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. Author manuscript; available in PMC: 2014 Mar 21.
Published in final edited form as: J Fish Dis. 2013 Jan 24;36(8):721–733. doi: 10.1111/jfd.12073

Antibody screening identifies 78 putative host proteins involved in Cyprinid herpesvirus 3 infection or propagation in common carp, Cyprinus carpio L.

M Gotesman 1, H Soliman 1, M El-Matbouli 1
PMCID: PMC3961710  EMSID: EMS57316  PMID: 23347276

Abstract

Cyprinid herpesvirus 3 (CyHV-3) is the aetiological agent of a serious and notifiable disease afflicting common and koi carp, Cyprinus carpio L., termed koi herpesvirus disease (KHVD). Significant progress has been achieved in the last 15 years, since the initial reports surfaced from Germany, USA and Israel of the CyHV-3 virus, in terms of pathology and detection. However, relatively few studies have been carried out in understanding viral replication and propagation. Antibody-based affinity has been used for detection of CyHV-3 in enzyme-linked immunosorbent assay and PCR-based techniques, and immunohistological assays have been used to describe a CyHV-3 membrane protein, termed ORF81. In this study, monoclonal antibodies linked to N-hydroxysuccinimide (NHS)-activated spin columns were used to purify CyHV-3 and host proteins from tissue samples originating in either CyHV-3 symptomatic or asymptomatic fish. The samples were next analysed either by polyacrylamide gel electrophoresis (PAGE) and subsequently by electrospray ionization coupled to mass spectrometry (ESI-MS) or by ESI-MS analysis directly after purification. A total of 78 host proteins and five CyHV-3 proteins were identified in the two analyses. These data can be used to develop novel control methods for CyHV-3, based on pathways or proteins identified in this study.

Keywords: electrospray ionization mass spectrometry, immunochemistry, koi herpesvirus, polyacrylamide gel electrophoresis, protein purification

Introduction

Cyprinid herpesvirus 3 (CyHV-3), formerly known as koi herpesvirus (KHV), is the aetiological agent of a serious and notifiable disease afflicting common and koi carp, Cyprinus carpio L., termed koi herpesvirus disease (KHVD), and it has spread via carp trade and koi shows (Pikarsky et al. 2004; Pokorova et al. 2005; Ilouze et al. 2008). CyHV-3 has been isolated from kidney, gill, spleen, intestine, liver and brain of dead fish and causes a high mortality rate, between 80 and 100% (Hedrick et al. 2000). The most common clinical signs of the disease are white patches, sunken eyes, enlargement of the spleen and kidney, and necrosis in the gills in infected fish (Hedrick et al. 2005). CyHV-3 is known to infect carp at temperatures between 15 and 25 °C (Gilad et al. 2003). The genome of CyHV-3 consists of approximately 295 thousand base pairs (kB), coding for 156 novel putative proteins (Aoki et al. 2007).

Several studies have used immunochemistry, which takes advantage of affinity based on antibody avidity to an antigen, in investigations into CyHV-3 (Rosenkranz et al. 2008; Soliman & El-Matbouli 2009). In a recent study, polyclonal antibodies against CyHV-3 were used for viral detection purposes (Soliman & El-Matbouli 2009), and the technique may be more sensitive than other traditional PCR-based methods (Gilad et al. 2002; Bercovier et al. 2005; El-Matbouli, Rucker & Soliman 2007; Bergmann et al. 2010; Soliman & El-Matbouli 2010). A sensitive enzyme-linked immunosorbent assay (ELISA) was developed for the detection of CyHV-3 by Adkinson, Oren & Hendrick (2005), and a different monoclonal antibody against ORF68 has also been developed by Aoki et al. (2011). Another monoclonal antibody, derived against ORF81, localized to cytoplasmic regions of cells, including endoplasmic reticulum or Golgi apparatus probably during protein synthesis, and into the viral envelope in mature virons (Rosenkranz et al. 2008).

Knowledge about protein interactions can be used to understand how viruses enter host cells and propagate during infection (Guo et al. 2011; Blondot et al. 2012). Although some genes in CyHV-3 maintenance and replication have been identified (Fuchs et al. 2011), little is known about the protein interactions in viral propagation and even less is known whether this virus interacts with any endogenous common carp proteins. A recent report (Michel et al. 2010) used polyacrylamide gel electrophoresis (PAGE) and chemical-based protein purification techniques to identify 40 CyHV-3 proteins incorporated into mature virons. Eighteen fish proteins were also identified in the study including one common carp protein (Michel et al. 2010). Although a number of methods for protein purification have been described (de Souza et al. 2008; Moen et al. 2011), purification by antibody-based avidity is a powerful technique to identify in vivo protein binding partners (Tuxworth et al. 2005; Gotesman, Hosein & Gavin 2010; Gotesman, Hosein & Gavin 2011).

During mass spectrometry, samples are ionized and filtered based on mass, charge and shape, and subsequently, a detector measures the sample’s mass-to-charge ratio (Cameron 2012). Analysis of samples containing polymers, peptides and proteins of molecular weights beyond 20 kDa using an electrospray ionization source coupled with a quadruple mass spectrometer (ESI-MS) was first proposed by Fenn et al. (1989). Since then, ESI-MS has developed to be a sensitive tool for detecting samples at femtomolar concentrations in nanomole quantities and a routine method for characterizing non-volatile and thermally labile bio-molecules that are not amenable to analysis by other conventional techniques (Ho et al. 2003). Over the last decade, ESI-MS has emerged as an important technique in proteomics for confirmation of amino acid sequence and for characterization of post-translational modifications (Griffiths et al. 2001). We used antibody purification in conjunction with ESI-MS analysis to identify novel proteins that may be used as potential targets for the inhibition of CyHV-3 replication.

Materials and methods

Fish specimens

Two naturally infected fish, which showed signs of severe necrosis of the gills and skin, and loss of mucus on the skin, were used as CyHV-3 asymptomatic samples. One fish that lacked any signs of CyHV-3 was used as a control.

Tissue preparation

Tissue extracts (pooled from gill, liver, kidney, spleen and brain) were separately sampled aseptically from two different CyHV-3 symptomatic and one asymptomatic koi carp, and the samples were separately homogenized in minimum essential medium. Part of each homogenate was used for DNA extraction using a DNeasy Blood Tissue Kit (Qiagen) in accordance with the manufacturer’s instructions. Another aliquot of the homogenate was used for propagation of CyHV-3 on common carp brain cell line (CCB). Extracted DNA was subjected to conventional PCR according to Bercovier et al. (2005) and real-time PCR according to Gilad et al. (2004). CyHV-3 from symptomatic fish was successfully propagated on CCB cells and subsequently confirmed by PCR amplification of target DNA segments by conventional (Fig. 1) and real-time PCR as previously described. However, neither conventional PCR nor real-time PCR detected CyHV-3 in the asymptomatic fish sample. The remaining aliquots were separately used to prepare tissue lysate for electrospray ionization mass spectrometry (ESI-MS) analysis.

Figure 1.

Figure 1

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PCR analysis of infected fish samples. Lane 1: DNA extract from the symptomatic fish that was later used for PAGE and subsequent ESI-MS analysis. Lane 2: DNA extract from the symptomatic fish that was later used directly for ESI-MS analysis. Lane 3: empty. Lane 4: DNA extract from the asymptomatic fish that was later used directly for ESI-MS analysis. Lane 5: empty. Lane 6: DNA extract from CyHV-3 positive control. Lane 7: empty. Fifteen microlitres of sample was added to lanes 1–7. Lane 8: 1 μL of Bio-Rad 1 kb DNA Ladder.

Tissue lysate preparation

Each homogenate from the previous step was separately lysed in a 1:1 ratio with a non-denaturing lysis buffer: 50 mm Tris–HCl (pH 8.0), 150 mm NaCl, 20 mm ethylene diamine tetraacetic acid (EDTA), 1% Na-deoxycholate, 1% Triton X-100 (Williams 2000) and protease inhibitor cocktail (50 μL mL−1 of lysis buffer). Subsequently, each lysate was vigorously vortexed and centrifuged at 16 000 g for 15 min. The supernatant was transferred to a fresh 1.5-mL Eppendorf tube and recentrifuged at 16 000 g for an additional 15 min. Supernatant from the second centrifugation for each fraction was separately used for affinity purification as described in the following sections.

Preparation of monoclonal antibody-linked spin columns

Monoclonal antibodies against CyHV-3 glycoprotein (KHV/10A9 #171103, Insel Riems, Germany) were conjugated to N-hydroxysuccinimide (NHS)-activated 33-mg-capacity agarose spin column (Thermo Fisher Scientific) according to the manufacturer’s instructions. Briefly, 50 μL of CyHV-3 monoclonal antibody was resuspended into 350 μL of phosphate-buffered saline (PBS): 13.7 mm NaCl, 0.27 mm KCl, 10 mm Na2HPO4, 0.2 mm KH2PO4, pH 7.4 to make a total solution of 400 μL, and incubated overnight at 4 °C with mild shaking of 300 rpm in an Eppendorf Thermomixer Comfort. The spin columns were emptied and washed twice with PBS after overnight incubation at 4 °C with mild shaking. Subsequently, the spin columns were quenched with 400 μL of 1 m ethanolamine (pH 7.4) by incubation for 1 h at 4 °C with mild shaking. Finally, the spin columns were emptied by centrifugation at 16 000 g for 30 s and washed six times with PBS, and optical density at 280 (OD280) of the final flow-through was zero.

Protein purification

Non-denatured whole-cell extracts from CyHV-3 tissue samples originating in either symptomatic or asymptomatic fish were separately incubated overnight at 4 °C with mild shaking at 300 rpm in monoclonal antibody-linked spin columns (previously described). Next, the spin columns were cleared, and the flow-through was saved for gel electrophoresis. The spin columns were washed eight times with PBS, and the OD280 of the final flow-through was zero. The spin columns were subsequently eluted with 500 μL of 0.1 m glycine (pH 3.0) by incubation with mild shaking for 1 h at 4 °C, and the pH was immediately neutralized by addition of 50 μL of 1 m Tris base (pH 8.0). The columns were immediately washed with PBS, and both the eluate and the first wash fraction were concentrated, using a dry vacuum concentrator (Eppendorf) to a final volume of 55 μL. Next, the samples were either analysed by ESI-MS or further concentrated to 10 μL and analysed by sodium dodecyl sulphate - polyacrylamide gel electrophoresis (SDS-PAGE).

SDS-PAGE

The 10 μL concentrated sample was mixed with 10 μL of 2× Laemmli sample buffer (100 mm Tris–HCl, pH 6.8, 200 mm 2-mercaptoethanol, 4% SDS, 0.2% bromophenol blue, 20% glycerol) and heated at 99 °C with mild shaking (300 rpm) for 10 min. Proteins were separated in 12% polyacrylamide resolving gel and 5% stacking gel prepared according to Sambrook & Russell 2001). Gels were run using the Mini-Protein Tetra Cell (Bio-Rad laboratories GmbH) at 120 V for 70 min in 1× running buffer (25 mm Tris, 250 mm glycine, pH 8.3, 0.1% SDS). Gel loading was equal for all lysates. Proteins were visualized by staining with Coomassie Brilliant Blue R-250 (Sigma-Aldrich). The broad range (10–200 kDa) ProSieve Unstained Protein Marker II (Lonza), which contain 11 bands, 10, 15, 20,30, 40, 50, 70, 100, 120, 150 and 200 kDa, was used to estimate the molecular weight of the separated proteins.

Electrospray ionization mass spectrometry (ESI-MS) analysis

After staining with Coomassie Blue, the PAGE-gel was cut at sites corresponding to regions where bands of interest were visualized and sent for electrospray ionization coupled to mass spectrometry (ESI-MS) analysis. The other samples, which consisted of the entire antibody-purified products originating in either symptomatic or asymptomatic fish, were also separately analysed by ESI-MS. Electrospray ionization mass spectrometry analysis was performed by the DKFZ, The German Cancer Research Centre in the Helmholtz Association (Heidelberg, Germany).

Results

Protein purification and gel electrophoresis

Protein purification by affinity was used to analyse host and viral proteins involved in CyHV-3 infection. Tissue samples originating in CyHV-3 symptomatic and asymptomatic fish were processed by anti-CyHV-3 monoclonal antibody conjugated to spin columns (as described in the Materials and methods). The two symptomatic samples were eluted with respective optical densities of 0.264 and 0.552 at 280 (OD280). After concentration, the OD280 of the samples was above 3.0. The third sample, originating from asymptomatic and PCR-negative fish, was also purified, and the eluate had an OD280 of 0.012. The sample with an original OD of 0.264 was analysed on sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). One band corresponding to ~10 kDa and another band corresponding to ~60 kDa were visualized (Fig. 2).

Figure 2.

Figure 2

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PAGE analysis of immunoprecipitation. Lane 1: 5 μL of the ProSieve Unstained Protein Marker II (By Lonza), 10–200 kDa was used, which consists of 11 bands: 10, 15, 20, 30, 40, 50, 70, 100, 120, 150 and 200 kDa. The 120, 150 and 200 kDa did not separate well and appear as 1 band. Lane 2: Flow-through of non-denatured lysate (see methods). Lane 3: Monoclonal antibody–purified peptides. Two bands are observed: one at ~10 kDa and another band corresponding to ~60 kDa. Lane 4: First phosphate-buffered saline wash after elution with glycine (pH 3.0). Forty microlitres of the respective samples was run in lanes 2–4.

ESI-MS analysis

Two separate samples of anti-CyHV-3 monoclonal antibody–purified CyHV-3 tissue samples originating in symptomatic fish and a tissue sample originating in asymptomatic, and PCR-negative fish were analysed by electrospray ionization coupled to mass spectrometry (ESI-MS) analysis.

The first sample, which had an original OD280 of 0.264 after purification, consisted of two slices that showed bands in Coomassie-stained gel as previously described. The other sample, which had an original OD280 of 0.552 after purification, was analysed without any modifications except for concentration. The two analyses identified a total of five CyHV-3 proteins, two of which were overlapping (40%), and a total of 78 carp proteins, 28 of which were overlapping (36%). In the sample originating from asymptomatic fish, only one CyHV-3 was identified in conjunction with the 12 carp proteins identified, of which eight (66%) were identical to ones identified in the gel separation.

Gel separation

In the gel separation sample, 56 proteins were identified by ESI analysis of anti-CyHV-3 monoclonal antibody–purified tissue samples originating in CyHV-3 symptomatic fish. Two CyHV-3 proteins were identified: the major capsid protein and glycoprotein (Table 1a). The remaining 54 proteins, corresponding to proteins originating from carp were classified into seven different groups (Table 2a). Five of the carp proteins or their homologs were previously identified in Michel et al.’s (2010) report, in conjunction with beta-actin (Kuznetsov, Langford & Weiss 1992), which was grouped with three other cytoskeletal proteins. Also, seven proteins involved in host defence regulation in addition to 12 proteins that are involved in protein modification were identified. Fifteen haem-harbouring proteins in conjunction with three transferrins were also identified, and the remaining eight proteins were unclassified.

Table 1.

CyHV-3 proteins identified by ESI-MS analysis

Accession no. Protein description Score Mass [Da] Matches Coverage [%]
(a) Two CyHV-3 proteins identified by eiectrospray analysis from gei slices originating in symptomatic fish samples
 gi|65306693 Major capsid protein [Cyprinid herpesvirus 3] 278 140437 7 4.8
 gi|129560573 Glycoprotein [Cyprinid herpesvirus 3] 43 100710 2 2
(b) Five CyHV-3 proteins identified by electrospray analysis from unmodified lysate solution originating in symptomatic fish samples
 gi|61696095 Major capsid protein [Cyprinid herpesvirus 3] 25 140436 1 0.9
 gi|l29560573 Glycoprotein [Cyprinid herpesvirus 3] 24 100710 1 0.9
 gi|l29560669 0RF150 [Cyprinid herpesvirus 3] 23 70515 1 1.1
 gi|l31840052 0RF22 [Cyprinid herpesvirus 3] 22 67563 1 1
 gi|l31840053 ORF23 [Cyprinid herpesvirus 3] 26 38808 1 2.1
(c) One CyHV-3 proteins identified by electrospray analysis from unmodified lysate solution originating in asymptomatic fish
 gi|129560573 Glycoprotein [Cyprinid herpesvirus 3] 24 100710 1 1.1
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CyHV-3, Cyprinid herpesvirus 3.

Table 2.

Cyprinus carpio proteins identified by ESI-MS analysis

Accession no. Protein description Score Mass [Da] Matches Coverage [%]
(a) Fifty-four C. carpio proteins identified by electrospray analysis from gel slices originating in symptomatic fish samples grouped into 7 aforementioned categories
 Michel et al. 2010 or homolog proteins
  gi|28628941 Elongation factor 1-alpha 128 50 325 3 6.1
  gi|l7369826 Lactate dehydrogenase A chain 115 36 539 2 5.4
  gi|l5628189 Heat shock protein 90 alpha 28 25 336 1 5.3
  gi|l4388583 Warm-temperature acclimation-related 65-kDa protein 78 50 640 3 6.6
  gi|218511593 RAB8A 23 23 823 1 5.3
 Cytoskeletal proteins
  gi|42560193 Beta-actin 496 42 068 24 25.1
  gi|1703140 Alpha-actin-1 326 42 274 14 18.8
  gi|13365501 Integrin beta-2 chain 173 87 597 5 7.3
  gi|6226787 Vimentin 56 52 516 4 3.5
 Host defence proteins
  gi|4126587 Complement C3-H1 223 184 985 6 4
  gi|9453863 Complement C4-1 67 193 004 1 0.8
  gi|9453865 Complement C4-2 98 194 643 3 1.9
  gi|3399699 Natural killer cell enhancing factor 44 22 410 1 5.5
  gi|297718575 Granzyme A/K 26 29 149 2 4.3
  gi|7939556 Lysozyme C 49 17 241 2 11.7
  gi|2829695 Granulin-3 47 6995 2 24.6
 Protein modification enzymes or inhibitors
  gi|439153 Serine protease inhibitor 288 45 960 12 13.4
  gi|416561 Alpha-1-antitrypsin homolog 372 42 044 22 23.7
  gi|56709493 Putative glyceraldehyde-3-phosphate dehydrogenase 21 33 540 2 2.2
  gi|4027925 Creatine kinase M1-CK 37 42 980 1 3.4
  gi|73762632 Carbonic anhydrase 80 28 716 3 11.9
  gi|373503076 Glutamate dehydrogenase 910 54 750 46 47.1
  gi|112901127 Glutathione S-transferase rho 35 26 700 1 6.6
  gi|152925970 Ubiquitin fusion protein 279 14 987 19 41.4
  gi|91798526 Proteasome activator PA28 subunit 37 28 579 1 2.8
  gi|217272706 Monoamine oxidase 442 59 621 13 16.3
  gi|6690508 Janus kinase 3 22 131 078 1 0.7
  gi|300676317 Catalase 21 49 187 1 3.1
 Globins
  gi|2208883 Alpha-globin 529 15 421 35 52.4
  gi|2208891 Alpha-globin 488 15 566 35 54.5
  gi|2208889 Alpha-globin 390 15 574 28 61.5
  gi|122392 Alpha-globin 595 15 437 45 62.2
  gi|6009727 Alpha-2-macroglobulin-1 631 162 274 22 11.8
  gi|6009729 Alpha-2-macroglobulin-2 541 157 876 18 10
  gi|6009731 Alpha-2-macroglobulin-3 434 88 297 14 15.1
  gi|140368180 Alpha-2-macroglobulin 4 368 101 340 12 11.4
  gi|22135548 Beta-globin 584 16 520 41 63.3
  gi|2208895 Beta-globin 732 16 617 67 87.2
  gi|1644253 Beta-globin 718 16 645 63 87.2
  gi|1644251 Beta-globin 706 16 542 68 87.2
  gi|3953518 Immunoglobulin heavy chain 1053 50 331 54 47.6
  gi|55700034 Immunoglobulin heavy chain 78 40 300 4 6.4
  gi|85067845 Myoglobin isoform 2 29 16 278 1 5.4
 Transferrins
  gi|18034630 Transferrin variant A 1882 75 595 100 47.1
  gi|189473163 Transferrin variant F 986 75 036 45 26
  gi|189473165 Transferrin variant G 1003 75 083 47 26.9
 Unclassified
  gi|29501366 Fetuin short form 43 34 332 2 7
  gi|13445027 Apolipoprotein A-I 83 20 797 4 16.1
  gi|435737 Glial fibrillary acidic protein 119 24 944 69 9.3
  gi|151558991 Vitellogenin B1 2221 148 683 100 39.6
  gi|52782167 Vitellogenin B2 964 179 640 48 13.4
  gi|14009437 Mitochondrial ATP synthase 32 59 699 1 2.2
  gi|47605558 Mitochondrial synthase subunit beta 71 55 327 2 4.1
  gi|10566900 Myeloid protein-1 35 17 849 1 7.5
(b) Fifty-two C. carpio proteins identified by electrospray analysis from unmodified lysate solution originating in symptomatic fish samples grouped into 7 aforementioned categories
 Michel et al. 2010 or homologs
  gi|28628941 Elongation factor 1-alpha 97 50 325 5 6.7
  gi|l7369826 Lactate dehydrogenase A chain 110 36 539 2 6.3
  gi|55669145 Lactate dehydrogenase B-type subunit 150 36 669 3 9
  gi|33598990 Constitutive heat shock protein HSC70-2 329 70 779 12 12.3
  gi|218511593 RAB8A 26 23 823 1 5.3
 Cytoskeletal proteins
  gi|42560193 Beta-actin 457 42 068 15 24.8
  gi|6226787 Vimentin 127 52 516 6 3.5
 Host defence proteins
  gi|4126587 Complement C3-H1 362 184 985 9 5.2
  gi|4126593 Complement C3-S 179 185 561 5 2.9
  gi|3399699 Natural killer cell enhancing factor 93 22 410 3 9.5
  gi|84569882 Natural killer cell enhancing factor 251 22 014 10 47.7
  gi|165880803 MIF 26 12 525 1 14.8
  gi|47117021 Lysozyme g 88 20 425 3 10.8
 Protein modification enzymes or inhibitors
  gi|439153 Serine protease inhibitor 83 45 960 2 9
  gi|416561 Alpha-1-antitrypsin homolog 261 42 044 7 15.6
  gi|56709493 Putative glyceraldehyde-3-phosphate dehydrogenase 146 33 540 2 9.2
  gi|4027925 Creatine kinase M1-CK 34 42 980 1 1.6
  gi|73762632 Carbonic anhydrase 285 28 716 7 40.4
  gi|373503074 Glutamate dehydrogenase 63 54 737 2 3.1
  gi|112901127 Glutathione S-transferase rho 51 26 700 1 4.4
  gi|95832156 Pi-class glutathione S-transferase 69 23 794 2 10.1
  gi|300676299 Glutathione peroxidase 1 37 19 182 1 10
  gi|343481094 Glutathione synthetase 22 41 342 1 2.5
  gi|353351682 Trypsin 1 114 26 940 1 6.6
  gi|117518748 Enolase 21 16 206 1 4.1
 Globins
  gi|2208891 Alpha-globin 527 15 566 44 73.4
  gi|2208883 Alpha-globin 494 15 421 35 69.2
  gi|2208887 Alpha-globin 492 15 449 36 69.2
  gi|2208889 Alpha-globin 372 15 574 24 69.2
  gi|122392 Alpha-globin 659 15 437 56 81.1
  gi|1644251 Beta-globin 652 16 542 47 78.4
  gi|2208895 Beta-globin 568 16 617 46 70.9
  gi|22135548 Beta-globin 485 16 520 26 54.4
  gi|3953518 Immunoglobulin heavy chain 177 50 331 5 12.8
  gi|85067845 Myoglobin isoform 2 86 16 278 2 12.9
 Transferrins
  gi|18034630 Transferrin variant A 420 75 595 17 12.3
  gi|189473165 Transferrin variant G 321 75 083 12 10.2
 Unclassified
  gi|29501366 Fetuin short form 64 34 332 3 7.3
  gi|13445027 Apolipoprotein A-I 226 20 797 7 32.2
  gi|435737 Glial fibrillary acidic protein 417 24 944 41 28
  gi|435739 Glial fibrillary acidic protein 504 24 531 44 33.2
  gi|49356890 Glia-derived neurotrophic factor 25 11 004 1 8.6
  gi|2252655 Nephrosin precursor 340 31 629 13 27.5
  gi|281333450 Heart-type fatty-acid-binding protein 156 14 704 5 21.8
  gi|15778562 Vitellogenin 294 148 794 12 7.8
  gi|84569880 Translationally controlled tumour protein 84 19 152 2 8.2
  gi|281429776 Adipocyte fatty-acid-binding protein 124 15 278 2 16.4
  gi|1173446 Somatoliberin 28 4976 1 17.8
  gi|585105 Ependymin 385 24 433 67 41.9
  gi|40549335 Cytochrome P4501C1 24 59 583 1 1.3
  gi|219935425 Mineralocorticoid receptor 24 107 380 1 0.5
  gi|169674670 Liver-basic fatty-acid-binding protein b 24 14 255 1 15.9
 (c) Twelve C. carpio proteins identified by electrospray analysis from unmodified lysate solution originating in asymptomatic fish samples grouped into 7 aforementioned categories
 Michel et al. 2010 or homolog proteins
  gi|32394421 Muscle-specific heat shock protein Hsc70-1 51 70 660 2 6.1
 Cytoskeletal proteins
  gi|42560193 Beta-actin 78 42 068 4 8.8
  gi|1703140 Alpha-actin-1 77 42 274 3 9
  gi|6226787 Vimentin 32 52 516 1 1.5
 Host defence proteins
  gi|7939556 Lysozyme C 264 17 241 20 43.4
  gi|313509543 Toll-like receptor 22 30 109 136 3 0.7
  gi|2829694 Granulin-2 43 7215 1 17.5
 Protein modification enzymes or inhibitors
  gi|152925970 Ubiquitin fusion protein 99 14 987 5 24.2
 Globins
  gi|122392 Alpha-globin 88 15 437 4 16.1
  gi|85067845 Myoglobin isoform 2 25 16 278 1 5.4
 Unclassified
  gi|435737 Glial fibrillary acidic protein 80 24 944 30 5.1
  gi|162423638 Cyclin B 27 45 068 1 2
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MIF, migration inhibitory factor.

The 54 and 52 identified proteins from either gel slices or unmodified samples, respectively, were classified into seven groups that include: Michel et al. 2010, cytoskeletal, host defence, protein modification or inhibitors, globins, transferrins, unclassified. Proteins that showed up in both analyses are in bold.

In solution

The second sample, which had an original OD of 0.552 at 280, was analysed directly by ESI-MS, and 57 proteins were identified. In addition to the two CyHV-3 proteins identified in the previous section, three additional CyHV-3 proteins were identified: ORF22, ORF23 and ORF150 (Table 1b). Additionally 52 carp proteins were identified, which were classified into seven different groups as in the previous section (Table 2b). Similarly, five of the carp proteins or their homologs were previously identified in Michel et al. (2010) in conjunction with beta-actin, which was grouped with vimentin, another cytoskeletal protein. Also, six proteins involved in host defence regulation in addition to 12 proteins that are involved in protein modification were identified. Ten haem-harbouring proteins in conjunction with two transferrins were also identified, and the remaining 15 proteins were unclassified.

Asymptomatic fish group

The third sample, which consisted of anti-CyHV-3 monoclonal antibody–purified tissue samples originating in CyHV-3 asymptomatic fish, identified only one CyHV-3 protein, glycoprotein, ORF56 (Table 1c), and 12 carp proteins that include one homolog to the Michel et al. (2010) study, three cytoskeletal proteins, two host defence–related proteins, one protein involved in protein modification, two haem-harbouring proteins and three unclassified proteins (2c).

Discussion

The importance of understanding proteomics such as protein–protein interactions is fundamental in understanding biological systems (Watson James 2003). In conjunction with the traditional tools used to describe protein interactions, such as immunochemistry and yeast two-hybrid system, analysis by mass spectrometry has become a very useful tool to study protein interactions (Cameron 2012). We used antibody-based purification coupled with mass spectrometry to identify protein targets during Cyprinid herpesvirus 3 infection that could ultimately be used to develop novel methods for CyHV-3 control in koi and in common carp. During this study, 78 host proteins involved in Cyprinid herpesvirus 3 infection or propagation and five potential immunogenic CyHV-3 protein targets were identified in CyHV-3 diseased fish. Additionally, four host proteins were identified in CyHV-3 asymptomatic fish. The major capsid protein identified in this study corresponds to ORF92 previously identified in Michel et al. (2010); however, the remaining four proteins, ORF22, ORF23, glycoprotein (ORF56) and ORF150, were previously undetected. To enhance our understanding of the CyHV-3 proteins, Prosite (Sigrist et al. 2010) and Simple Modular Architect Research Tool (SMART) by Letunic, Doerks & Bork (2012) were used to elucidate important domain structures from the five CyHV-3-identified proteins (Table 3).

Table 3.

Description of CyHV-3 proteins identified. Prosite (Sigrist et al. 2010) and Simple Modular Architect Research Tool (SMART) by Letunic et al. (2012) were used to identify important domains in the 5 CyHV-3 proteins identified in this study. Amino acid number is referred to by the abbreviation aa

Accession no. Protein description Protein length Domain of interest Spanning region
(a) Glycoprotein and 0RF150 modelled using Prosite
 gi|129560573 Glycoprotein 897 aa Putative AMP binding 173-184 aa
 gi|129560669 0RF150 628 aa Zinc finger Ring-type 24-66 aa
(b) The remaining three proteins, 0RF22, 0RF23 and 0RF92 (major capsid protein), modelled by SMART
 gi|65306693 Major capsid protein 1268 aa MHC II beta 461-513 aa
 gi|131840052 0RF22 588 aa Transmembrane domain 22-217 aa
 gi|131840053 0RF23 335 aa Interleukin-10 (IL-10) 24-163 aa
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We compared proteins identified in this purification to the ones identified by Michel et al. (2010) to assess the validity of our assay. The comparison is complicated because the data set used in the previous study was composed before the entire genome for C. carpio was available, and therefore, all but one of the proteins (elongation factor) listed in the previous report come from either zebra fish, Danio rerio, or Atlantic salmon, Salmo salar. However, two proteins (elongation factor, and lactate dehydrogenase) were identical matches for proteins identified in the aforementioned studies. Rab8a was also included in as a match because of the high sequence homology amongst the Rab family of proteins (Bright et al. 2010). Similar logic was used to include heat shock proteins amongst the proteins also identified in the Michel et al.’s (2010) study. Although glutathione S-transferase rho was identified in both isolations and is a ras-related protein, it was not included in the Michel et al. (2010) list because ClustalW alignment did not show significant homology between the two proteins. Interestingly, our study identified several cytoskeletal proteins including actin (Hosein et al. 2003; Williams et al. 2006) and vimentin, and the Michel et al. (2010) study also identified cytoskeleton proteins that include two of the ubiquitous dynamic cytoskeletal proteins tubulin and actin (Gavin 1997), and a cofilin-like protein that may act as a regulator of actin dynamics (De La Cruz 2009; Shiozaki et al. 2009).

Electrospray analysis of samples originating in symptomatic fish identified seven proteins from the in-gel analysis and six proteins from the in-solution analysis that are involved in the host defence pathway. Two of the proteins, natural killer cell enhancing factor (Fujiki et al. 1999) and complement C3-H1 (Nakao et al. 2000), were observed in each of the two samples originating in symptomatic fish; therefore, a total of 11 proteins were identified. In addition to the C3-H1 protein, three other proteins (complements C3-S, C4-1 and C4-2) were identified in the complement host defence pathway (Kato et al. 2004). Of the remaining five host defence proteins, granzyme A/K is a member of the serine protease family (Huang et al. 2010) along with natural killer cell enhancing factor, which is a component of natural kill cells, and macrophage migration inhibitory factor (MIF) is a member of the cytokine signalling pathway (Bernhagen et al. 1993). Belcourt et al. (1995) demonstrated that granulin-1 localizes to macrophages in common carp and gold fish, Carassius auratus auratus, and granulin-3 which was identified in the gel analysis also is likely part of the host defence pathway. Lysozyme C and lysozyme G were also identified in this analysis; ClustalW alignment did not show significant homology between the two proteins and are phylogenetically unlinked (Savan, Aman & Sakai 2003). Overexpression of endogenous lysozyme C has antimicrobial and antiviral activities in grouper, Epinephelus coioides, spleen cells (Wei et al. 2012). Although lysozyme C and lysozyme G share very low sequence homology in the grass carp, Ctenopharyngodon idellus, they share similar expression profiles and were both shown to have antimicrobial activities, which may indicate that lysozyme G also has antiviral activities consistent with the previous study (Ye et al. 2010).

Protein modification is the final method in protein regulation, such as in the modifying gene regulation via the histone code (Jenuwein & Allis 2001); the ubiquitin code and other proteins modifiers are used to activate, repress, target, stabilize or degrade proteins (Sims & Reinberg 2008; Harper & Schulman 2006; Williams & Schwarzbauer 2009). A total of 17 proteins involved in protein modification were identified in this study. Seven of the proteins overlapped in both the gel and unmodified solution analysis. Two members of the protein degradation pathway, an ubiquitin fusion protein along with a proteasome activator subunit, were identified in the gel solution (Table 2a), and accordingly, one of the CyHV-3 proteins identified (Table 3a) has a RING-finger domain, which is known to mediate E3 ubiquitin-ligase activity (Joazeiro & Weissman 2000). Both analyses identified glutathione S-transferase rho; in addition, four other glutathione-related proteins were also identified in the unmodified solution (Table 2b), and these proteins are known to regulate inflammatory response and modulate cytoskeletal proteins (Zhang et al. 1995). Interestingly, alpha-1-antitrypsin homolog was identified in both analyses, and trypsin 1 was identified in the untreated sample; one may wonder whether interaction of these proteins is modulated during CyHV-3 disease.

Cyprinid herpesvirus 3 is also known as KHV, which was once called carp interstitial nephritis and gill necrosis virus (CNGV) because the disease was initially described to affect the kidneys and gills (Hutoran et al. 2005; Pokorova et al. 2005; Ilouze et al. 2008). Recently, there has been debate as to whether the skin, especially at points of abrasion, is the site for viral entry (Costes et al. 2009; Raj et al. 2011; Fournier et al. 2012). However, we are unaware of a study that describes how the virus spreads to other major organs such as liver, spleen and brain (Gilad et al. 2004; Dishon et al. 2007). Abundance of blood-related proteins such as the globins and transferrins may lead one to speculate whether CyHV-3 propagates via the blood transport system.

Amongst the unclassified list, a total of 21 proteins were grouped. Three proteins were identified in both analyses, the gel solution contained an additional six proteins, and the unmodified solution contained an additional 12 proteins (Table 3). CyHV-3 is known to affect kidneys, spleen and brain of carp (Ronen et al. 2003; Pikarsky et al. 2004; Miyazaki et al. 2008); several proteins that localize to CyHV-3-affected areas were identified including nephrosis precursor, fetuin short form and three other glial-associated proteins (Kálmán 1998; Tsai et al. 2004).

Initial attempts to control CyHV-3 outbreaks were carried out by a short exposure to CyHV-3 at permissible temperature (i.e. 15–25 °C) and subsequent treatment at non-permissible temperature (30 °C) to stimulate an immunogenic response against CyHV-3 (Ronen et al. 2003). However, several studies indicate that CyHV-3 stays dormant in survivors of CyHV-3 disease (Bretzinger et al. 1999; Bergmann et al. 2009; Eidea et al. 2011) and that CyHV-3 can even resurface to propagate and infect naïve fish (St-Hilaire et al. 2005; St-Hilaire et al. 2009). The third sample that was analysed came from a fish with no signs of CyHV-3 disease and was free of CyHV-3 according to analysis by conventional PCR (Bercovier et al. 2005) and real-time PCR (Gilad et al., 2004); however, ESI-MS detected glycoprotein (ORF56), indicating that the sample may have been a latent carrier for CyHV-3 disease (Table 1c). Interestingly, in the asymptomatic carrier, four novel proteins were identified in conjunction with the 8 proteins previously identified. The four novel proteins include an additional homolog to heat shock proteins, termed Hsc70-1 (Wu et al. 2011), and a cell cycle regulator cyclin B in addition to the two host defence proteins. Granulins share very high sequence homology (Belcourt et al. 1995), and therefore, granulin-2 may not be a novel finding in the asymptomatic study. However, Toll-like receptor 22 (TLR-22) was not detected in the other two analyses and has been demonstrated to have antiviral function (Lv et al. 2012).

A few regions of the world are relatively free of the Cyprinid herpesvirus such as India and Australia (McColl et al. 2007; Rathore et al. 2009); however, CyHV-3 has become an epidemic threat to the world carp industry, especially in Europe, Israel, Indonesia and the largest aquaculture carp producer, China (Bergmann et al. 2006; Bondad-Reantaso, Sunarto & Subasinghe 2007; Dishon et al. 2007; Sunarto et al. 2007; Dong et al. 2011). Several studies have explored the method of infection, latency and detection of CyHV-3 (Bergmann et al. 2009, 2010; Costes et al. 2009). However, relatively few studies have explored CyHV-3 on a molecular level (Michel et al. 2010; Fuchs et al. 2011). We used anti-body-based purification followed by electrospray ionization analysis to investigate molecular interactions during CyHV-3 infections. Seventy-eight endogenous host proteins along with five putative immunogenic CyHV-3 proteins involved in CyHV-3 replication and propagation were identified in this investigation. Additionally, four endogenous proteins were identified in samples originating from CyHV-3 asymptomatic fish. CyHV-3 infection has been recently shown to modulate gene expression in the proteasome degradation pathway, granulin, and other protein modifying enzymes identified in this report (Rakus et al. 2012). The antibody against glycoprotein (ORF56) was able to isolate CyHV-3 proteins in a sample that was KHV negative according to PCR methods detecting the TK gene. The glycoprotein may also be highly immunogenic and a good target for detection of CyHV-3 in asymptomatic fish by ELISA or even by PCR analysis. The diversity of proteins identified in this study opens the door for further analysis of host proteins involved in CyHV-3 replication, propagation and repression. For example, Toll-like receptor 22 appears to be involved in suppressing CyHV-3 disease in infected fish. Therefore, stimulating the overexpression of TLR-22 may be an alternative method of controlling CyHV-3 disease.

Acknowledgements

Monoclonal antibodies were kindly provided by Dr Bergmann and Dr Fischtner; Friedrich Loeffler Institute (FLI), the National Research Centre for Animal Health in Greifswald, Germany. We would like to thank the Proteomics Core Facility of the German Cancer Research Centre, Heidelberg, for their excellent work regarding protein identification by ESI-MS. Technical assistance was provided by Dr Andrea Dressler.

Funding for this project was provided by the Austrian Science Fund (FWF), grant no. P 23550.

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