The IL-10 homologue encoded by cyprinid herpesvirus 3 is essential neither for viral replication in vitro nor for virulence in vivo
© Ouyang et al.; licensee BioMed Central Ltd. 2013
Received: 15 April 2013
Accepted: 10 June 2013
Published: 16 July 2013
Cyprinid herpesvirus 3 (CyHV-3), a member of the family Alloherpesviridae, is the causative agent of a lethal disease in common and koi carp. CyHV-3 ORF134 encodes an interleukin-10 (IL-10) homologue. The present study was devoted to this ORF. Transcriptomic analyses revealed that ORF134 is expressed as a spliced gene belonging to the early-late class. Proteomic analyses of CyHV-3 infected cell supernatant demonstrated that the ORF134 expression product is one of the most abundant proteins of the CyHV-3 secretome. To investigate the role of ORF134 in viral replication in vitro and in virulence in vivo, a deleted strain and a derived revertant strain were produced using BAC cloning technologies. The recombinant ORF134 deleted strain replicated in vitro comparably to the parental and the revertant strains. Infection of fish by immersion in water containing the virus induced comparable CyHV-3 disease for the three virus genotypes tested (wild type, deleted and revertant). Quantification of viral DNA by real time TaqMan PCR (in the gills and the kidney) and analysis of carp cytokine expression (in the spleen) by RT-qPCR at different times post-infection did not revealed any significant difference between the groups of fish infected with the three virus genotypes. Similarly, histological examination of the gills and the kidney of infected fish revealed no significant differences between fish infected with ORF134 deleted virus versus fish infected with the control parental or revertant strains. All together, the results of the present study demonstrate that the IL-10 homologue encoded by CyHV-3 is essential neither for viral replication in vitro nor for virulence in common carp.
Koi herpesvirus (KHV), also known as cyprinid herpesvirus 3 (CyHV-3; species Cyprinid herpesvirus 3, genus Cyprinivirus, family Alloherpesviridae, order Herpesvirales), is the etiological agent of an emerging and mortal disease in common (Cyprinus carpio carpio) and koi (Cyprinus carpio koi) carp [1, 2]. Since its emergence, in the late 1990s, this highly contagious and dreadful disease has caused severe economic losses in both common and koi carp culture industries worldwide [3–5].
The genome of CyHV-3 comprises a linear double-stranded DNA sequence of ~295 kbp , similar to that of cyprinid herpesvirus 1 and 2 (CyHV-1 and CyHV-2) [7, 8], but larger than those of other members of the order Herpesvirales which generally range from 125 to 240 kbp. Phylogenetic analysis of the CyHV-3 genome sequence led to its classification in the new family Alloherpesviridae encompassing herpesviruses of fish and amphibians [9, 10]. The CyHV-3 genome contains 155 potential protein-coding open reading frames (ORFs), some of which have relatives in other herpesviruses, and a few of which have relatives in poxviruses, iridoviruses and other large DNA viruses [6, 8, 11]. Interestingly, CyHV-3 genome encodes proteins potentially involved in immune evasion mechanisms such as, for example, TNF receptor homologues (encoded by ORF4 and ORF12) and an IL-10 homologue (encoded by ORF134) .
Cellular IL-10 has been described in a wide range of vertebrate species, including fish [12, 13]. It is a pleiotropic immunomodulatory cytokine with both immunostimulating and immunosuppressive properties ; however, IL-10 is generally described as an immunosuppressive cytokine. It inhibits expression of a large number of cytokines as, for example, TNF-α, IFN-γ, IL-1β, IL-2, IL-3, IL-6, and MHC class II [15–17]. Many viruses exploit the immunosuppressive properties of IL-10 to evade immune recognition either by up-regulation of host IL-10 or by expression of virally encoded IL-10 homologues (vIL-10s) [14, 18, 19].
Virally encoded IL-10 homologues have been reported in members of the Poxviridae family and the Herpesvirales order [19–21]. Among the Herpesvirales order, vIL-10s have been described in members of the Herpesviridae (e.g. human cytomegalovirus [HCMV] and Epstein-Barr virus [EBV]) and more recently in the family Alloherpesviridae (Anguilid herpesvirus 1 [AngHV-1] and CyHV-3) . While the role of vIL-10s has been demonstrated in the pathogenesis of one Poxviridae and one Herpesviridae[23–25]; this has not yet been investigated in the family Alloherpesviridae. However, a very recent study suggested that the IL-10 homologue encoded by CyHV-3 ORF134 could play a role in the pathogenesis. Firstly, it has been demonstrated that this ORF is transcribed in infected fish maintained at permissive and even restrictive temperature . Secondly, it has been shown that injection of CyHV-3 ORF134 mRNA into zebrafish embryos increased the number of lysozyme-positive cells to a similar degree as zebrafish IL-10 ; an effect that was inhibited by down regulation of the IL-10 receptor long chain using a specific morpholino .
The present study was devoted to CyHV-3 ORF134 encoding an IL-10 homologue. In vitro studies demonstrated that ORF134 is expressed as a spliced early-late gene and that its expression product is the second most abundant viral protein in the CyHV-3 secretome. Taking advantage of the recent BAC cloning of CyHV-3 as an infectious bacterial artificial chromosome (BAC), a strain deleted for ORF134 and a derived revertant strain were produced. Comparison of these strains demonstrated that ORF134 is essential neither for CyHV-3 replication in vitro nor for virulence in common carp.
Materials and methods
Cells and viruses
Cyprinus carpio brain cells (CCB) were cultured in minimum essential medium (MEM) (Invitrogen, Merelbeke, Belgium) containing 4.5 g/L glucose (D-glucose monohydrate; Merck, Darmstadt, Germany) and 10% fetal calf serum (FCS). Cells were cultured at 25 °C in a humid atmosphere containing 5% CO2. The CyHV-3 FL strain was isolated from the kidney of a fish that died from CyHV-3 infection (CER, Marloie, Belgium) .
Determination of ORF134 kinetic class of transcription
These experiments were performed as described elsewhere . Briefly, monolayers of CCB cells in 24-well plates were pre-incubated for 2 h before infection with cycloheximide (CHX) (100 μg/mL) (Sigma-Aldrich, Saint Louis, Missouri, USA) or phosphonoacetic acid (PAA) (300 μg/mL) (Sigma-Aldrich), the inhibitors of de novo protein synthesis or viral DNA polymerase, respectively. After removal of the medium, cells were infected with CyHV-3 FL strain at a multiplicity of infection (MOI) of 0.1 plaque forming unit (PFU) per cell in presence of inhibitors. After an incubation of 2 h, cells were overlaid with fresh medium containing the inhibitors. At 6, 8 and 12 h after inoculation cells were harvested and treated for RT-PCR analysis of gene expression (see below). CyHV-3 ORF3 (immediate early [IE]), ORF55 (early [E]) and ORF78 (late [L]) were used as reference gene of the three kinetic classes [29, 30].
Transcriptional analysis by RT-PCR
Primers and probes.
Sequence (5’- 3’)
Accession n°/ reference
Primers for PCR and RT-PCR
Primers for amplification of recombination cassettes
H1-gal K-H2 cassette
134 gal K F
Warming et al. 
134 gal K R
Primers and probes for real-time TaqMan PCR quantification of CyHV-3 genome
Primers for RT-qPCR analysis of carp gene expression
TNF-α1 and 2
TNF-α1 and 2-F
TNF-α1 and 2-R
Production of concentrated cell supernatant
CCB cells were infected with CyHV-3 FL strain at a MOI of 0.05 PFU per cell using serum free culture medium. Cell supernatants were collected 72 h post-inoculation and then submitted to two cycles of centrifugation at 4 °C (clarification at 2000 g for 15 min followed by pelleting of viral particles at 100 000 g for 2 h through a 30% sucrose gradient). The supernatant was then concentrated 25-fold by centrifugation (2000 g, 75 min, 4 °C) through an Amicon Ultra-15 centrifugal filter unit (3K NMWL; Merck Millipore, Billerica Massachusetts, USA) and stored at −80 °C until use.
2D-LC MS/MS proteomic approach
Proteomic analyses were performed using 2D-LC MS/MS workflow as described previously . Briefly, proteins were reduced at 4 °C for 1 h with 10 mM DTT and alkylated by incubation with 25 mMiodoacetamide at 4 °C for 1 h in the dark. Proteins were recovered through acetone precipitation and digested with trypsin at an enzyme:substrate ratio of 1:50 in 50 mM NH4HCO3 overnight at 37 °C. Tryptic peptides (25 μg) were analysed by bidimensional (SCX-RP) chromatography and online MS/MS, as described elsewhere , except that only 3 successive salt plugs of 25, 100 and 800 mM NH4Cl were used. Peptides were analyzed using the “peptide scan” option of an HCT ultra ion Trap (Bruker, Evere, Belgium), consisting of a full-scan mass spectrometry (MS) and MS/MS scan spectrum acquisitions in ultrascan mode (26 000 m/z sec-1). Peptide fragment mass spectra were acquired in data-dependent AutoMS (2) mode with a scan range of 100–2,800 m/z, three averages, and 5 precursor ions selected from the MS scan 300–1500 m/z. Precursors were actively excluded within a 0.5 min window, and all singly charged ions were excluded. Peptide peaks were detected and deconvoluted automatically using Data Analysis 2.4 software (Bruker). Mass lists in the form of Mascot Generic Files were created automatically and used as the input for Mascot MS/MS Ions searches of the NCBInr database release 20120809 using an in-house Mascot 2.2 server (Matrix Science). The default search parameters used were: Taxonomy = Bony vertebrates or Cyprinivirus; Enzyme = Trypsin; Maximum missed cleavages = 1; Fixed modifications = Carbamidomethyl (C); Variable modifications = Oxidation (M); Peptide tolerance ± 1.2 Dalton (Da); MS/MS tolerance ± 0.6 Da; Peptide charge = 2+ and 3+; Instrument = ESI-TRAP. All data were also searched against the NCBI bony vertebrate database in order to detect host proteins. Only proteins identified with p value lower than 0.05 were considered, and single peptide identifications were systematically evaluated manually. In addition, the emPAI  was calculated to estimate protein relative abundance in the culture supernatant.
Production of CyHV-3 ORF134 recombinants
Southern blot analysis of recombinant viruses was performed as described previously [27, 35]. PCRs were performed to produce ORF55 probe (primers ORF55InF and ORF55stopR) and ORF134Del probe (primers ORF134InF and ORF134InR) using the CyHV-3 FL genome as a template (Table 1).
Multi-step growth curves
Triplicate cultures of CCB cells were infected at a MOI of 0.5 PFU per cell. After an incubation period of 2 h, cells were washed with phosphate-buffered saline (PBS) and then overlaid with Dulbecco’s modified essential medium (DMEM, Invitrogen) containing 4.5 g of glucose/liter and 10% FCS. Supernatant of infected cultures was harvested at successive intervals after infection and stored at −80 °C. The amount of infectious virus was determined by plaque assay on CCB cells as described previously .
Common carp (Cyprinus carpio carpio) (CEFRA, University of Liège, Belgium), were kept in 60-liter tanks at 24 °C. Microbiological, parasitical and clinical examinations of the fish just before the experiments demonstrated that these fish were fully healthy.
CyHV-3 inoculation of carp
For viral inoculation mimicking natural infection, fish were kept for 2 h in water containing CyHV-3. At the end of the incubation period, fish were returned to larger tanks. In some experiments, fish that survived the primary infection were challenged 42 days after inoculation by cohabitation with fish that were infected by immersion in water containing 200 PFU/mL of the FL strain just before their release into the tank to be challenged. Two freshly infected fish were released per tank to be challenged. The animal study was accredited by the local ethics committee of the University of Liège, Belgium (Laboratory accreditation N°1610008, protocol N°810).
Quantification of virus genome copies in organs by real-time TaqMan PCR
Virus genome quantitation was performed by real-time TaqMan PCR as described elsewhere . The primers and the probes used are presented in Table 1. Two sets of primers were used to amplify fragments of CyHV-3 ORF89 and carp glucokinase genes. The amplicons were cloned into the pGEM-T Easy vector and the resulting plasmids were used to generate standard curves by running reactions with 101 to 1010 plasmid molecules. DNA was isolated using a DNA mini kit (Qiagen) from 25 mg of organs stored at −80 °C in RNAlater® (Invitrogen). The reaction mix contained 1 × iQSupermix (Bio-Rad), 200 nM of each primer, 400 nM of fluorescent probe and 250 ng of DNA. The analyses were performed using a C1000 Touch Thermal cycler (Bio-Rad). All real-time TaqMan PCRs for CyHV-3 DNA were run with equal amounts of DNA estimated by the real-time TaqMan PCR performed on carp glucokinase gene.
Quantification of carp gene expression in spleen by RT-qPCR
Total RNA was isolated from spleens stored at −80 °C in RNALater® (Ambion®, Invitrogen, Merelbeke, Belgium) using TRI reagent® (Ambion®, Invitrogen), including DNase I digestion and RNA purification using RNeasyMinElute Cleanup Kit (Qiagen). cDNA was synthetized from 1 μg of RNA using iScriptcDNA Synthesis Kit (Bio-Rad). The primers used for RT-qPCR were described previously  and are listed in Table 1. The RT-qPCR master-mix was prepared as follows: 1 × IQ™ SYBR® Green Supermix (Bio-Rad), 200 nM of each primer, 5 μL of 25 × diluted cDNA and sterile water to a final volume of 25 μL. The amplification program included an initial denaturation at 95 °C for 10 min, followed by 40 cycles with denaturation at 95 °C for 15 s, annealing at 58 °C for 30 s and elongation at 72 °C for 30 s. At the end, the dissociation stage was performed (95 °C for for 10 s) and the melt curve was obtained by increasing the temperature from 60 °C to 95 °C with a rate of 0.5 °C per 5 s. Fluorescence data from RT-qPCR experiments were analyzed using the CFX96 real-time system and exported to Microsoft Excel. The threshold cycle (Ct) was determined using the Auto method for all runs. The expression of analyzed genes was calculated using the 2-ΔΔCt method . The 40S ribosomal protein S11 was used as a reference gene.
Organs from mock-infected or infected carp were fixed in 4% buffered formalin and embedded in paraffin blocks. Sections of 5 μm were stained with haematoxylin and eosin prior to microscopic analysis .
Multi-step growth curves data expressed as mean titer ± standard deviation (SD) were analyzed for significance of differences (p< 0.05) using one-way ANOVA. The differences in mortality induced by the CyHV-3 strains tested were analyzed using Kaplan and Meier survival analysis. Significant differences (p< 0.05) in virus load between fish infected with the different CyHV-3 strains at each sampling point were assessed using one-way ANOVA followed by Holm-Sidak test when data were normally distributed, or with the non-parametric Kruskal–Wallis test followed by Tukey test when they were not. Significant differences (p< 0.05) in RT-qPCR gene expression between CyHV-3 infected and mock-infected fish, as well as between fish infected with different CyHV-3 strains at each sampling point were assessed using one-way ANOVA followed by Holm-Sidak test in cases where the data were normally distributed, or with the non-parametric Kruskal–Wallis test followed by Dunn’s test when they were not
CyHV-3 ORF134 kinetic class of expression
CyHV-3 and host proteins identified by 2D-LC MS/MS in the supernatant of CyHV-3 infected CCB cells.
Predicted MW (kDa)
No. of matching spectra
ORF12, TNF receptor superfamily homologue
ORF134, Interleukin 10 homologue
ORF116, predicted membrane glycoprotein
ORF119, putative uncharacterized protein containing an hydrophobic region
ORF52, predicted membrane glycoprotein
Host proteins (species origin)
Novel protein (zgc:103659) (Danio rerio)
Trypsin (Sus scrofa)
Predicted keratin, type II cytoskeletal 1-like isoform 6 (Macaca mulatta)
Enolase 1, alpha (D. rerio)
Predicted collagen alpha-3(VI) chain-like (D. rerio)
Fibronectin 1b (D. rerio)
Pigment epithelium-derived factor precursor (D. rerio)
Beta-2-microglobulin (Cyprinus carpio)
Serum albumin (Bos taurus)
Predicted keratin, type II cytoskeletal 1 (Pongo abelii)
Albumin (B. taurus)
Pgd protein (D. rerio)
Gelatinase (Paralichthys olivaceus)
Fructose-bisphosphate aldolase C (Carassius auratus)
Fibronectin 1 (D. rerio)
Procollagen type I alpha 1 chain (D. rerio)
Cadherin 11, osteoblast (D. rerio)
Fructose-bisphosphate aldolase C (Homo sapiens)
Mutant beta-actin (beta'-actin) (H. sapiens)
Unnamed protein product (H. sapiens)
Pre-serum amyloid P component (H. sapiens)
Histone H4 (H. sapiens)
Carp C1q-like molecule (C. carpio)
Ubiquitin (Salmo sp.)
Alpha-2-HS-glycoprotein precursor (B. taurus)
Myristoylated alanine-rich C kinase substrate 2 (D. rerio)
N-cadherin precursor (D. rerio)
Wnt inhibitory factor 1 precursor (D. rerio)
Prion-like protein 1 (C. carpio)
Collagen type I alpha 3 (C. auratus)
14-3-3 protein beta/alpha-B (D. rerio)
Nucleoside diphosphate kinase-Z1 (D. rerio)
Chain A, Solution Structure Of Calcium-Calmodulin N-Terminal Domain (H. sapiens)
FK506 binding protein 1A, 12kDa (D. rerio)
Predicted neuroblast differentiation-associated protein AHNAK (D. rerio)
Alpha-tubulin (H. sapiens)
Tissue inhibitor of metalloproteinase 2b (D. rerio)
Heart fatty acid binding protein (Anguilla japonica)
Tissue inhibitor of metalloproteinases, Type-2 (H. sapiens)
Fibrillin-2 (H. sapiens)
Osteopontin (D. rerio)
Cold inducible RNA binding protein isoform 2 (D. rerio)
Fstl1b protein (D. rerio)
Triosephosphate isomerase B (D. rerio)
Clusterin (D. rerio)
Unnamed protein product (Tetraodon nigroviridis)
The MS data presented above demonstrate that CyHV-3 ORF134 encodes a protein that is abundantly secreted in the extracellular medium by infected cells. This observation is consistent with the hypothesis that ORF134 may be a functional IL-10 homologue playing a role in CyHV-3 pathogenesis .
Production and characterization of CyHV-3 ORF134 recombinant strains
Effect of ORF134 deletion on viral growth in vitro
Effect of ORF134 deletion on CyHV-3 pathogenesis
Real-time TaqMan PCR results demonstrated that fish infected with the three viral strains had statistically comparable viral loads in the gills and the kidney throughout the course of the experiment (Figure 10). Using the approach described in Figure 9, PCR reactions were performed on randomly selected fish demonstrated that each tank was infected with the correct strain and confirmed the absence of viral spread between tanks (data not shown). Together, these results suggested that ORF134 deletion has no effect on viral load during primary acute infection.
The spleen is one of the organs in which CyHV-3 is the most abundant during the course of acute infection . It is also considered as one of the major lymphoid organ in teleost . In order to study the effect of CyHV-3 ORF134 on the carp immune response, the kinetics of gene expression of the cytokines IFN-γ2, TNFα1, TNFα2, IL-1β, IL-6, CXCa and IL-10 were analyzed in spleen from fish infected with FL BAC revertant, FL BAC revertant ORF134 deleted and FL BAC revertant ORF134 Rev strains (Figure 11). Samples were collected over a period of 2 to 8 days post-infection and analyzed by RT-qPCR. The kinetics of expression of studied cytokines showed similar patterns to those observed previously . Taking mock-infected fish as a reference, expression of several cytokines (IFN-γ2, IL-1β, IL-6, and IL-10) was up-regulated as early as day 3 post-infection. The most pronounced up-regulation was observed for IL-1β and IFN-γ2. We observed a moderate and late (day 6 and day 8 post-infection) up-regulation of TNFα1 and TNFα2. The expression level of CXCa in infected fish was comparable to mock-infected fish or even down-regulated for some strains at some time points. Importantly, the results presented in Figure 11 demonstrate that there is almost no difference in the expression levels of the cytokines studied between carp infected with three virus strains. The only significant differences observed between virus strains were for IFN-γ2 at day 4 post-inoculation and for IL-6 at day 8 post-inoculation. The expression level of IFN-γ2 at day 4 post-inoculation was significantly higher in fish infected with FL BAC revertant ORF134 deleted as compared to FL BAC revertant and FL BAC revertant ORF134 Rev strains. However, this difference was rather small and was not observed for the other sampling points, suggesting that it could reflect data variation rather than the expression of ORF134 biological activities. Supporting the latter hypothesis, the expression level of IL-6 at day 8 post-inoculation was significantly higher in the FL BAC revertant group as compared to the FL BAC revertant ORF134 Rev group. The absence of cross-contamination between tanks was controlled using the approach described in Figure 9 (data not shown). Together, these results suggested that ORF134 does not significantly affect the carp immune response under the experimental conditions used.
The present study was devoted to CyHV-3 ORF134, which encodes a potential vIL-10. We confirmed that ORF134 is transcribed as a spliced E-L gene (Figure 2). We also demonstrated for the first time that it is one of the most abundant proteins of the CyHV-3 secretome (Table 1) and that ORF134 is essential neither for viral replication in vitro nor for virulence in vivo. The latter conclusion relied on the observations that an ORF134 deleted strain could not be differentiated from its parental and revertant strains based on induced clinical signs and mortality rate (Figure 8), kinetic of viral load in gills and kidney (Figure 10), kinetic of cytokine expression in the spleen (Figure 11) and histological examination of gill and kidney (Figure 12).
As described in the introduction, cellular IL-10 is a pleiotropic immunomodulatory cytokine with both immunostimulatory and immunosuppressive properties . Virally encoded IL-10 homologues have been reported in several members of the Poxviridae family and the Herpesvirales order [19–21]. Numerous molecular and in vitro studies suggest that there has been adaptive evolution of viral IL-10 following capture through positive selection to retain properties most beneficial for the virus life cycle. However, very few studies have addressed the role of viral IL-10 in vivo by comparison of a wild type strain and derived deleted and revertant strains. This approach, which is the only one that can test the in vivo biological relevance of a gene, has been performed for only two viruses: rhesus cytomegalovirus (rhesus CMV) and Orf virus (ORFV) [23–25]. For both viruses, deletion of viral IL-10 induced virus attenuation and modulation of the host anti-viral innate immune response.
The results of the present study demonstrate that the IL-10 homologue encoded by CyHV-3 does not affect significantly its virulence in common carp (Figure 8) or the host innate immune response (Figure 11). However, a recent study based on an in vivo artificial model suggested that CyHV-3 ORF134 encodes a functional vIL-10 . As IL-10 is known to induce a transient neutrophilia and monocytosis in addition to T cell suppression , these authors tested the in vivo functionality of CyHV-3 encoded IL-10 by injection of zebrafish embryos with mRNA encoding CyHV-3 ORF134 and analysis by whole-mount in situ hybridization (using a pan-leukocyte marker lysozyme at 56 hours post-fertilization before development of T cells). A slight but statistically significant increase in the number of lysozyme positive cells was observed in embryos injected with CyHV-3 ORF134 mRNA compared to control embryos. The effect observed was inhibited by down regulation of the IL-10 receptor long chain by a specific morpholino. These data suggested that CyHV-3 ORF134 encodes a functional vIL-10. Importantly, the ORF134 sequence used in this study is identical to the sequence encoded by the CyHV-3 strain used in the present study. Various hypotheses could explain the apparent paradox between the functional effect reported by Sunarto et al. and the lack of effect of deleting ORF134 described in the present study .
Firstly, it is possible that the slight effect observed by Sunarto et al. using optimal artificial conditions (overexpression of ORF134, no inflammatory stimulation by the viral infection, a rather immature host immune system) has no significant biological relevance during a real viral infection of carp. Secondly, it is possible that the role of ORF134 is strictly restricted to latency and viral reactivation. This hypothesis is inconsistent with the higher level of ORF134 expression observed during acute infection compared to those observed during latency and reactivation . However, experiments are in progress to determine whether ORF134 deletion affects viral load during latency and/or the ability of the virus to reactivate and to be excreted. Thirdly, it may be that ORF134 expression product has a biological activity in zebrafish but not in common carp. This hypothesis is related to the still unknown origin of CyHV-3. Indeed, the rapid emergence of CyHV-3 in the common and koi carp population during the late 90s and the relatively low polymorphism existing between CyHV-3 isolates suggest that CyHV-3 is the consequence from a recent host-jump from a yet unidentified fish species to common and koi carp. According to this evolutionary scenario, it could be that ORF134 is functional in the CyHV-3 original host species and closely related species but not in the recently colonized common and koi carp species.
In conclusion, the present study addressed for the first time the in vivo role of a vIL-10 encoded by a member of the family Alloherpesviridae. It demonstrates that CyHV-3 ORF134 does not contribute significantly to viral growth in vitro or to virulence in vivo under the conditions tested. However, it is possible that this protein is important under circumstances that were not recapitulated in the present laboratory setting.
Anguilid herpesvirus 1
Bacterial artificial chromosome
Cyprinus carpiocarpio brain cell
Dulbecco’s modified essential medium
Enhanced green fluorescent protein
Multiplicity of infection
Open reading frame
Phosphate buffered saline
Plaque forming unit
- Rhesus CMV:
Reverse transcription PCR
Real-time quantitative PCR
Virally encoded IL-10 homologues.
PO is a research fellow of the Chinese Scholarship Council. This work was supported by a grant from the University of Liège and by grants of the “Fonds National Belge de la Recherche Scientifique” (FNRS) (R.FNRS.2165, -2697). KR and AV are members of the BELVIR consortium (IAP, phase VII) granted by the Belgian Science Policy Office (BELSPO) (Belgium).
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