Flow cytometry assay for intracellular detection of Infectious Pancreatic Necrosis virus (IPNV) in Atlantic salmon (Salmo salar L.) leucocytes

Fish & Shellfish Immunology 33 (2012) 1292e1302

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Flow cytometry assay for intracellular detection of Infectious Pancreatic Necrosis virus (IPNV) in Atlantic salmon (Salmo salar L.) leucocytes Anita Rønneseth*, Eirin Fausa Pettersen, Heidrun I. Wergeland Department of Biology, University of Bergen, Bergen High-Technology Center, PO Box 7803, NO-5020 Bergen, Norway

a r t i c l e i n f o

a b s t r a c t

Article history: Received 16 August 2012 Received in revised form 20 September 2012 Accepted 20 September 2012 Available online 29 September 2012

Infectious Pancreatic Necrosis virus (IPNV) is traditionally detected in adherent leucocytes using immunofluorescence labelled specific antibodies, PCR or by further cultivation of infected cell material in cell lines. We present a flow cytometry (FCM) assay for detection of intracellular IPNV in salmon leucocytes, where each single cell is analysed for presence of virus. The method is established using in vitro challenge of salmon leucocytes and CHSE-214 cells. For detection of intracellular virus antigen the Cytofix/Cytoperm kit from BD is optimal compared with paraformaldehyde or acetone/methanol for cell permeabilisation. This is combined with labelling procedures allowing both internal virus antigen labelling and external antibody labelling of cell markers to identify B-cells and neutrophils. The secondary antibodies were Alexa Fluor 647 for the internal labelling and RPE for the external labelling of bound cell subtype specific antibodies. The presences of virus within cells are also demonstrated by confocal and light microscopy of infected cells. IPNV is successfully detected in blood and head kidney leucocyte samples. IPNV is found both in B-cells and neutrophils as well as in other types of leucocytes that could not be identified due to lack of cell-specific antibodies. Serial samples from cultivation of in vitro infected leucocytes and CHSE-214 cells analysed by flow cytometry showed that number of infected cells increased with increasing number of days. The flow cytometry protocol for detection of intracellular IPNV is verified using CHSE-214 cells persistently infected with IPNV. These analyses are compared with virus titre and virus infected naive CHSE-214 cells. The detection of IPNV in persistently infected cells indicates that carrier fish can be analysed, as such cells are considered to have virus titres similar to carriers. Ó 2012 Elsevier Ltd. All rights reserved.

Keywords: Flow cytometry Intracellular virus IPNV Leucocytes Atlantic salmon

1. Introduction Flow cytometry is frequently used for detection of virus in human medicine for clinical diagnostic and research purposes. The method allows phenotypic and functional characteristics of the cells to be simultaneously measured. The main advantage of the method is the fast and multi parametric analysis of single cells in suspension. Flow cytometry is powerful because one can analyse thousands of cells in a few seconds. The method can be used for a wide variety of applications, from distinguishing cell populations to intracellular staining and signal profiling, to date 20 colours can be simultaneously analysed from one cell [1e4]. In this study we have established a 2-color protocol for analysing virus infection in Atlantic salmon leucocytes. Infectious pancreatic necrosis virus

* Corresponding author. Tel.: þ47 55584421 (office); fax: þ47 55584450. E-mail address: [email protected] (A. Rønneseth). 1050-4648/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.fsi.2012.09.020

(IPNV) is used as a model since this virus has been reported to hide and multiply within salmon leucocytes [5e12]. IPNV is the causative agent of the viral disease IPN. It is a widespread aquatic virus, affecting mainly salmon and rainbow trout (Onchorhynchus mykiss), but disease caused by IPNV has also been reported on species like Atlantic halibut (Hippoglossus hippoglossus) [13e16], turbot (Pecten maximus) [17e20] and Atlantic cod (Gadhus morhua) [17,21e24]. IPNV are affecting mainly the young fish and can cause severe mortality in Atlantic salmon fry and in smolt shortly after seawater transfer. The disease is since 2008 not listed as a notifiable disease in Norway. IPNV is one of the best studied viruses affecting Atlantic salmon, but still the economic and ethical impact of mortality and reduced growth of carrier fish are serious problems. IPNV has been shown to reside and replicate within the leucocytes of Atlantic salmon [6,7]. The presence of virus within the immune cells themselves might have significant impact on cell functions involved in immunity necessary to control or evade this virus during the infection. Elucidating the leucocyte populations

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harbouring the virus may help clarify why it has been so difficult to obtain proper protection in vaccinated fish, and in this way give a better understanding of what types of cells and immune functions that needs to be stimulated in order to prime the immune system. Detection and quantification of IPNV can be done by a series of methods; usually it is performed by virus isolation in cell cultures or by molecular detection techniques. The use of flow cytometry will introduce a method where virus infections in cells can be analyzed using blood samples, and it allows studies of the virus cell interactions during the course of an infection. Further, flow cytometry has the potential of being used for evaluating responses to vaccines by analysing various parameters of subtypes of cells as a response to vaccination and subsequent challenge of fish. Such analyses can include stimulation based assays and specific markers of leucocyte subpopulations. The present study shows recording of two signalling events from individual cells, allowing cell surface marker expression to be correlated with intracellular presence of IPNV. The sensitivity of the method and the high numbers of cells analysed give means to identify infections that appear in few cells of the total leucocyte population from individual fish. Therefore, using flow cytometry, one will likely be able to identify the low number of cells sheltering the virus in carrier fish. Further, this method will have potential use to investigate which leucocyte populations that carry the virus in acute stages of the disease and if particular cell populations harbour the virus in carrier fish. In the presented flow cytometry experiments, leucocytes from head kidney (HK) and peripheral blood (PB) were in vitro infected with IPNV. Different fixation and permeabilisation strategies were tested, and the optimal procedure for labelling with antibodies was identified. For comparisons of infection, IPNV in Chinook salmon embryo cells (CHSE-214 cells), commonly used for propagation of the virus, was also analysed.

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discontinuous Percoll gradients as described by Pettersen et al. [26]. Isolated leucocytes were diluted to 1 ml in L-15þ medium and analysed in a CASY-TT cell counter (Innovatis) measuring viable cells ml1 and the aggregation factor. Cell suspensions were diluted in L-15þ medium containing 2% (v/v) FCS to 5  106 cells ml1 based on counts of viable cells ml1 in the CASY-TT. Viable peripheral blood leucocytes (PBL) were generally 13.3e15 mm in size and viable head kidney leucocytes were 7e16 mm. Leucocytes were cultivated in 24 well plates (Nunclon) at 2.5  106 cells ml1, the volume was 500 ml in each well. For incubation, the wells sealed with sealing tape (Nunc), covered by the lid and wrapped in plastic. PBL and HKL were collected from eight fish per trial. Each sample of PBL and HKL were distributed into six wells. For each sample, the leucocytes in three parallel wells were infected with IPNV and cells in three wells were supplied with the same volume of L-15 medium. Incubation was done at gentle low speed tilting (Vari-Mix Thermolyne) at 14  C. The cells were harvested at 5 h, 10 h and 24 h post infection. For each individual fish the cell numbers from infected cells were compared to the cell number from parallel non-infected cells as well as the number of cells seeded to each well, all analysed using the CASY-TT. Leucocytes present in the supernatant and adherent leucocytes in each well were analysed separately. To harvest adherent leucocytes, 100 ml trypsin (Trypsin-Versene (EDTA) (Bio Whittaker)) was added to the wells. After approximately 1 min 100 ml L-15 was supplied and the cells were gently scraped from the bottom using a bent glass pipette. The harvested supernatants from individual wells were supplied to new clean wells and supplied with 500 ml fresh L-15 media. The adherent cells were supplied with 500 ml fresh L-15 media. Similarly cultivation of leucocytes in 10 ml closed tubes (Nunc) was investigated. Total volume in the tubes was 2 ml and the concentration of cells 2.5  106 cells ml1. The leucocytes were incubated for 5 h, 10 h and 24 h at gentle tilting as described above, before being harvested and analysed in the CASY-TT.

2. Materials and methods 2.3. Virus stock and propaganation 2.1. Fish and rearing conditions The fish were healthy non-vaccinated Atlantic salmon and kept at the rearing facilities at Industrilaboratoriet (ILAB) located at Bergen High Technology Center, western Norway. Upon arrival a statistical sufficient group of the fish was screened by RT-PCR for various disease agents, including IPNV. No positives were identified. No mortality occurred throughout the experimental period. Rearing conditions included a temperature of 12  C, salinity of 25 ppm and the water flow was regulated to maintain 70% oxygen saturation in the outlet water. The photoperiod light/dark cycle was (LD) 24:0. Fish sampled for analysis had average bodyweights in the range 100e200 g. 2.2. Isolation and cultivation of leucocytes Blood was collected from Vena caudalis using a syringe and immediately transferred to heparinised containers. 700 ml of collected blood from each sample was diluted to a total volume of 2 ml in L-15þ medium previously described by Øverland et al. [25] with slight modifications, L-15 (Bio Whittaker) (adjusted to 380 mOsm by adding 5% (v/v) of a solution consisting of 0.41 M NaCl, 0.33 M NaHCO3 and 0.66% (v/v) D-glucose, supplemented with 100 mg ml1 gentamicin sulphate (Bio Whittaker), 2 mM Lglutamin (Bio Whittaker), 10 U ml1 heparin (Sigma),15 mM HEPES and 5% (v/v) FBS Australian origin (BioWhittaker)). Leucocyte suspensions from head kidney was collected by gently forcing tissue from anterior kidney through a 100 mm cell strainer (Becton Dickinson) and diluting to 2 ml using L-15þ medium. Leucocytes from the blood and head kidney samples were isolated on

The virus isolate NVI-011 (NS-IPNV 17) was provided by professor Øystein Evensen at the Norwegian School of Veterinary Science (NVH), the isolate has previously been identified as highly virulent [27,28] and was passed once on Rainbow trout gonad (RTG-2) cells prior to storage on glycerol stock at 80  C. Further virus production was preformed by diluting the virus suspension 1:10 in cell culture medium and passing the suspension once on RTG-2 cells and once on Chinook salmon embryo cells (CHSE-214) cells. The RTG-2 cells were cultivated in Leibovitz’s L-15 medium (Bio Whittaker) supplied with 10% FBS (Cambrex), 2% (v/v) L-glutamine (Cambrex) and 0.1% (v/v) Gentamicine (Cambrex) at 20  C without CO2 in 75 cm2 plastic cell culture flasks (Nunclon). For infection of the cells with IPNV, the supernatant was removed and the cells were added 2 ml diluted virus suspension in maintenance medium (cell culture medium containing 2% (v/v) FBS). After 2 h 8 ml maintenance medium was added to each flask. The cells were incubated at 15  C until cytopathic effect (CPE) was observed. The resulting cell culture supernatant were centrifuged at 2500  g for 10 min at 4  C (Beckman GPR centrifuge) and filtered using a 45 mm syringe filter and a syringe. The CHSE-214 cells used for the second virus passage were cultivated in MEM Eagle with Earle’s BSS and HEPES (Cambrex) supplemented with 10% (v/v) FBS, 4 mM glutamine, 50 mg ml1 gentamicin and 1% (v/v) MEM Eagle non-essential amino acid solution (NEAA 100x) (Cambrex). The cells were incubated at 20  C without CO2 in 75 cm2 plastic cell culture flasks (Nunclon). Infectious supernatant from the RTG-2 cells was diluted 1:2, and infection of the CHSE-214 cells was done as described above. The virus was titrated at ten fold

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dilutions on monolayers of CHSE cells seeded on 24 well plates (Nunclon). Six wells were analysed for each dilution from 105 to 1010. The TCID50 was calculated by the Kärber method [29]. 2.4. In vitro challenge of leucocytes Challenge of leucocytes was performed in 10 ml centrifugation tubes (Nunclon), before the cells were transferred to well plates (Nunclon) or 10 ml centrifugation tubes (Nunclon). The infectious dose was approximately 500 ml IPNV (2  105 TCID50) supplied to 2 ml cell suspension containing 5  106 cells. Different incubation times of leucocytes with virus were investigated 2 h, 3 h and 4 h. The tubes were incubated at gentle low speed tilting (Vari-Mix Thermolyne) at 14  C. Post challenge the tubes were filled up with L-15þ medium and centrifuged at 193  g (Beckman Coulter AllegraÒ X-15R Centrifuge) for 10 min at 4  C. The supernatant was discarded and cells were resuspended in L-15þ to a concentration of 2.5  106 cells ml1. The cells were further incubated in 24 well plates (Nunclon), 1000 ml/well, or in 10 ml centrifugation tubes (Nunclon) 5000 ml/tube. Different times, 2 he24 h and 1 de5 d, of cultivation were tested. 2.5. Preparation of cells, fixation and permeabilization To access intracellular antigens leucocytes must be fixed and permeabilized. Antigens present in the cell cytoplasm can be reached by relative mild permeabilization reagents such as saponin, while nuclear antigens such as DNA are best measured after alcohol dehydration [30,31]. Fixation will make the cells more rigid, but on the other hand unfixed samples often have less cell aggregates, debris and there are less autofluorescence from the cells. Three different fixation and permeabilisation strategies were tested. First, freshly mixed acetone:methanol (1:1) for 90 s were supplied to tubes at volume 500 ml/200 ml of 2.5  106 cells ml1. The cells were centrifuged and washed in PBS supplemented with 1% (w/v) BSA and 25 mM EDTA, pH ¼ 7.3 (PBSþ). Second, freshly made 3.5% (w/v) formaldehyde solution from paraformaldehyde supplied at volume 500 ml/200 ml of 2.5  106 cells ml1 for 10 min. The cells were centrifuged and washed in PBS before adding 500 ml of freshly made 0.01% Triton x100 for 30 min. Third, Becton Dickinson (BD) Cytofix/CytopermÔ Fixation/Permeabilization kit which is based on paraformaldehyde (4%) fixation and saponin for permeabilization. The cell pellet of 1  106 cells ml1 was resuspended in 50 ml of PBS (380 OsM) and supplied with 500 ml fixative. The tubes were incubated for 20 min on ice with gentle cycles on a Grant-bio PS-M3D. The cells were centrifuged at 400  g for 4 min, the supernatant discarded, the pellet supplied with 900 ml of the perm wash solution. Cell suspensions were centrifuged at 400  g and resuspended in the perm wash solution at every step except for the last resuspension before the flow cytometry analysis where PBSþ was used. 2.6. CHSE-214 cells To obtain persistently infected cells, monolayers of CHSE-214 cells were infected with the same virus supernatant as used for infection of leucocytes. The CHSE-214 cells were incubated until full cytopathic effect was observed, the supernatant was removed and the few cells that remained intact on the bottom of the flasks, were gently washed once with PBS (Bio Whittaker) and added 10 ml of maintenance medium. These cells were further grown and passed several times to obtain persistently infected cells. A separate protocol for flow cytometry analysis was developed for the CHSE-214 cell line mainly due to the differences in size, autofluorescence and granularity from leucocytes. For CHSE-214

cells the forward scatter (FSC) voltage was set to E-1 which reduces the signal of large events. 10.000 cells were recorded from each sample. For infection of naïve CHSE-214 cells 2 ml virus at a concentration of 105 TCID50 ml1 diluted in maintenance medium was supplied to each 75 cm2 flask and incubated for 2 h. Control flasks were supplied with 2 ml of maintenance medium. Medium containing the unattached virus was discarded and the cells supplied with 10 ml cultivation medium. The cells were incubated at 15  C and sampled every 12 h. Negative controls were uninfected CHSE-214 cells and persistently infected CHSE-214 cells were used as positive controls. Infected, uninfected and persistently infected cells from 75 cm2 monolayers in cell culture flasks (Nunclon) were sampled. The maintenance medium was discarded and cells were gently washed twice with PBS (Bio Whittaker) and supplied with 1 ml TrypsinVersene (Bio Whittaker). Detached cells were suspended in 5 ml maintenance medium and the cell suspension transferred to 10 ml centrifuge tubes (Nunclon). They were centrifuged at 400  g for 5 min at 4  C (Beckman Coulter AllegraÒ X-15R Centrifuge). The supernatant was discarded and the cell pellets were resuspended in 1 ml maintenance medium. The cell numbers and viability were analysed by Casy-TT. The cells were diluted to a final concentration of 5  106 cells ml1 in PBS and 200 ml was added to each flow cytometry tube, centrifuged at 400  g for 5 min and resuspended in 50 ml PBS (380 OsM). To each tube 500 ml of the BD fix solution containing 4% paraformaldehyde were added and the tubes incubated for 20 min on ice at at 10 rpm and gentle tilting (PS-M3D multi-function 3D rotator Grant-bio). The tubes were centrifuged at 400  g for 4 min at 4  C, the supernatant discarded and the pellet resuspended in 900 ml perm/wash solution containing saponin for washing. The cell pellet was resuspended in 200 ml perm/wash solution and added 250 ml rabbit anti-IPNV antibody diluted 1:5000 in perm/wash solution. The samples were incubated for 1 h on ice at gentle tilting, washed two times and the pellets were resuspended in 150 ml Alexa Fluor 647 conjugated goat anti-rabbit antibody diluted 1:400 in perm/wash solution. The samples were incubated for 45 min on ice at with gentle tilting, followed by two washings and the pellet was resuspended in 500 ml PBS (Bio Whittaker) prior to flow cytometry analysis. 2.7. Flow cytometry For flow cytometry, a two step procedure using a fluorescent labelled secondary antibody could be preformed to identify both cells and virus since the antibody for cell surface was a mouse monoclonal antibody (MAb) while for IPNV a polyclonal rabbit antiserum, kindly provided by professor Øystein Evensen at NVH, was used. This labelling procedure enhanced the signalling strength from the cells. Other less time consuming procedures were also tested, using mouse IgG1 Ab for both external and intracellular staining, then one secondary antibody to mouse Ig had to be directly conjugated to the MAb. The monoclonal antibody directed towards VP2 of IPNV (Anti-Infectious Pancreatic Necrosis virus (IPNV) monoclonal antibody, Aquatic Diagnostics) was directly conjugated using Alexa FluorÒ647 MAb Labeling Kit (A-20186) (Molecular Probes) and purified following the producer’s instructions. Conjugated antibodies were tested at concentrations 0.25, 0.5, 1, 2, 3 and 4 mg ml1 for 200 ml of 2.5  106 cells ml1. The cell surface antibodies were also attempted labelled with R-phycoerythrin (R-PE) using ZenonÒMouse IgG Labeling Kit (Molecular Probes) to obtain a 3:1 molar ratio of labelled Fab fragments to antibody target at the producer’s instructions. When choosing flourophores we considered the fluorophores absorbance spectrum according to the lasers in our BDFacs Calibur (Red diode Laser (635-nm) and Argon Laser (488-nm)) and that the

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emission fell within the detector filter sets in the instrument. The fluorophore on the conjugated antibody for intracellular staining must be small enough to be able to penetrate the fixed and permeabilised cell. Considering these options we chose to use Alexa Fluor 647 which is a small, photo stable and bright fluorophore, for intracellular staining, while we used R-phycoerythrin which is also a bright molecule for extracellular staining. These two fluorophores are excited by individual lasers and their emission spectra do not overlap to a great degree, limiting the need for compensation which could reduce signals from cells low in virus content. The Alexa Fluor 647Ò fluorophore was supplied to the leucocyte suspension as a 1:400 dilution of F(ab0 )2 fragments of goat antirabbit IgG (H þ L) (Invitrogen Molecular ProbesÒ) in perm/wash buffer. The R-phycoerythrin was supplied as as a 1:400 dilution of goat anti-rabbit IgG (H þ L) (Invitrogen Molecular ProbesÒ) in perm/wash buffer. Different combinations of the internal antibodies were tested and optimal concentrations of both primary and secondary antibodies selected on the basis of titration curves. For the external antibodies we used, for 200 ml of 2.5  106 cells ml1, E3D9 (neutrophilic granulocytes) 2 mg ml1, C7G7 (B-cells) 2 mg ml1, G2H3 (B-cells) 1 mg ml1 and 4D11 (isotypic control)

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1 mg ml1 as described by Pettersen et al. [26]. For the intracellular antibodies different concentrations were evaluated for the rabbit anti-IPNV antibody; 1:500, 1:1000, 1:3000 and 1:5000. Applying the 1:5000 dilution of rabbit anti-IPNV antibody, we also performed a time lapse of infected leucocytes to investigate if we could detect virus replication after in vitro infection. 10.000 cells were recorded in each sample. HKL and PBL was analysed for forward scatter (FS) and sideward scatter (SS) dividing C7G7þ and E3D9þ cells according to size (FS) and granularity (SS) in a scatter plot. For the flow cytometry analysis of leucocytes the FSC voltage was set to E00 which multiplies the signal by 100. Further analyses were done in FCSExpress Version 3. 2.8. Immunohistochemistry Immunohistochemistry of in vitro infected leucocytes and noninfected controls from different times post infection were performed to visualize the internalization of virus observed in the flow cytometer. The leucocytes were exposed to the virus in vitro for two, three or four hours and then the excess virus was washed away. Cell suspensions from PBL and HKL were diluted to

Fig. 1. Exposure time for optimal IPNV infection of leucocytes in vitro. Flow cytometry histograms show percent IPNV positive (IPNVþ) leucocytes in HKL and PBL from representative fish at two and four hours post in vitro infection. Cytospin preparations of IPNVþ leucocytes from infected leucocyte culture are shown in photos, scale bars ¼ 10 mm. Leucocytes appear green and are stained, by rabbit-antiserum to TO cells, while IPNV (yellow/red) are detected by use of rabbit-anti IPNV antibodies. Fluorochrome labelled secondary antibodies were used and nuclei are DAPI stained. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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1  106 cells ml1 and cytospin preparations of 100 ml cell suspensions were made by centrifugation for 3 min at 1000 rpm in a Shandon cytospinn centrifuge. The preparations were left to air dry in the dark for 24 h. The cells were encircled with a PAP penn (DAKO) and the preparations were fixed with 3.7% (w/v) freshly made formaldehyde for 10 min. The slides were washed in PBS bath for 2  5 min. Incubation with 0.5% (w/v) BSA (bovine serum albumin) in PBS for 30 min were performed to block background staining. Antibodies were supplied by droplets of 50 ml containing E3D9 (30 mg ml1), C7G7 (4 mg ml1), 4D11 (4 mg ml1), G2H3 (4 mg ml1) or rabbit anti-TO (stain all leucocytes [32]) (1:1000). Incubation was done in the dark in humidity chambers for 1 h. The slides were washed in PBS baths for 3  5 min. AlexaFluor488 conjugated goat anti-mouse or goat anti-rabbit at dilution 1:400 were used as secondary antibodies. Incubation was done in the dark in humidity chambers for 45 min before washed in PBS baths for 3  5 min. The cells were permeabilized by applying 0.1% Triton x100 for 3e5 min, and then washed in PBS baths for 2  5 min. Each slide were supplied with a droplet of 50 ml of antibodies for internal antigens, either MAb anti-IPNV (Aquatic Diagnostics) (3 mg ml1), Rabbit anti-IPNV (1:5000), MAbB anti-IPNV (hybridoma supernatant) or rabbit preserum (1:5000). Incubation was done in the dark in humidity chambers for 1 h. The slides were washed in PBS baths for 3  5 min. As secondary antibodies we used Alexa Fluor 555 or 647 conjugated goat anti-rabbit/mouse antibodies. Incubation was done in the dark in humidity chambers for 45 min. The slides were washed in PBS baths for 3  5 min. The cell preparations were mounted with Pro Long GoldÒ antifade reagent containing DAPI. Directly conjugation of primary antibodies using zenon kits and cocktail staining was also tested, but these procedures usually gave low fluorescence and were therefore abandoned. For immunohistochemistry of IPNV infected CHSE-214 cells, uninfected CHSE-214 cells and persistent CHSE-214 cells were seeded onto chamber slide flasks (Nunclon) and incubated until 90% confluent monolayers. The CHSE cells were infected by procedures described above. Uninfected cells were included as controls. The cells were washed gently in PBS and covered with 1000 ml BD fix for 20 min. The cells were gently washed in PBS bath before permeabilisation with 0.1% (v/v) Triton x100 for 3e5 min. The cells were washed in PBS baths and covered with PBS containing 0.5% (w/v) BSA for blocking of background staining. The

slides were supplied with 1 ml of MAbB anti-IPNV and incubated for 1 h. The cells were washed for 3  5 min in PBS baths before covered by a 1:400 dilution of a Rabbit anti-mouse 488 conjugate (Invitrogen) or a Rabbit anti-mouse 647 conjugate (Invitrogen). The slides were incubated for 45 min in the dark. The cells were washed for 3  5 min in PBS baths the chambers removed and coverslips mounted with Pro Long GoldÒ antifade reagent containing DAPI. 2.9. Microscopy Slide preparations were examined in a Zeiss Axioskop 2 plus (Carl Zeiss Germany) and photographs captured using a Nikon DS Camera Head DS-5M. Further processing of the pictures was done in Photoshop CS2 (Adobe Systems Incorporated, San Jose, USA). Some cell-line preparations were examined in a Leica DM 2500 with a TCS SPE confocal unit. Confocal microscopy of leucocytes took place at the Molecular Imaging Centre (MIC) at the University of Bergen on a Leica SP2 AOBS Confocal microscope. For the confocal preparations the cells were cultivated on sterilized coverslide glass (MenzelGlaser Ø 12 mm # 1.5) in 24 well plates. Further processing of the pictures was done in Imaris version 6.1.5 (Bitplane). 3. Results 3.1. Leucocyte cultivation Leucocytes could be cultivated in both wells and tubes and all leucocyte samples used in experiments had a viability count of at least 97% and an aggregation factor of less than 2. Cultivation in tubes was preferred since this method implies less handling and treatment of the cells and the viability was therefore generally highest in tubes. The method for cultivation in wells was used for separate analysis of adherent and non-adherent cells. 3.2. In vitro challenge of leucocytes For in vitro infection of the leucocytes an exposure time of 3 h was sufficient to internalize the virus in the leucocytes (Fig. 1). An infectious dose of 500 ml IPNV (2  105TCID50) supplied to 2 ml cell suspension containing 5  106 cells was found to be suitable for infection.

Fig. 2. Flow cytometry scatter plots of HKL and PBL according to size and granularity, after fixation with formaldehyde, acetone:methanol (1:1) or BD Cytofix/Cytoperm and unfixed leucocytes. Instrument settings are adjusted for the different treatments. The plots provide information on sample debris and separations of the cells in the leucocyte population after the fixation treatment.

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3.3. Virus quantification IPNV is a virus which is easily grown in a number of different cell lines, but different cells give different virus yield. Due to the problem of attenuation after growth of IPNV in cell cultures [33] we chose to pass the virus once in RTG-2 cells and once in CHSE-214 cells. The virus yield after propagation on CHSE-214 cells was in the range 106e108 TCID50. 3.4. Fixation and permeabilization analyses The BD Cytofix/Cytoperm kit was found to be the most suitable of the three procedures tested (Fig. 2). This method gave less

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autofluorescence and the lowest loss of cells. Saponin removes cholesterol from the cell membrane, making it permeable to antibodies by creating tiny holes in the cell membrane. Since formaldehyde is a cross linking fixative it will keep the cell constituents, including virus, from leaking out of the cell. The formaldehyde/Triton x-100 method resulted in considerable loss of cells. Triton x-100 extract lipids from the cell membrane, but it may also extract proteins along with the lipids. This procedure also gave considerable difference in scatter properties between PBL and HKL after fixation. The organic solvent method, Acetone:Methanol, gave acceptable results on both scatter properties and binding of antibodies, but reduced the number of cells in the solution.

Fig. 3. Histogram overlays of non-infected (filled) and in vitro infected (red line) leucocytes (10.000 leucocytes per analysis) from PBL (A) and HKL (B) 3 days post challenge. The leucocytes (PBL and HKL) in density plots and histograms are from the same fish and show that there are a change in scatter properties, size and granularity of infected cells in both PBL and HKL. The scatter plots from PBL and HKL are the same cells as the density plots from the IPNV infected leucocytes, and IPNVþ leucocytes are identified by red dots and noninfected leucocytes are identified by black dots. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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3.5. Flow cytometry detection of intracellular IPNV If mouse primary monoclonal antibodies with different specificity are used in the same assay they have to be conjugated directly with fluorochromes. Calculations of labelling of primary

monoclonal antibodies with Alexa Fluor 647 Labeling Kit showed only 1.84 mol of Alexa Fluor 647 per mol of MAb (of Anti-Infectious Pancreatic Necrosis virus (IPNV) monoclonal antibody, Aquatic Diagnostics) after incubation overnight at 4  C. The manufacturer states 3e7 mol as an optimal degree of labelling. If directly

Fig. 4. Flow cytometry quadrant plots of PBL and HKL in vitro infected with IPNV from day 1 to day 3 (D1, D2, D3) post infection and non-infected controls. Intracellular IPNV are detected by rabbit anti-IPNV antibody. The quadrants are termed upper right region (UR), lower right region (LR), lower left region (LL) and upper left region (UL). Non-infected control samples are stained by rabbit anti-IPNV and Alexa Fluor 647 they are also supplied with the secondary antibody for external antibodies; Alexa Fluor RPE (additional control plots are supplied in supplementary). Positive cells will appear in UL. Less than 1% positive cells were detected in UL of the non-infected cells from day 1 to day 3 in all samples. A: UR: Neutrophils (E3D9þ) with IPNV. LR: Neutrophils (E3D9þ) without IPNV. LL: Leucocytes except neutrophils without IPNV. UL: IPNVþ leucocytes except neutrophils. B: UR: B-cells (C7G7þ) with IPNV. LR: B-cells (C7G7þ) without IPNV. LL: Leucocytes except B-cells without IPNV. UL: IPNVþ leucocytes except B-cells. Percent positive leucocytes in LR and LL are designated in or beneath the actual quadrants.

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conjugated a concentration of 0.25 mg ml1 of conjugated MAb was suitable, but because of the rather low conjugation ratio achieved and the fact that the polyclonal anti-IPNV antibody provided a stronger signal, the two step polyclonal antibody staining procedure was preferred. The Rabbit anti-IPNV antiserum was titrated to find the appropriate concentration for the flow cytometry analyses. Dilutions evaluated were 1:500, 1:1000, 1:3000 and 1:5000. 1:5000 were found to be the most suitable concentration. Conjugation of Fab fragments to the external antibodies using the Alexa Fluor staining kit (Invitrogen) generally gave weak signals and the method was abandoned. 3 days post in vitro infection the percentage IPNV positive (IPNVþ) leucocytes of the total cell population, in representative fish, was 11.5% in PBL (Fig. 3A) while in HKL it was 9.1% (Fig. 3B). The size and granularity of infected cell populations are different from non-infected cells and infected cells are present as both high and low sizes and granularities (Fig. 3A,B). An increase in percentage positive leucocytes post infection with increasing time would indicate virus replication. In PBL there were about 18% neutrophils each day, and no apparent rise in IPNVþ neutrophils was observed (Fig. 4A). A slight rise in other IPNVþ cells could be observed from day 1 to day 2. In HK at day 3 all IPNVþ cells are neutrophils. Other cells that are IPNVþ increase from day 1 to day 2, but are absent at day 3 (Fig. 4A). The percentage of cells that are both IPNVþ and C7G7þ has decreased in PBL at day 3 (Fig. 4B). From day 1-3 there was an increase in other cells that are IPNVþ. In HKL a slight increase in C7G7þ IPNVþ cells are observed at day 3. In both PBL and HKL other cells that are IPNVþ are also observed at day 3 (Fig. 4B). 3.6. CHSE-214 cells By the infectious titres described we could not detect IPNV positive CHSE-214 cells by flow cytometry before 72 h post infection. At this time 3.2% of the cells were virus positive (Table 1). The percentages increased rapidly after this. At 96 h post infection 67.5% of the cells were positive, at 106 h 76.3% and at 121 h 83.5%. In the persistently infected cells the percentage of IPNV positive cells was in the range of 49.6e62.7% (Table 1). As indicated in Fig. 5 the mean of fluorescence intensity is higher from the infected CHSE cells after 96 h indicating more virus particles in each positive cell than in the persistently infected cells at this sampling. It is also interesting to observe that the infected cells has two peaks of cells with fluorescence intensity representing infected cells, one population low in virus and one high in virus showing cells at different stages of infection present in the population. The persistently infected cells are in between these populations. By titration of filtrated supernatant from persistently infected cells a yield of 106 TCID50 was obtained. Obtained virus yield from one passage on CHSE-cells (post virus propagation) was in the range 108e109 TCID50. Persistantly infected CHSE cells are visualized in Fig. 5B. One infected CHSE cell are visualized in Fig. 5C. 3.7. Immunohistochemistry Internalized IPNV could be observed 12 h post infection. At this time both IPNV positive B-cells and neutrophils were found, but they were scarce in the preparations. At this time point IPNVpositive cells were easier observed in PBL than in HKL. Two days post in vitro infection the leucocyte preparations had a much denser appearance of virus positive cells, especially in HKL. Most positive cells were mononuclear, large and had a high amount of virus. IPNV was not detected in samples from non-infected control cells, neither in slides of infected cells stained with rabbit preserum.

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Table 1 Percent IPNVþ CHSE-214 cells at different times post infection (p.i.) and in two preparations of CHSE-214 cells that have become persistently infected with IPNV. Sample

Number of gated cells

%IPNVþ of gated cells

Uninfected control CHSE cells 24 h p.i. CHSE cells 48 p.i. CHSE cells 72 h p.i. CHSE cells 96 h p.i. CHSE cells 106 h p.i. CHSE cells 121 h p.i. Persist. Inf. CHSE cells (2)a Persist. Inf. CHSE cells (9)a

10,000 10,000 10,000 10,000 10,000 10,000 10,000 10,000 10,000

1.03 0.71 0.36 3.23 67.54 76.29 83.46 49.63 62.65

a (2) And (9) are initially infected with a IPNV stock that had previously been passed two and nine times on CHSE-214 cells, respectively.

In the CHSE-214 cell line a higher density of virus positive cells were observed in the in vitro infected than in the persistent infected cells. The density of virus within each positive cell was also higher in the in vitro infected cells (Fig. 5A,B). Scanning confocal microscopy verified the presence of virus within both B-cells and neutrophils (Fig. 6). 4. Discussion In the present study, we have described a 2-color method for detection of virus within leucocyte subpopulations from Atlantic salmon. The development of this method is based on analysis of cultivated leucocytes from PBL and HKL. All leucocyte suspensions included in the experiments had a high viability count and low aggregation factor. By use of the presented protocols, both leucocytes and CHSE cells were analysed and found positive for IPNV, and virus replication was observed as increasing numbers of IPNV positive cells with increasing time post infection. This was also observed by immunohistochemistry. Persistently infected cell lines with IPNV have previously been derived from both susceptible cell lines [34e37] and from non susceptible cell lines [38]. The strong signals from our persistently infected CHSE cells indicate that good sensitivity is obtained with the presented flow cytometry protocol. Detection of intracellular viral antigens by flow cytometry requires a fixation and permeabilisation protocol that will allow large molecules of fluorochrome conjugated antibodies to enter the cell and access the targeted antigen, but at the same time pores must not allow the virus to be released from the cell. The disadvantage of fixing the cells with aldehydes is that it reacts with a variety of amines found in cells to produce fluorescent material resulting in high autofluorescence. Measuring small amounts of virus within the cells, this autofluorescence can make detection difficult and likely impossible. By the formaldehyde/saponin method, the scatter properties of the salmon leucocytes were maintained and at the same time it allowed internalization of primary and secondary antibodies. The small permeabilization pores will close once saponine is removed from the solutions and thereby trapping antigens and antibodies inside the cells once the staining procedure is finished. The preserum control will show if unattached antibodies are trapped within the cells, which would give false positives. Protein structures on the cell wall may be altered after fixation. For this reason one often stain the cell surface antigens before fixing the cells. For staining the cell surface, we used antibodies directed towards neutrophilic granulocytes and B-cells at concentrations determined in previous studies [26]. Of the different internal antibodies tested, the rabbit anti-IPNV antibody, having a broad epitope affinity, gave the strongest signal and the two step labelling procedure will be preferred in further studies due to this. The amount of antigens within the cells is expected to be quite low, and

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dealing with the autofluorescence of fixed cells, it was important to choose a bright and small fluorochrome to enter the cell. Only few protocols for intracellular virus detection in fish cells are described. To our knowledge this is the first flow cytometry protocol for analysis of internal antigen and leucocyte specific receptors simultaneously in fish. Of identified leucocyte populations, the results revealed IPNV within both B-cells and neutrophils. Flow cytometry has previously been employed to detect virus in leucocytes and cell lines, but specific leucocyte antibodies were not included. A flow cytometry method used for detection of fish iridovirus in a fish cell culture used paraformaldehyde-tween-20 for fixation and permeabilisation [39]. Flow cytometry analysis performed on rainbow trout (Onchorhyncus mykiss) leucocytes from IPNV carrier fish and in vitro infected CHSE cells [9,40] applied a 3.7% solution of formaldehyde in PBS for 15 min and permeabilisation with a 0.01% solution of Triton x-100 for 1 min. The CHSE cells were fixed cold 80% acetone at 20  C. The differences might be due to fish species variations. Different types of negative controls, like leucocytes from another species, instead of preimmune serum to define the threshold between positive and negative cells will likely have an impact. The most consistent histopathological finding in fish suffering from IPN is degeneration and necrosis in exocrine pancreas tissue [41]. However, surviving fish may become asymptomatic carriers and shedders of the virus. The titres in these fish are sometimes low and IPNV is most efficiently detected in the HK which is a haematopoietic tissue in Atlantic salmon. For this reason, leucocytes have

been suggested to harbour IPNV [5,7,10e12] and adherent leucocytes from HK have been proposed as the primary leucocyte target of the virus [6,7]. An understanding of how IPNV spreads in vivo and identification of the leucocyte subpopulations involved will help in developing strategies to prevent infection. Based on the obtained results, the presented flow cytometry method may be used to follow the impact of the viral infection on the different leucocyte populations during the acute stages of the infection and later in carriers. Since we could detect virus within the persistently infected cell line, it is likely that we also can detect IPNV-positive leucocytes in carrier fish, as these are supposed to have approximately the same amount of internalized virus [35]. Defected interfering (DI) viral particles have been suggested to be responsible for survival of the persistent carrier cell cultures [34,37,42]. These particles are genetically altered and not themselves virulent, but dependent on the wild type virus for replication. Once introduced they have a replication advantage relative to the wild-type virus in competition for limited essential replication factors, and hence inhibit wild-type replication and permits survival of the host cell [43]. Commonly, DI particles arise after serial passages of virus in vitro at high multiplicities of infection (MOI). The DI particles contain normal viral structural proteins and will therefore likely be detected by polyclonal antibodies provided the target epitopes exists. Therefore it is not likely that the fluorescence observed in the persistently infected CHSE-214 cells can be explained by the presence of DI particles alone. In addition, naïve CHSE-214 cells were re infected by filtrated supernatants from the

Fig. 5. Overlays flow cytometry histograms of CHSE-214 cells that are non-infected (control), in vitro infected (24e121 h post infection) and persistently infected with IPNV. Persistent CHSE IPNV infected cells are shown for two different virus passages (2 and 9) to show virulence abilities. The threshold (1%) between IPNV- negative and IPNV þ are set by using non-infected cells incubated, with both the primary antibody to IPNV and the secondary antibody conjugate. A: Picture from confocal microscopy (mid section) show representative persistently infected CHSE cells with IPNV (red), scale bar ¼ 10 mm. B: Inverted microscope picture of cells culture showing one IPNVþ (green) CHSE cell four days post challenge, scale bar ¼ 10 mm. Virus are stained by use of rabbit-anti IPNV antibodies and fluorochrome labelled secondary antibodies while nuclei are DAPI stained. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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Fig. 6. Confocal scanning microscopy of a B-cell (C7G7þ) (A) and a neutrophilic granulocyte (E3D9þ) (B) from PBL with internalized IPNV two days post in vitro infection, scale bars ¼ 5 mm. A: C7G7þ cells (yellow) and virus (red). B: E3D9þ cells (green) and virus (red). A (1e6) and B (1e6) are comprised of selected z-stack slices prepared to overlays in Adobe Photoshop Elements 2.0. C: Confocal microscopy picture of a higly IPNVþ neutrophil, identified by Mab E3D9, are shown. Surface rendering is performed using Imaris version 6.1.5 (Bitmap). A 3D animation sequence is presented in Supplementary; Video 1. IPNV was detected by rabbit-anti IPNV antibodies and fluorochrome labelled antibodies to Mab and rabbit antibodies were used. Nuclei are stained by DAPI. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

established persistant infected cells and this verify that infective virus particles were present in the persistant cell cultures. To date one does not know how the IPN virus achieves the clinical quiescence in carrier fish or whether there are periods of more active replication. Flow cytometry analyses can provide information on the distribution of infected leucocytes in the different stages of in vivo infection that might help resolve this question. Acknowledgements Paul Løvik, University of Bergen, is thanked for technical assistance and Professor Øystein Evensen at the Norwegian School of Veterinary Science is thanked for providing the IPNV isolate (NI011) and the rabbit anti-IPNV antibodies. The confocal imaging of leucocytes was performed at the Molecular Imaging Center (Fuge, Norwegian Research Council), University of Bergen. Appendix A. Supplementary data Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.fsi.2012.09.020.

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