mM EDTA, 1 % (w/v) SDS, pH 8·0], extraction with phenol/chloroform/isoamyl alcohol and ethanol precipitation (Sambrook et al., 1989
). The DNA was eluted in TE (50 mM Tris/HCl, 1 mM EDTA, pH 7·5) and the concentration was determined by spectrophotometry. DNA preparations were stored at 20 °C.
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In addition, a region of the DNA polymerase gene was amplified by PCR from KHV-U, CyHV-1 and CyHV-2 by using primers whose 3' ends were based on two regions conserved among the DNA polymerases of herpesviruses, poxviruses and iridoviruses (Ito & Braithwaite, 1991) (5'-CGGAATTCTAGAYTTYGCNWSNYTNTAYCC-3' and 5'-CCCGAATTCAGATCTCNGTRTCNCCRTA-3'; 497 bp product). BLASTP searches of the sequences again revealed close relationships between the three cyprinid viruses, with the next closest neighbours being the corresponding portions of the DNA polymerases of IcHV-1, Anguillid (eel) herpesvirus 1 and Ranid (frog) herpesvirus 1 (RaHV-1). The sequence data generated by using the degenerate primers facilitated the construction of KHV-specific primers (5'-GACTTTGCCAGCCTGTACCCCAGC-3' and 5'-CCGTGTCGCCGTACACGACGGTCA-3'; 496 bp product). The 448 bp sequence (i.e. the 496 bp product lacking the primer-binding sites) generated for the 12 KHV isolates in Table 1
was identical to the sequence determined previously by us for KHV-U (GenBank accession no. AY572853).
The data obtained thus far indicated that KHV is related most closely to CyHV-1 and CyHV-2 and more distantly to herpesviruses of non-cyprinid fish and of frog. Subsequently, data accumulating during KHV-U and CyHV-1 genome-sequencing projects being carried out in Tokyo, Japan, and Davis, CA, USA, respectively, permitted extension of the partial information obtained by PCR to the entire helicase, triplex protein and DNA polymerase genes (Fig. 1ac). KHV-U and KHV D-132 were found to be identical in the helicase gene and a single, synonymous nucleotide difference was noted in the triplex protein gene. In interpreting the results, it should be registered that the capsid proteins, including those constituting the intercapsomeric triplex, are specific to herpesviruses and that their conservation constitutes strong evidence for evolution from a common ancestral herpesvirus. Moreover, capsid proteins of fish and frog herpesviruses exhibit no detectable sequence similarity to their counterparts in higher-vertebrate herpesviruses (Davison & Davison, 1995
). In contrast, helicases and DNA polymerase genes are ubiquitous in organisms and acquisition by horizontal transfer rather than vertical inheritance is a significant possibility. Therefore, further data were extracted from the genome-sequencing projects for another herpesvirus-specific gene, encoding the major capsid protein (ORF39 in IcHV-1). The pattern of relationships for this gene was similar to that observed for the others (Fig. 1d
). In summary, KHV, CyHV-1 and CyHV-2 are related closely in each of the four genes analysed and are related more distantly to IcHV-1 and, where data are available, to other lower-vertebrate herpesviruses. Development of a detailed phylogenetic scheme is anticipated when more sequence data for a larger set of viruses become available.
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Our study has shown that four complete KHV genes of substantial size are conserved in lower-vertebrate herpesviruses. Two of these encode structural proteins that contribute to the capsid architecture conserved among all herpesviruses, and are detectably similar in sequence only among lower-vertebrate herpesviruses. The other two encode proteins involved in DNA replication and, even though they have relatives in many other organisms, are related most closely to counterparts in fish and frog herpesviruses. The comparisons demonstrate that KHV is related closely to, but distinguishable from, CyHV-1 and CyHV-2 at each locus examined. The three cyprinid viruses are also different from each other in their biological properties. These factors substantiate the case for continued inclusion of CyHV-1 and CyHV-2 in the family Herpesviridae and prompt us to propose that KHV should join them in this family as Cyprinid herpesvirus 3 (CyHV-3).
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ACKNOWLEDGEMENTS |
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Received 21 February 2005;
accepted 28 February 2005.