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Short Communication |
1 Institute of Molecular Biology, Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, 17493 Greifswald-Insel Riems, Germany
2 Institute of Infectology, Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, 17493 Greifswald-Insel Riems, Germany
Correspondence
Walter Fuchs
walter.fuchs{at}fli.bund.de
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; Hedrick et al., 2000; Neukirch et al., 1999). Since the causative agent exhibited a herpesvirus-like morphology it had been designated koi herpesvirus (KHV; Hedrick et al., 2000). Alternatively, it has been described as carp nephritis and gill necrosis virus (CNGV) because of the typical pathological effects (Ronen et al., 2003).
The virus possesses an unusually large double-stranded DNA genome of approximately 295 kbp including 22 kbp direct repeat sequences present at both genome ends (Hutoran et al., 2005). Complete sequence analysis of three different isolates revealed the presence of 156 common, presumably protein-encoding open reading frames (Aoki et al., 2007). Many of the predicted genes of KHV possess significantly conserved homologues in other herpesviruses of fish and amphibians, such as channel catfish virus (ictalurid herpesvirus 1, IcHV-1) and ranid herpesviruses 1 and 2 (Davison, 1992; Davison et al., 2006). In particular, the deduced amino acid sequences of selected genes of two previously described pathogens of cyprinids, carp pox herpesvirus (cyprinid herpesvirus 1) and haematopoietic herpesvirus of goldfish (cyprinid herpesvirus 2) exhibit high degrees of identity with the corresponding gene products of KHV, which has therefore been designated cyprinid herpesvirus 3 (Waltzek et al., 2005). In contrast, detectable sequence homologies to mammalian herpesviruses are essentially limited to genes encoding enzymes involved in DNA replication, which are conserved in all organisms. Therefore, it has been proposed by the Herpesviridae Study Group to assign the herpesviruses of fish and amphibians to a new family Alloherpesviridae, which should be grouped together with herpesviruses of shellfish (Malacoherpesviridae), and herpesviruses of mammals, birds and reptiles (Herpesviridae) in the new order Herpesvirales (McGeoch et al., 2006).
The lack of sequence identity to herpesviruses of humans and other mammals precludes predictions about the functions of most KHV genes. However, this information is urgently needed to obtain better tools for the diagnosis and combat of an important fish disease. In particular, it would be interesting to ascertain which of the predicted 27 membrane proteins of KHV (Aoki et al., 2007) are major components of the virion envelope and relevant for virus–host cell interactions, as well as for the host immune response. However, up to now, no gene product of KHV has been characterized or even identified.
One of the putative membrane proteins of KHV is encoded by ORF81. This gene is a positional homologue of ORF59 of IcHV-1, which is considered to encode the major envelope glycoprotein of this virus (Aoki et al., 2007; Davison & Davison, 1995; Kucuktas et al., 1998). The deduced ORF81 gene product of KHV consists of 256 aa and, like pORF59 of IcHV-1, specifies four predicted trans-membrane helices (Fig. 1c; Hirokawa et al., 1998). The C-terminal domain of the type III membrane protein is very hydrophilic (Fig. 1c), correlating with a high predicted antigenic index (Jameson & Wolf, 1988). Therefore, in the present study ORF81 was cloned, its product (pORF81) was expressed in pro- and eukaryotic cells, and a monospecific antiserum was prepared and tested for its suitability for the detection of KHV in infected cells and carp tissues.
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). It was propagated in common carp brain (CCB) cells (Neukirch et al., 1999) and grown at 25 °C in minimum essential medium (MEM) supplemented with 10 % fetal calf serum (both purchased from Invitrogen). After complete cytolysis of infected cells, KHV virions were purified by consecutive centrifugation through a 35 % sucrose cushion, and a discontinuous (30, 40 and 50 %) sucrose gradient as described for pseudorabies virus (PrV; Böttcher et al., 2007). Virion DNA was prepared (Fuchs & Mettenleiter, 1996), and viral DNA fragments generated by digestion with BamHI were cloned into the plasmid vector pBluescript SK(-) (Stratagene). The complete ORF81 was recloned from pBl-KB4455 containing a genomic 4455 bp BamHI fragment of KHV, as a 1109 bp PshAI/XhoI fragment into the eukaryotic expression vector pcDNA3 (Invitrogen; Fig. 1b
). Furthermore, a 555 bp BsaI/XhoI subfragment of pBl-KB4455 was recloned in vector pGEX-4T-3 (Amersham; Fig. 1b). The resulting construct permitted abundant expression of a 36 kDa fusion protein consisting of glutathione S-transferase (GST) and the hydrophilic C-terminal part of pORF81 (aa 177–257; Fig. 1c) in Escherichia coli. The GST–pORF81 fusion protein was purified and used for immunization of a rabbit as described previously (Fuchs et al., 2002).
The rabbit serum obtained after five immunizations with 100 µg each of the fusion protein was used for Western blot analyses of CCB cells harvested 2 days after infection with KHV at a multiplicity of 1 p.f.u. per cell, or after transfection (TransFectin lipid reagent; Bio-Rad) with pcDNA-KO81 (Fig. 1b). Lysates of 104 cells, or 2 µg purified virion proteins per lane were separated, transferred to membranes, and consecutively incubated with the anti-pORF81 serum (dilution 1 : 50 000) and horseradish peroxidase-conjugated secondary antibodies as described previously (Fuchs & Mettenleiter, 1999). The antiserum specifically reacted with a viral protein exhibiting an apparent molecular mass of 26 kDa (Fig. 2a), which was not found in uninfected CCB cells (Fig. 2a), and also was not detected by the preimmune serum (Fig. 2b). Thus, the size of the ORF81 protein of KHV closely matches the predicted molecular mass of 28.25 kDa (Aoki et al., 2007). The marginally lower apparent mass of pORF81 is presumably not caused by the removal of an N-terminal signal peptide, since no corresponding consensus sequences (von Heijne, 1986) are present in the deduced translation product. Similar, 26 kDa proteins could be detected in cells transfected with pcDNA-KO81 (Fig. 2a), and also after in vitro transcription and translation of this plasmid (data not shown). These findings indicate the absence of extensive post-translational modifications of pORF81, which is in agreement with the absence of putative N-glycosylation sites (Kornfeld & Kornfeld, 1985) in the deduced amino acid sequence (Fig. 1c). Western blot analysis of purified KHV particles indicated that pORF81 is efficiently incorporated into virions (Fig. 2a).
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). Confocal laser scan microscopy (Zeiss LSM510) revealed a granular cytoplasmic fluorescence of infected cells (Fig. 2c), indicating the expected association of the predicted membrane-spanning protein with cytoplasmic organelles, such as endoplasmic reticulum or Golgi apparatus. A similar, predominantly cytoplasmic localization of pORF81 was detectable in cells transfected with pcDNA-KO81 (Fig. 2d), but not in cells transfected with a control plasmid (Fig. 2e). Although no pronounced accumulation of pORF81 at cytoplasmic membranes could be observed (Fig. 2c, d), the antiserum raised against the C-terminal part also showed weak, but clearly specific reactions with non-permeabilized cells that had been fixed with 3 % paraformaldehyde after infection or transfection (data not shown). Thus, although according to topology predictions (Krogh et al., 2001) the location of the C terminus of pORF81 inside the cytosol is more likely, our experimental results suggest that this protein part might represent an ecto-domain.
To determine the location of pORF81 in virions, native KHV particles were prepared for immunoelectron microscopy as described previously (Veits et al., 2003). After incubation with the diluted anti-pORF81 serum, specific binding could be detected using goat anti-rabbit antibodies tagged with 10 nm gold particles (British BioCell Int.). Examination with an electron microscope (Tecnai 12, Philips) revealed the accumulation of gold particles only along the surface of intact KHV particles (Fig. 2f). In contrast, no label was found after incubation of KHV virions with irrelevant antisera (data not shown), or after incubation of similarly prepared PrV particles with the anti-pORF81 serum (Fig. 2g). Thus, pORF81 is incorporated into the viral envelope, and the antigenic C-terminal domain is presumably localized at the outside of the particle.
For in vivo studies, juvenile common carp (body mass approx. 5 g) were infected by immersion in water containing 105 p.f.u. l–1 KHV for 1 h and kept at 22 °C. All animals developed typical clinical signs of KHV infections like apathy, inappetence, dyspnoea, uncoordinated movement, gill necroses and skin lesions (Haenen et al., 2004; Pikarsky et al., 2004), and died between 7 and 11 days after infection. At necropsy the infected carp demonstrated mild to moderate branchial necrosis, haemorrhages of the gills and mild focal cutaneous ulcerations. For further investigations, organs were fixed for 24–48 h in 4 % neutral buffered formaldehyde and embedded in paraffin.
Histopathological investigation of haematoxylin and eosin (H&E) stained 2 µm sections revealed a partial loss of architecture of the gills with blunting of lamellae, and moderate to severe mixed cellular interstitial inflammatory infiltration in a large number of the filaments (Fig. 3a). Congestion of the gills' central sinusoids was also present as multifocal detachment of the gill epithelium. Numerous large ovoid to polygonal basophilic cells with large pale basophilic intranuclear inclusion bodies (arrows) were scattered throughout the capillaries of the filaments or within larger vessels. The kidneys (Fig. 3d) showed moderate to severe diffuse interstitial infiltration with lymphocytes and numerous large polygonal to round cells with abundant basophilic cytoplasm and large pale basophilic nuclei with marginated chromatin and intranuclear inclusions (arrows). Similar large ‘karyomegalic’ cells were seen in the hepatopancreas within areas of degenerate and necrotic pancreatic acinar cells. A comparable inflammatory infiltrate was also present multifocally in the skin and subcutis (data not shown).
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), in cells infiltrating the renal interstitium (Fig. 3e), the submucosa of the intestine, in dermis and subcutis, spleen, periosteum and pancreas. In addition, few acinar cells of the exocrine pancreas and singular or groups of neurons in spinal cord and brain were also positive for KHV antigen. No specific reactivity was observed in tissues of non-infected carp (data not shown), or of infected carp incubated with the respective preimmune serum (Fig. 3c, f).
Thus, the presence of pORF81 correlates with the observed lesions in KHV-infected animals and is in general agreement with the distribution of viral antigens described in earlier pathogenesis studies (Pikarsky et al., 2004). Since the anti-pORF81 serum showed no detectable cross-reactivity with cellular proteins, precise investigations of organ-tropism of KHV and kinetics of virus spread might be further facilitated. If pORF81 is sufficiently immunogenic in naturally infected carp, the recombinant ORF81 proteins expressed in eukaryotic cells or bacteria might also be used as diagnostic antigens for the detection of KHV-specific serum antibodies by immunofluorescence tests or ELISAs. Furthermore, the ORF81 expression constructs might be suitable for the development of subunit- or DNA-vaccines against KHV.
In summary, we describe here the identification of the first KHV protein, using an antiserum of known specificity. The monospecific rabbit antiserum against the envelope protein pORF81 is suitable for sensitive detection of KHV infection and KHV particles in vitro and in vivo.
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ACKNOWLEDGEMENTS |
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Received 5 November 2007;
accepted 3 January 2008.
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