Cells and virus
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 . Cells were cultured at 25°C in a humid atmosphere containing 5% CO2. The KHV FL BAC 136 LUC TK revertant strain of CyHV-3 was described previously . This recombinant strain encodes a firefly luciferase (LUC) expression cassette inserted in the intergenic region between open reading frame (ORF) 136 and ORF137. The KHV FL BAC recovered strain of CyHV-3 was described previously . This recombinant strain encodes an enhanced green fluorescent protein (EGFP) expression cassette inserted at the end of ORF55.
Koi carp (Cyprinus carpio koi) (Hazorea Aquatics, Kibbutz Hazorea, Israel) and common carp (Cyprinus carpio carpio) (CEFRA, University of Liège, Belgium), with an average weight of 16 g, 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.
Physical treatments of the skin
Four physical treatments were applied on a defined area of the carp epidermis (disc shape, diameter of 15 mm): rubbing with a soft tissue paper (TORK premium, Goteborg, Sweden), rubbing with a cotton swab (Swube Applicator, Becton Dickinson Microbiology system, Maryland, USA), brushing with a rotary electric tooth brush (Philips Sensiflex HX 1513, Anderlecht, Belgium) for 2 s or rubbing with sandpaper (average particle diameter of 265 μm, Medium p60, LUX Wermelskirchen, Germany).
Histochemistery and microscopy analysis
Fish skin explants were fixed by immersion in Carnoy solution (ethanol 6: acetic acid 1: chloroform 3, v/v/v) for 2 h at 4°C. After dehydration with ethanol, samples were embedded in paraffin . Five μm thick sections were stained by a combined Alcian Blue (AB) and Periodic acid-Schiff (PAS) staining . Mounted samples were observed using a Nikon Eclipse TE 2000-S microscope equipped with a DC 300F charge-coupled device (CCD) camera (Leica, Heerbrugg, Switzerland).
Culture of tail fin explants
Fish were euthanized using benzocaine (100 mg/L of water) (Sigma-Aldrich, Saint Louis, Missouri). The ventral lobe of the tail fin was clipped with forceps before section. Fin fragments maintained in forceps were immerged in a vertical position in minimum essential medium (GIBCO, Invitrogen, Paisley, UK) containing 4.5 g/liter glucose (D-glucose monohydrate; Merck, Damstadt, Germany) and 10% fetal calf serum (FCS) (Greiner Bio One, Frickenhausen, Germany). Tail fin explants were cultured at 25°C in a humid atmosphere containing 5% CO2.
CyHV-3 inoculation of carp
For viral inoculation mimicking natural infection, fish were kept for 2 h in water containing 103 plaque forming unit (PFU)/mL of the KHV FL BAC 136 LUC TK revertant strain. At the end of the incubation period, fish were returned to larger tanks. To avoid removal of skin mucus, fish were caught using a container rather than a fish net, and they were manipulated with great care wearing humidified latex gloves. The animal study was accredited by the local ethics committee of the University of Liège, Belgium (Laboratory accreditation N°1610008, protocol N°810).
Imaging of firefly (Photinus pyralis) LUC was performed using an "in vivo imaging system" (IVIS) (IVIS®spectrum, Xenogen, Caliper LifeSciences, Hopkinton, Massachusetts, USA) as described previously . For in vivo analysis, fish were anesthetized with benzocaine (50 mg/L of water). Ten minutes before bioluminescence analysis, D-luciferin (150 mg/kg body weight) (Xenogen, Caliper LifeSciences, Hopkinton, Massachusetts, USA) was administrated by intraperitoneal injection. Each fish was analyzed lying on its left and right side. For analysis of tail fin explants cultured ex vivo, culture medium was replaced by fresh medium containing D-luciferin (150 μg/mL) ten minutes before bioluminescence analysis. All the images presented in this study were acquired using a field view of 15 cm, a 1 min exposure time, a binning factor of 4 and a f/stop of 1. Relative intensities of transmitted light from bioluminescence were represented as a pseudocolor image ranging from violet (least intense) to red (most intense). Corresponding grey-scale photographs and color luciferase images were superimposed using the LivingImage analysis software (Xenogen, Caliper LifeSciences, Hopkinton, Massachusetts, USA).
Transmission electron microscopy
Samples were fixed in 0.1% glutaraldehyde (Sigma-Aldrich, Saint Louis, Missouri, USA). Epon blocks and sections were prepared as described elsewhere . Sections were analyzed using a Tecnai Spirit transmission electron microscope (FEI, Eindhoven, The Netherlands), and electron micrographs were taken using a bottom-mounted 4-by-4 K Eagle camera (FEI).
Collection of carp epidermal mucus and production of clarified mucus extract
Epidermal mucus was collected from common carp (average weight of 5 kg) kept at 22°C (CEFRA, University of Liège, Belgium). Immediately after euthanasia, epidermal mucus was collected by gentle scraping of fish flanks using a soft rubber spatula. Mucus samples were pooled and stored on ice. Clarified mucus extract (CME) was then prepared as follows. Mucus was first clarified by centrifugation (2000 g for 10 min at 4°C). Clarified mucus was diluted five times in MEM on ice. To enhance mucus solubilisation, β2-mercaptoethanol (Sigma-Aldrich) was added at the final concentration of 5 mM. The sample was then processed five times through a 7 mL Dounce homogenizer (tight pestle, VWR, Chicago, USA). After an incubation of 30 min on ice, the sample was ultracentrifuged at 100 000 g for 30 min at 4°C. The supernatant was collected and sterilized by filtration through a 0.45 μm filter (0.45 μm filter PES, VWR). Finally, the sample was concentrated five times by centrifugation through an Amicon Ultra 3K column (Millipore). The resulting product, hereafter called CME, was stored at -80°C until use. The CME used in the present study had an estimated protein concentration of 0.95 mg/mL as determined with the non-interfering protein assay (GBiosciences, St Louis, USA).
CyHV-3 neutralisation assay by CME
The KHV FL BAC recovered strain of CyHV-3 was diluted in MEM to reach a concentration of 5.104 plaque forming unit (pfu)/mL. The effect of CME on CyHV-3 infectivity was tested under two conditions hereafter called pre-incubation and post-incubation addition of CME. For pre-incubation addition of CME, the virus suspension was mixed with adequate volumes of CME and MEM supplemented with 5 mM β2-mercaptoethanol to reach CME final concentrations (vol/vol) of 1/2, 1/4, 1/8, 1/16 and 1/32. Samples were then incubated at 25°C for 2 h. For post-incubation addition of CME, the samples were processed as described above with the exception that the CME volumes were added after the 2 h incubation period. A negative control (NC) sample consisted of incubating the viral suspension with an equal volume of MEM supplemented with 5 mM β2-mercaptoethanol before the 2 h incubation period. All samples were then diluted 200 times in MEM and CyHV-3 infectivity was titrated on CCB monolayers grown in 24 well plates (BD, Erembodegen, Belgium) as described elsewhere . Viral plaques were counted 3 days post-infection (dpi) using an epifluorescent microscope (Eclipse TE2000-S, Nikon). Statistical analyses of the results were performed by post hoc tests on least squares means for pair wise group comparisons. These analyses were done using SAS version 9.1.