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Induction of viral interference by IPNV-carrier cells on target cells: A cell co-culture study
Fish & Shellfish Immunology 58 (2016) 483e489
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Fish & Shellfish Immunology journal homepage: www.elsevier.com/locate/fsi
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Induction of viral interference by IPNV-carrier cells on target cells: A cell co-culture study ~ o, Susana Torres, Lucía Almagro, Melissa Bello -Pe rez, Amparo Estepa, Ricardo Parren * Luis Perez ndez, Av. Universidad s/n, 03202 Elche, Spain Instituto de Biología Molecular y Celular, Universidad Miguel Herna
a r t i c l e i n f o
a b s t r a c t
Article history: Received 12 July 2016 Received in revised form 20 September 2016 Accepted 26 September 2016 Available online 28 September 2016
IPNV is a salmonid birnavirus that possesses the ability to establish asymptomatic persistent infections in a number of valuable fish species. The presence of IPNV may interfere with subsequent infection by other viruses. In the present study we show that an IPNV-carrier cell line (EPCIPNV) can induce an antiviral state in fresh EPC by co-cultivating both cell types in three different ways: a “droplet” culture system, a plastic chamber setup, and a transmembrane (Transwell®) system. All three cell co-culture methods were proven useful to study donor/target cell interaction. Naïve EPC cells grown in contact with EPCIPNV cells develop resistance to VHSV superinfection. The transmembrane system seems best suited to examine gene expression in donor and target cells separately. Our findings point to the conclusion that one or more soluble factors produced by the IPNV carrier culture induce the innate immune response within the target cells. This antiviral response is associated to the up-regulation of interferon (ifn) and mx gene expression in target EPC cells. To our knowledge this is the first article describing co-culture systems to study the interplay between virus-carrier cells and naive cells in fish. © 2016 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Keywords: IPNV VHSV Viral interference Cell co-culture Interferon
1. Introduction Various viruses have been identified as the etiological agents of diseases of farmed fish. Amongst them, viral hemorrhagic septicemia virus (VHSV) is an enveloped virus belonging to the family Rhabdoviridae that poses a threat to salmonid fish farming worldwide. VHS is listed as a notifiable disease by the World Organization for Animal Health [1]. Up to date, there are no treatments or vaccines for VHSV commercially available. Infectious Pancreatic Necrosis Virus (IPNV) is a double-stranded RNA genome nonenveloped virus (family Birnaviridae, genus Aquabirnavirus) that has been isolated from marine and fresh water fish species [2]. IPNV often establishes an asymptomatic carrier state in fish [3,4]. We have previously shown the inhibition of VHSV replication in EPCIPNV cells, a cyprinid cell line persistently-infected with IPNV [5]. Our initial hypothesis was that IPNV replication in some of the cells within the persistently-infected culture line would cause a stimulation of the interferon-mediated response. This would lead to the synthesis and secretion of antiviral factors to the cell culture
* Corresponding author. E-mail address: [email protected] (L. Perez).
medium and the establishment of an antiviral state in the whole IPNV-carrier culture. This can be verified by subsequent challenge with the heterologous virus VHSV, showing that EPCIPNV cells exhibit a blockade of VHSV replication [6]. Interference between IPNV and other fish viruses has been also found in vivo [7e9]. Fish can respond to viral infection by activating the innate immune response genes, in particular the type I interferon (IFN) signaling pathway [10e13]. IFN production is triggered in response to molecular patterns associated to pathogens, such as double stranded RNA. Production of IFN finally results in the transcription of IFN stimulated genes (ISGs). Genes coding for the Mx proteins are one of those ISGs playing a major role in the establishment of an antiviral state in the cell [9,14]. The interferon response to IPNV has been observed both in cell culture [15] as well as in vivo [3,4,16]. The presence of interferon or interferon-like activity in supernatants of IPNV-infected rainbow trout cells has been reported earlier [17]. To get a better understanding of the viral interference phenomenon and the establishment of the antiviral state in naive cells exposed to IPNV virus-carrier cells we have set-up here three different co-culture systems where two distinct cell populations can be maintained physically apart from each other, but they are connected by sharing the growth medium. The co-culture setup
http://dx.doi.org/10.1016/j.fsi.2016.09.052 1050-4648/© 2016 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/ 4.0/).
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allowed us to analyze the interaction between the EPCIPNV carrier cells and fresh EPC cells. In the present study we show that exposure of target EPC cells to soluble factors released by the IPNVinfected cells renders them resistant to subsequent challenge by VHSV, which goes in parallel to the upregulation of interferon and mx gene expression. To our knowledge there have been no studies of this type to evaluate the interaction between a virus-carrier cell line and the original cell line in fish. Results presented here show for the first time a co-culture system analysis of viral interference in fish. 2. Materials and methods 2.1. Cell culture and viral isolates The epithelioma papulosum cyprini (EPC) cells, a cell line originated from fathead minnow Pimephales promelas, was purchased from the European Collection of Authenticated Cell Cultures (ECACC no 93120820). EPC cells were cultured in RPMI Dutchmodified medium with 20 mM HEPES (Gibco BRL-Invitrogen), fetal bovine serum 10% (FBS, Linus-Cultek) glutamine 2 mM (Gibco BRL), sodium pyruvate 1 mM, gentamicin 50 mg/ml (Gibco BRL), and amphotericin 1.2 mg/ml (Gibco BRL). Viruses used in this study are infectious pancreatic necrosis virus (IPNV, Sp strain) and viral hemorrhagic septicemia virus (VHSV07.71 strain). 2.2. Cell culture in droplets To seed the donor (EPCIPNV) cells a 150 ml droplet containing 9 104 cells is placed on one side of a 60 mm plate. On the opposite side of the plate another 150 ml drop containing the target (EPC) cells is placed. Cells are allowed to attach to the plastic surface of the plate overnight, before removing the droplets by aspiration. Finally, all cells in the culture dish were overlaid with 2 ml growth medium. After 24 h of incubation at 21 C the cells were infected with VHSV at a moi of 0.05 and incubated at 14 C. At 6 days postinfection the cells were fixed and stained with 10% crystal violet in formaldehyde. 2.3. Rectangular chambers co-culture system A plastic grid with three rectangular compartments is placed on a 60 mm culture dish (see Fig. 3A). Donor and target cells are seeded on the left and right compartments, respectively, leaving the middle one empty. After cell growth for one day, the plastic grid is removed and the two cells populations are placed under the same medium for 24e48 h before challenge with VHSV at a m.oi ¼ 0.01 TCID50/cell. At 8 days post-infection the cells were fixed and stained with 10% crystal violet in formaldehyde. 2.4. Transmembrane (Transwell®) co-culture assay In this system cell-cell contact is prevented by using an insert with a 0.4 m pore size polycarbonate membrane (Transwell®, Costar) in a 24-well plate. In these experiments, target EPC cells were cultured below the insert, whereas donor cells (EPCIPNV) were placed in the insert at z90% confluence. Unless stated otherwise, the donor cells/target cells were allowed to cocultivate for approximately 48 h, followed by removal of the insert and challenge of the target cells in the lower chamber with VHSV to assess the induction of antiviral state. VHSV-induced cytopathic effect was visualized by staining with a 5 mg/ml Giemsa solution. Cells were observed with a Nikon Eclipse TE2000-U microscope. A duplicate plate of the experiment was prepared to harvest the cells in lysis
buffer for RNA extraction and quantitative real-time RT-PCR analysis. 2.5. Quantitative real-time RT-PCR Total RNA was extracted from cultured cells using the HP Total RNA kit (Omega).The RNA concentration was determined in a Nanodrop 1000 spectrophotometer (Thermo Scientific). 300 ng of RNA were converted to cDNA by reverse transcription using random hexamers as primers and Moloney murine leukemia virus reverse transcriptase (M-MLV RT, Gibco). Quantitative PCR was performed with 750 nM of primers and SYBR Green Universal PCR Master mix (Applied Biosystems) and amplified by 40 cycles of 95 C 15 s and 60 C 1 min in a 7300 Real Time PCR System (Applied Biosystems). For each sample qPCR was done in triplicate. Primers for EPC interferon (ifn) gene amplification were: forward primer, 50 -ACAGGCAGTCGTCGGAACTTA-30 ; reverse primer, 50 GAAGTGCCTTTTTATCTTAATCT-30 . Primers for EPC mx gene amplification: forward, 50 -GGAGAAGAGGTTAAATGTGGATCAG-3; reverse, 50 -TGAAGTGCCTTTTTATCTTAATCT-30 . Primers for EPC ifn gene amplification were designed from a sequence obtained from a DNA fragment retrieved from EPC cells by amplification with primers for crucian carp (Carassius auratus) ifn gene, and cloned in eukaryotic expression vectors. The EPC ifn gene sequence has accession number FN178457.1 in the Genbank [18]. Primers for EPC mx gene were designed in our laboratory following the same strategy: RTPCR amplification of a DNA sequence from EPC cells using primers from Carassius auratus mx1 gene (GenBank acc. no. AY303813.1). The PCR cDNA was cloned into a pCRII-TOPO plasmid, and each clone was confirmed by nucleotide sequence. The EPC mx sequence can be found in supplementary material no. 2. Primers were designed using Primer Express 3.0 and verified with Primer3 software v0.4.0. Selection criteria included product length that were <200bp with optimal melting temperatures of 57e63 C. The cycle threshold (Ct) values were converted into relative expression values normalized against 18S rRNA expression (Eukaryotic 18S rRNA VIC-TAMRA, Thermo Scientific). The results are presented as fold differences between the expression level of ifn/mx gene in control cells and the expression in cells exposed to each treatment. Mean values of the relative expression of ifn and mx genes and standard deviations were calculated by using the Microsoft Excel software from datasets of two independent experiments (n ¼ 3). 2.6. Statistical analysis Datasets from gene expression experiments were imported to Graph Pad Prism 5.0 software to conduct a one-way analysis of variance (ANOVA) with one independent variable (gene expression) and >2 conditions (different treatments of the EPC cells). The comparison of gene expression between control EPC cells and the different experimental groups were obtained by Dunnet's Multiple Comparison Test. A value of p < 0.05 was considered to indicate a significant difference between the control group and the experimental group. 3. Results 3.1. Anti-VHSV state in mixed EPC/EPCIPNV cell cultures In a first set of experiments the IPNV-carrier cells were mixed with fresh EPC cells at different ratios and then seeded on a multiwell plate. The mixed cultures were challenged with VHSV and the resistance to infection was evaluated by examining virus-induced cell lysis at 6 days post-infection. When 50% (1:1 ratio) or at least
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20% of the cells (1:4 ratio) were EPCIPNV cells the whole culture was still resistant to VHSV superinfection (Fig. 1). At 1:10 or 1:20 EPCIPNV to EPC ratios the mixed culture showed clear signs of VHSVinduced cytopathic effect, thus suggesting that at the lowest EPCIPNV:EPC ratios the number of cells harbouring IPNV was not sufficient to produce an effective antiviral state on the whole culture. 3.2. Determination of resistance to VHSV superinfection in a drop cell culture setup In the drop co-culture system two 150 ml droplets of growth medium containing z90000 cells are placed separated by z 0.5 cm on 6 well plates. The droplets maintain their position and shape by surface tension allowing the cells to attach to the bottom of the well, forming a roughly circular pattern. Then the drops are carefully vacuum sucked and donor and target cells can be put into contact by sharing the growth medium. After 24 h of cocultivation the cells were infected with VHSV and the cytopathic effect was evaluated by crystal violet staining and visual inspection. While the droplets of EPC cells virtually disappeared after infection with VHSV, we consistently observed that EPC cells became resistant to VHSV challenge after co-cultivation with EPCIPNV cells (Fig. 2). 3.3. Interaction between virus-carrier and naive cell populations in a plastic grid culture system Next we set up a co-culture system were the two types of cells were physically separated during seeding and growing. In order to do so, we used a plastic rectangular grid which is placed on a 60 mm plate (Fig. 3A). Cells are seeded on the left and right chambers, leaving the central one empty. Once the cells reach confluence the plastic grid can be removed, and the two cell types put into contact by sharing the medium. This co-culture system allows for separate treatment of the cells in one chamber, such as in Fig. 3A where one of the chambers contained EPC cells pre-treated with the synthetic dsRNA poly I:C to induce the interferon response. EPC cells sharing growth medium with the poly I:C treated cells developed an antiviral state against VHSV challenge (Fig. 3B). Likewise, EPC cells kept in contact with EPCIPNV cells showed a much-decreased cytopathic effect after superinfection with VHSV, indicating that some soluble factor produced by the IPNV-carrier cells reached the target EPC cells provoking an antiVHSV response. 3.4. Analysis of EPC: EPCIPNV interaction in a Transwell® co-culture setup In the following series of experiments we employed a
Fig. 1. VHSV challenge of mixed EPC/EPCIPNV cultures. EPC and EPCIPNV cells were mixed at the indicated ratios and seeded on 48-well plate. After a 24 h incubation period the mixed cultures were infected with a m.o.i. of 0.1 VHSV. At 6 days postinfection the cells were fixed and stained with crystal violet to visualize the virusinduced cytopathic effect.
Fig. 2. EPC cells infected with VHSV in a cell droplets co-culture system. Drops of EPC and EPCIPNV cells were placed on 6-well plates as indicated. Donor and target cells were grown sharing the medium for 24 h and subsequently challenged with VHSV (1.5 105 TCID50/well). At 6 days p.i. the cells were fixed and stained with crystal violet. Mock: non-infected cells.
commercially available co-culture system: Transwell®. In this system the cells are grown in a compartment with a porous bottom that can be inserted in a multiwell plate. Molecules secreted to the medium can pass through the porous membrane and reach the cells in the outer chamber. In our setup, donor cells are placed within the insert, and the target cells are seeded on the bottom of the well (Fig. 4.). We chose to do it this way to better visualize the target cells after VHSV challenge (as shown in Fig. 5). At 4 days post-infection VHSV-infected EPC cells showed a distinct cytopathic effect while EPCIPNV cells were refractory to VHSV infection as expected. We found that EPC cells co-cultured with an upper compartment containing EPCIPNV cells prior to challenge with VHSV did not show any sign of virus infection. Direct exposure to poly I:C led to protection of EPC cells to VHSV challenge. However, poly I:C pretreated donor cells induced almost negligible protection against VHSV superinfection in the target EPC cells.
3.5. Measuremet of ifn and mx gene expression in EPC target cells The Transwell® culture system allows independent RNA extraction from donor and target cells for quantitative analysis of gene expression. Thus, it is a well suited system to measure changes in gene expression by factors passing through the membrane and reaching the target cells. To analyze the role of innate immune response in the establishment of interference against VHSV, interferon (ifn) and Mx protein (mx) gene expression was measured in EPC cells exposed to different stimuli by quantitative real-time RTPCR (Fig. 6). Direct exposure to conditioned medium from EPCIPNV cells appears to be the best ifn and mx inducer on EPC cells. Treatment with poly I:C as well as co-cultivation with poly I:C treated cells led to the up-regulation of the mx gene, but not the ifn gene. One additional control sample (EPC cells exposed to 100 TCID50 IPN virus) was included to show that small amounts of IPN virus produced by the IPNV-carrier cells do not exert any effect on the target cells. This issue had to be checked to rule out that any effect we were seeing was due to the passage of IPNV particles through the membrane pores. EPCIPNV cells showed an upregulation of both ifn and mx gene expression relative to control EPC cells,
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Fig. 3. Rectangular chambers co-culture system. A. Image of a 60 mm diameter culture plate with the three-chamber plastic grid showing the culture of target and donor cells. The size of each chamber is 1 2 cm. Once the plastic grid is removed, the two types of cells can be incubated under the same medium for the desired amount of time before testing the antiviral state by VHSV challenge. 3B. Response to VHSV challenge by fresh EPC cells co-cultured with poly I:C treated (20 mg/ml poly I:C for 24 h) EPC or with EPCIPNV cells. Cells were incubated sharing the culture medium for 24 h before infection with VHSV (moi ¼ 0.01). In the rightmost image, EPC and EPCIPNV cells were kept separated by the plastic grid during growth and virus infection. Plates were incubated 8 days at 14 C and VHSV-induced cytopathic effect was revealed by crystal violet staining. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 4. Transwell® co-culture experimental setup. Transwell membrane inserts (0.4 mm pore size) were used to separate the donor cells (inner chamber) from naïve target cells (outer chamber). Cells seeded on wells with no transwell inserts were used as controls. Donor cells were usually IPNV-carrier EPC cells or EPC cells pretreated with poly I:C. Following 48 h of incubation the transwell inserts were removed and the target cells were challenged with VHSV. Once the cytopathic effect was evident in the virus-infected control, the cells were fixed and stained to determine cell survival to VHSV infection. A duplicate transwell plate of the experiment is set up to collect the cells for RNA extraction and measurement of gene expression.
as expected. Incubating EPC cells with donor EPCIPNV cells led to an increase of ifn and mx RNA levels (Fig. 6C,D). 4. Discussion Infectious pancreatic necrosis virus is capable of establishing a persistent infection both in vivo [3,4,19] as well as in vitro [5,17]. Previously we found that an IPNV carrier state could be achieved under certain conditions in the cyprinid cell line EPC [5]. We named this IPNV-carrier cell line EPCIPNV. A noteworthy feature of the EPCIPNV culture is that EPCIPNV cells display resistance to superinfection with heterologous viruses such as VHSV. Our previous studies determining the antiviral activity in EPCIPNV culture supernatants suggested that one or more soluble factors may be responsible of the establishment of the antiviral state in our EPC/ EPCIPNV cell system [6]. Our next step was to design experimental setups to study the interaction between the two cell populations: co-culture systems specifically aimed to investigate the effect of persistently infected cells over naive cells in our EPCIPNV model. We
first hypothesized that cells harbouring IPNV in the carrier culture could be producing some soluble antiviral factor. Therefore, reducing the number of IPNV-positive cells within the culture by “diluting” the EPCIPNV cells in fresh EPC cells would lead to the loss of the anti-VHSV state since the antiviral factor would not reach an effective concentration in the growth medium. Our data appear to support that hypothesis. This is in accordance with the previously reported anti-VHSV activity of the conditioned medium from EPCIPNV cells where the conditioned medium lost the antiviral activity when it was serially diluted in fresh medium [6]. The simplest co-culture system we tested was the cell culture in droplets. Here no special device is required, and the visualization and interpretation of the results is very straightforward. EPC cells should be destroyed by VHSV, whereas a drop of EPC cells placed next to a drop of EPCIPNV cells should show some sign of induced protection against subsequent VHSV infection. We proved that this was the case. Unfortunately, the cell droplet co-culture system has a number of drawbacks: the droplets can be perturbed by any movement during incubation and more important: it is not feasible
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Fig. 5. VHSV-induced cytopathic effect on EPC cells exposed to different donor cells in a transmembrane co-culture system. In brackets: donor cells in the inner chamber. Donor and target cells were co-cultured for 48 h before removing the transwell insert and infecting the cells in the outer chamber with 0.1 TCID50 VHSV/cell. At 4 days post-infection the cells were fixed with methanol and stained with Giemsa. 10 images of representative fields captured by the microscope are shown.
Fig. 6. Determination of ifn and mx gene expression by real-time RT-PCR in donor and target cells in a transwell co-culture setup. In brackets: cells seeded in the inner chamber. A and B: ifn and mx gene expression in experimental controls. IPNV (100): exposure to100 TCID50 units of IPNV; CM-EPCipnv: exposure to growth medium from EPCIPNV culture clarified by centrifugation and diluted 1:4 in fresh medium. Poly I:C: exposure to 10 mg/ml poly I:C. The normalized relative expression to untreated EPC cells is shown. C and D: ifn gene and mx gene expression in the target cells at the indicated donor/target cells matches. All data are presented as the mean ± sd from two independent cDNA samples with three replicates per sample. Statistical significance of gene expression compared to EPC control cells was determined by Dunnet's Multiple Comparison Test (*, p < 0.05).
to perform an independent harvesting of donor and target cells for RNA extraction and quantitative analysis of gene expression. The absence of a physical barrier between the two drops also precludes
to carry out any treatment (i.e: with an interferon inductor such as poly I:C) of one of the cell types apart from the other cell type. A better suited method for independent handling of two types
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of cells is the rectangular chamber grid co-culture system described in here: the two cell types are kept physically separated by a plastic grid until desired. That feature allowed us to test the effect of cocultivating EPC cells previously treated poly I:C with fresh EPC cells. This is something that could not be done with the drop culture system. Once the plastic grid is removed the two types of cells can interact just by overlaying the two cell patches with growth medium. At a later time in the experiment the plastic grid can be placed again over the plate in order to harvest the two rectangular patches of cells separately for RNA extraction and measurement of gene expression. In our hands RNA amounts of 0.5e1.5 mg could be obtained from one rectangular patch containing z6 105 cells (not shown). The limitations of the rectangular grid culture system are that it allows for a relatively small number of experimental points per assay, plus the high consumption of medium and reagents that the use of 60 mm dishes implies. The third co-culture system, the Transwell®, makes use of porous membrane inserts. The cells are grown in inserts with the permeable material at the bottom that are placed on the top (but not touching) the cell monolayer in the plate underneath. In our experimental designs the inserts contain the donor cells for convenience, since it was easier to visualize the effect of co-culture on the target cells if they were kept on the plastic bottom of the well. There are a number of examples of Transwell® system applications in the literature [20] but very few are used to analyze the interplay between virus-infected cells and naive cells. The Transwell® system is costly but it is very well adapted to our purposes due to its versatility. For instance, the cells in the inserts could be pretreated with poly I:C separately, and later on be placed on top the target cells. Likewise, time of contact between donor and target cells can be ended by simply removing the insert from the well, making the transwell a suitable system to perform time-course assays. Most important, it is the co-culture system best suited to analyze gene expression since it is easy to extract RNA from both donor and target cells independently at different time points along the experiment. Here we show how co-cultivation of EPC cells (outer chamber) with EPCIPNV (insert) leads to the protection of the EPC target cells against subsequent VHSV challenge. From our previous studies [6] we suspected the role of the interferon pathway in the establishment of the anti-VHSV state. Particularly, the production of the antiviral protein Mx (one of the interferon-stimulated genes) in response to virus infection has been observed in several fish species [21e23]. In fact, most fish viruses have some capacity to induce the interferon pathway in the diseased animal [24,25] including IPNV [26]. In the aforementioned works the role of the innate immune system in the protection of fish against viral challenge seems well proven. Here we show that following exposure to inserts containing IPNV-carrier cells the target EPC cells underwent an up-regulation of the ifn and mx gene expression, strongly supporting the role of the ifn-dependent immune response in the establishment of the antiviral state. Poly I:C is a good mx inducer on EPC cells. However we did not observe ifn induction by poly I:C treatment. Lack of ifn gene up-regulation by poly I:C has been reported before [14,27]. That may imply that an interferon-independent mx induction may be taking place in poly I:C treated cells. In summary, we have explored three different approaches to investigate the superinfection exclusion that IPNV exerts over VHSV in EPC cells. The co-culture systems employed along this work have proven useful to demonstrate the induction of an antiviral state by IPNV-carrier cells on naive cells by means of soluble factors. They will be most helpful in elucidating the molecular basis of broad range non-specific antiviral responses in fish cells, with potential implications in the design of novel strategies to control viral diseases of fish.
Acknowledgements n, This work was supported by Programa Estatal de Investigacio n Orientada a los Retos de la Sociedad of Spain Desarrollo e Innovacio (AGL2014-51773-C3-1R) and Consolider Ingenio 2010 CSD 2008 Aquagenomics. Lucía Almagro was a recipient of a Beca de n of Spanish MINECO (student fellowship nº 403486). Colaboracio Technical assistance from Beatriz Bonmatí and Irene Mudarra is acknowledged. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.fsi.2016.09.052. References [1] OIE, Manual of Diagnostic Tests for Aquatic Animals, 2012. http://www.oie. int/en/international-standard-setting/aquatic-manual/access-online/. [2] E. Gomez-Casado, A. Estepa, J.M. Coll, A comparative review on Europeanfarmed finfish RNA viruses and their vaccine, Vaccine 29 (2011) 2657e2671. [3] J. Garcia, K. Urquhart, A.E. Ellis, Infectious pancreatic necrosis virus establishes an asymptomatic carrier state in kidney leucocytes of juvenile Atlantic cod, Gadus morhua L. J. Fish. Dis. 29 (2006) 409e413. ~ ez, K. Maisey, [4] S. Reyes-Cerpa, F. Reyes-Lopez, D. Toro-Ascuy, J. Iban A.M. Sandino, M. Imarai, IPNV modulation of pro and anti-inflammatory cytokine expression in Atlantic salmon might help the establishment of infection and persistence, Fish. Shellfish Immunol. 32 (2012) 291e300. [5] I. Garcia, A. Galiana, A. Falco, A. Estepa, L. Perez, Characterization of an infectious pancreatic necrosis (IPN) virus carrier cell culture with resistance to superinfection with heterologous viruses, Vet. Microbiol. 149 (2011) 48e55. [6] M.T. Jurado, P. Garcia-Valtanen, A. Estepa, L. Perez, Antiviral activity produced by an IPNV-carrier EPC cell culture confers resistance to VHSV infection, Vet. Microbiol. 166 (2013) 412e418. [7] N. Byrne, J. Castric, J. Cabon, C. Quentel, Study of the viral interference between infectious pancreatic necrosis virus (IPNV) and infectious haematopoietic necrosis virus (IHNV) in rainbow trout (Oncorrhynchus mykiss), Fish. Shellfish Immunol. 24 (2008) 489e497. [8] H.J. Kim, N. Oseko, T. Nishizawa, M. Yoshimizu, Protection of rainbow trout from infectious hematopoietic necrosis (IHN) by injection of infectious pancreatic necrosis virus (IPNV) or poly(I: C), Dis. Aquat. Org. 83 (2009) 105e113. [9] R. Pakingking, Y. Okinaka, K. Mori, M. Arimoto, K. Muroga, T. Nakai, In vivo and in vitro analysis of the resistance against viral haemorrhagic septicaemia virus in Japanese flounder (Paralichtys olivaceus) precedingly infected with aquabirnavirus, Fish. Shellfish Immunol. 17 (2004) 1e11. [10] M. Adamek, K. Rakus, J. Chyb, G. Brodgen, A. Huebner, I. Irnazarow, D. Steinhagen, zInterferon type I responses to virus infections in carp cells: In vitro studies on Cyprinid herpesvirus 3 and Rhabdovirus carpio infections, Fish. Shellfish Immunol. 33 (2012) 482e493. [11] M.-X. Chang, J. Zou, P. Nie, B. Huang, Z. Yu, B. Collet, C.J. Secombes, Intracellular interferons in fish: a unique means to combat viral infection, PLoS Pathog. 9 (2013) e1003736. [12] V.G. Chinchar, O. Logue, A. Antao, G.D. Chinchar, Channel catfish reovirus (CRV) inhibits replication of channel catfish herpesvirus (CCV) by two distinct mechanisms: viral interference and induction of an anti-viral factor, Dis. Aquat. Org. 33 (1998) 77e85. [13] E.R. Verrier, C. Langevin, A. Benmasour, P. Boudinot, Early antiviral response and virus-induced genes in fish, Dev. Comp. Immunol. 35 (2011) 1204e1214. [14] C. Langevin, E. Aleksejeva, G. Passoni, N. Palha, J.P. Levraud, P. Boudinot, The antiviral innate immune response in fish: evolution and conservation of the IFN system, J. Mol. Biol. 425 (2013) 4904e4920. [15] S. Rodriguez Saint-Jean, A.I. de las Heras, S.I. Perez-Prieto, The persistence of infectious pancreatic necrosis virus and its influence on the early immune response. Vet. Immunol, Immunopathol 136 (2010) 81e91. [16] K. Julin, L.H. Johansen, A.-I. Sommer, J.B. Jorgensen, Persistent infections with infectious pancreatic necrosis virus (IPNV) of different virulence in Atlantic salmon, Salmo salar L. J. Fish. Dis. 38 (2014) 1005e1019. [17] S.B. Lin, C.H. Lin, Y.L. Hsu, Establishment of an antiviral activity assay and the identification and partial purification of interferon-like protein from rainbow trout gonadal cells (RTG-2), Zool. Stud. 40 (2001) 240e246. [18] S. Biacchesi, M. LeBerre, A. Lamoureux, Y. Louise, E. Lauret, P. Boudinot, M. Bremont, Mitochondrial antiviral signaling protein plays a major role in induction of the fish innate immune response against RNA and DNA viruses, J. Virol. 83 (2009) 7815e7827. [19] S.E. LaPatra, K.A. Lauda, G.R. Jones, Aquareovirus interference mediated resistance to infectious hematopoietic necrosis virus, Vet. Res. 26 (1995) 455e459. [20] Q.Y. Chen, Y.H. Liu, J.H. Li, Z.K. Wang, J.X. Liu, Z.H. Yuan, DNA-dependent activator of interferon-regulatory factors inhibits hepatitis B virus replication,
~ o et al. / Fish & Shellfish Immunology 58 (2016) 483e489 R. Parren World J. Gastroenterol. 18 (2012) 2850e2858. [21] C. Carballo, E. Garcia-Rosado, J.J. Borrego, C. Alonso, SJNNV down-regulates RGNNV replication in European sea bass by the induction of the type I interferon system, Vet. Res. 47 (2016) 6e16. [22] M.A. Fernandez-Trujillo, E. García-Rosado, M.C. Alonso, D. Castro, Mx1, Mx2 and Mx3 proteins from the gilthead seabream (Sparus aurata) show in vitro antiviral activity against RNA and DNA viruses, Mol. Immunol. 56 (2013) 630e636. [23] N.T.K. Vo, A.W. Bender, D.A. Ammendiola, J.S. Lumsden, B. Dixon, N.C. Bols, Development of a walleye spleen stromal cell line sensitive to viral hemorrhagic septicemia virus (VHSV IVb) and to protection by synthetic dsRNA, Fish. Shellfish Immunol. 45 (2015) 89e93. [24] H.C. Ingerslev, A. Ronneseth, E.F. Pettersen, H.I. Wergeland, Differential expression of immune genes in Atlantic salmon (Salmo salar L.) challenged
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intraperitoneally or by cohabitation with IPNV, Basic Immunol. 69 (2009) 90e98. [25] O. Kileng, M.I. Brundtland, B. Robertsen, Infectious salmon anemia virus is a powerful inducer of key genes of the type I interferon system of Atlantic salmon, but is not inhibited by interferon, Fish. Shellfish Immunol. (2007) 378e389. [26] K. Lockhart, A.J.A. McBeath, B. Collet, M. Snow, A.E. Ellis, Expression of Mx mRNA following infection with IPNV is greater in IPN-susceptible Atlantic salmon post-smolts than in IPN-resistant Atlantic salmon parr, Fish. Shellfish Immunol. 22 (2007) 151e156. [27] Y. Valero, P. Morcillo, J. Meseguer, F. Buonocore, M.A. Esteban, E. Chaves-Pozo, A. Cuesta, Characterization of the interferon pathway in the teleost fish gonad against the vertically transmitted viral nervous necrosis virus, J. Gen. Virol. 96 (2015) 2176e2187.
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Full length article
Induction of viral interference by IPNV-carrier cells on target cells: A cell co-culture study ~ o, Susana Torres, Lucía Almagro, Melissa Bello -Pe rez, Amparo Estepa, Ricardo Parren * Luis Perez ndez, Av. Universidad s/n, 03202 Elche, Spain Instituto de Biología Molecular y Celular, Universidad Miguel Herna
a r t i c l e i n f o
a b s t r a c t
Article history: Received 12 July 2016 Received in revised form 20 September 2016 Accepted 26 September 2016 Available online 28 September 2016
IPNV is a salmonid birnavirus that possesses the ability to establish asymptomatic persistent infections in a number of valuable fish species. The presence of IPNV may interfere with subsequent infection by other viruses. In the present study we show that an IPNV-carrier cell line (EPCIPNV) can induce an antiviral state in fresh EPC by co-cultivating both cell types in three different ways: a “droplet” culture system, a plastic chamber setup, and a transmembrane (Transwell®) system. All three cell co-culture methods were proven useful to study donor/target cell interaction. Naïve EPC cells grown in contact with EPCIPNV cells develop resistance to VHSV superinfection. The transmembrane system seems best suited to examine gene expression in donor and target cells separately. Our findings point to the conclusion that one or more soluble factors produced by the IPNV carrier culture induce the innate immune response within the target cells. This antiviral response is associated to the up-regulation of interferon (ifn) and mx gene expression in target EPC cells. To our knowledge this is the first article describing co-culture systems to study the interplay between virus-carrier cells and naive cells in fish. © 2016 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Keywords: IPNV VHSV Viral interference Cell co-culture Interferon
1. Introduction Various viruses have been identified as the etiological agents of diseases of farmed fish. Amongst them, viral hemorrhagic septicemia virus (VHSV) is an enveloped virus belonging to the family Rhabdoviridae that poses a threat to salmonid fish farming worldwide. VHS is listed as a notifiable disease by the World Organization for Animal Health [1]. Up to date, there are no treatments or vaccines for VHSV commercially available. Infectious Pancreatic Necrosis Virus (IPNV) is a double-stranded RNA genome nonenveloped virus (family Birnaviridae, genus Aquabirnavirus) that has been isolated from marine and fresh water fish species [2]. IPNV often establishes an asymptomatic carrier state in fish [3,4]. We have previously shown the inhibition of VHSV replication in EPCIPNV cells, a cyprinid cell line persistently-infected with IPNV [5]. Our initial hypothesis was that IPNV replication in some of the cells within the persistently-infected culture line would cause a stimulation of the interferon-mediated response. This would lead to the synthesis and secretion of antiviral factors to the cell culture
* Corresponding author. E-mail address: [email protected] (L. Perez).
medium and the establishment of an antiviral state in the whole IPNV-carrier culture. This can be verified by subsequent challenge with the heterologous virus VHSV, showing that EPCIPNV cells exhibit a blockade of VHSV replication [6]. Interference between IPNV and other fish viruses has been also found in vivo [7e9]. Fish can respond to viral infection by activating the innate immune response genes, in particular the type I interferon (IFN) signaling pathway [10e13]. IFN production is triggered in response to molecular patterns associated to pathogens, such as double stranded RNA. Production of IFN finally results in the transcription of IFN stimulated genes (ISGs). Genes coding for the Mx proteins are one of those ISGs playing a major role in the establishment of an antiviral state in the cell [9,14]. The interferon response to IPNV has been observed both in cell culture [15] as well as in vivo [3,4,16]. The presence of interferon or interferon-like activity in supernatants of IPNV-infected rainbow trout cells has been reported earlier [17]. To get a better understanding of the viral interference phenomenon and the establishment of the antiviral state in naive cells exposed to IPNV virus-carrier cells we have set-up here three different co-culture systems where two distinct cell populations can be maintained physically apart from each other, but they are connected by sharing the growth medium. The co-culture setup
http://dx.doi.org/10.1016/j.fsi.2016.09.052 1050-4648/© 2016 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/ 4.0/).
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allowed us to analyze the interaction between the EPCIPNV carrier cells and fresh EPC cells. In the present study we show that exposure of target EPC cells to soluble factors released by the IPNVinfected cells renders them resistant to subsequent challenge by VHSV, which goes in parallel to the upregulation of interferon and mx gene expression. To our knowledge there have been no studies of this type to evaluate the interaction between a virus-carrier cell line and the original cell line in fish. Results presented here show for the first time a co-culture system analysis of viral interference in fish. 2. Materials and methods 2.1. Cell culture and viral isolates The epithelioma papulosum cyprini (EPC) cells, a cell line originated from fathead minnow Pimephales promelas, was purchased from the European Collection of Authenticated Cell Cultures (ECACC no 93120820). EPC cells were cultured in RPMI Dutchmodified medium with 20 mM HEPES (Gibco BRL-Invitrogen), fetal bovine serum 10% (FBS, Linus-Cultek) glutamine 2 mM (Gibco BRL), sodium pyruvate 1 mM, gentamicin 50 mg/ml (Gibco BRL), and amphotericin 1.2 mg/ml (Gibco BRL). Viruses used in this study are infectious pancreatic necrosis virus (IPNV, Sp strain) and viral hemorrhagic septicemia virus (VHSV07.71 strain). 2.2. Cell culture in droplets To seed the donor (EPCIPNV) cells a 150 ml droplet containing 9 104 cells is placed on one side of a 60 mm plate. On the opposite side of the plate another 150 ml drop containing the target (EPC) cells is placed. Cells are allowed to attach to the plastic surface of the plate overnight, before removing the droplets by aspiration. Finally, all cells in the culture dish were overlaid with 2 ml growth medium. After 24 h of incubation at 21 C the cells were infected with VHSV at a moi of 0.05 and incubated at 14 C. At 6 days postinfection the cells were fixed and stained with 10% crystal violet in formaldehyde. 2.3. Rectangular chambers co-culture system A plastic grid with three rectangular compartments is placed on a 60 mm culture dish (see Fig. 3A). Donor and target cells are seeded on the left and right compartments, respectively, leaving the middle one empty. After cell growth for one day, the plastic grid is removed and the two cells populations are placed under the same medium for 24e48 h before challenge with VHSV at a m.oi ¼ 0.01 TCID50/cell. At 8 days post-infection the cells were fixed and stained with 10% crystal violet in formaldehyde. 2.4. Transmembrane (Transwell®) co-culture assay In this system cell-cell contact is prevented by using an insert with a 0.4 m pore size polycarbonate membrane (Transwell®, Costar) in a 24-well plate. In these experiments, target EPC cells were cultured below the insert, whereas donor cells (EPCIPNV) were placed in the insert at z90% confluence. Unless stated otherwise, the donor cells/target cells were allowed to cocultivate for approximately 48 h, followed by removal of the insert and challenge of the target cells in the lower chamber with VHSV to assess the induction of antiviral state. VHSV-induced cytopathic effect was visualized by staining with a 5 mg/ml Giemsa solution. Cells were observed with a Nikon Eclipse TE2000-U microscope. A duplicate plate of the experiment was prepared to harvest the cells in lysis
buffer for RNA extraction and quantitative real-time RT-PCR analysis. 2.5. Quantitative real-time RT-PCR Total RNA was extracted from cultured cells using the HP Total RNA kit (Omega).The RNA concentration was determined in a Nanodrop 1000 spectrophotometer (Thermo Scientific). 300 ng of RNA were converted to cDNA by reverse transcription using random hexamers as primers and Moloney murine leukemia virus reverse transcriptase (M-MLV RT, Gibco). Quantitative PCR was performed with 750 nM of primers and SYBR Green Universal PCR Master mix (Applied Biosystems) and amplified by 40 cycles of 95 C 15 s and 60 C 1 min in a 7300 Real Time PCR System (Applied Biosystems). For each sample qPCR was done in triplicate. Primers for EPC interferon (ifn) gene amplification were: forward primer, 50 -ACAGGCAGTCGTCGGAACTTA-30 ; reverse primer, 50 GAAGTGCCTTTTTATCTTAATCT-30 . Primers for EPC mx gene amplification: forward, 50 -GGAGAAGAGGTTAAATGTGGATCAG-3; reverse, 50 -TGAAGTGCCTTTTTATCTTAATCT-30 . Primers for EPC ifn gene amplification were designed from a sequence obtained from a DNA fragment retrieved from EPC cells by amplification with primers for crucian carp (Carassius auratus) ifn gene, and cloned in eukaryotic expression vectors. The EPC ifn gene sequence has accession number FN178457.1 in the Genbank [18]. Primers for EPC mx gene were designed in our laboratory following the same strategy: RTPCR amplification of a DNA sequence from EPC cells using primers from Carassius auratus mx1 gene (GenBank acc. no. AY303813.1). The PCR cDNA was cloned into a pCRII-TOPO plasmid, and each clone was confirmed by nucleotide sequence. The EPC mx sequence can be found in supplementary material no. 2. Primers were designed using Primer Express 3.0 and verified with Primer3 software v0.4.0. Selection criteria included product length that were <200bp with optimal melting temperatures of 57e63 C. The cycle threshold (Ct) values were converted into relative expression values normalized against 18S rRNA expression (Eukaryotic 18S rRNA VIC-TAMRA, Thermo Scientific). The results are presented as fold differences between the expression level of ifn/mx gene in control cells and the expression in cells exposed to each treatment. Mean values of the relative expression of ifn and mx genes and standard deviations were calculated by using the Microsoft Excel software from datasets of two independent experiments (n ¼ 3). 2.6. Statistical analysis Datasets from gene expression experiments were imported to Graph Pad Prism 5.0 software to conduct a one-way analysis of variance (ANOVA) with one independent variable (gene expression) and >2 conditions (different treatments of the EPC cells). The comparison of gene expression between control EPC cells and the different experimental groups were obtained by Dunnet's Multiple Comparison Test. A value of p < 0.05 was considered to indicate a significant difference between the control group and the experimental group. 3. Results 3.1. Anti-VHSV state in mixed EPC/EPCIPNV cell cultures In a first set of experiments the IPNV-carrier cells were mixed with fresh EPC cells at different ratios and then seeded on a multiwell plate. The mixed cultures were challenged with VHSV and the resistance to infection was evaluated by examining virus-induced cell lysis at 6 days post-infection. When 50% (1:1 ratio) or at least
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20% of the cells (1:4 ratio) were EPCIPNV cells the whole culture was still resistant to VHSV superinfection (Fig. 1). At 1:10 or 1:20 EPCIPNV to EPC ratios the mixed culture showed clear signs of VHSVinduced cytopathic effect, thus suggesting that at the lowest EPCIPNV:EPC ratios the number of cells harbouring IPNV was not sufficient to produce an effective antiviral state on the whole culture. 3.2. Determination of resistance to VHSV superinfection in a drop cell culture setup In the drop co-culture system two 150 ml droplets of growth medium containing z90000 cells are placed separated by z 0.5 cm on 6 well plates. The droplets maintain their position and shape by surface tension allowing the cells to attach to the bottom of the well, forming a roughly circular pattern. Then the drops are carefully vacuum sucked and donor and target cells can be put into contact by sharing the growth medium. After 24 h of cocultivation the cells were infected with VHSV and the cytopathic effect was evaluated by crystal violet staining and visual inspection. While the droplets of EPC cells virtually disappeared after infection with VHSV, we consistently observed that EPC cells became resistant to VHSV challenge after co-cultivation with EPCIPNV cells (Fig. 2). 3.3. Interaction between virus-carrier and naive cell populations in a plastic grid culture system Next we set up a co-culture system were the two types of cells were physically separated during seeding and growing. In order to do so, we used a plastic rectangular grid which is placed on a 60 mm plate (Fig. 3A). Cells are seeded on the left and right chambers, leaving the central one empty. Once the cells reach confluence the plastic grid can be removed, and the two cell types put into contact by sharing the medium. This co-culture system allows for separate treatment of the cells in one chamber, such as in Fig. 3A where one of the chambers contained EPC cells pre-treated with the synthetic dsRNA poly I:C to induce the interferon response. EPC cells sharing growth medium with the poly I:C treated cells developed an antiviral state against VHSV challenge (Fig. 3B). Likewise, EPC cells kept in contact with EPCIPNV cells showed a much-decreased cytopathic effect after superinfection with VHSV, indicating that some soluble factor produced by the IPNV-carrier cells reached the target EPC cells provoking an antiVHSV response. 3.4. Analysis of EPC: EPCIPNV interaction in a Transwell® co-culture setup In the following series of experiments we employed a
Fig. 1. VHSV challenge of mixed EPC/EPCIPNV cultures. EPC and EPCIPNV cells were mixed at the indicated ratios and seeded on 48-well plate. After a 24 h incubation period the mixed cultures were infected with a m.o.i. of 0.1 VHSV. At 6 days postinfection the cells were fixed and stained with crystal violet to visualize the virusinduced cytopathic effect.
Fig. 2. EPC cells infected with VHSV in a cell droplets co-culture system. Drops of EPC and EPCIPNV cells were placed on 6-well plates as indicated. Donor and target cells were grown sharing the medium for 24 h and subsequently challenged with VHSV (1.5 105 TCID50/well). At 6 days p.i. the cells were fixed and stained with crystal violet. Mock: non-infected cells.
commercially available co-culture system: Transwell®. In this system the cells are grown in a compartment with a porous bottom that can be inserted in a multiwell plate. Molecules secreted to the medium can pass through the porous membrane and reach the cells in the outer chamber. In our setup, donor cells are placed within the insert, and the target cells are seeded on the bottom of the well (Fig. 4.). We chose to do it this way to better visualize the target cells after VHSV challenge (as shown in Fig. 5). At 4 days post-infection VHSV-infected EPC cells showed a distinct cytopathic effect while EPCIPNV cells were refractory to VHSV infection as expected. We found that EPC cells co-cultured with an upper compartment containing EPCIPNV cells prior to challenge with VHSV did not show any sign of virus infection. Direct exposure to poly I:C led to protection of EPC cells to VHSV challenge. However, poly I:C pretreated donor cells induced almost negligible protection against VHSV superinfection in the target EPC cells.
3.5. Measuremet of ifn and mx gene expression in EPC target cells The Transwell® culture system allows independent RNA extraction from donor and target cells for quantitative analysis of gene expression. Thus, it is a well suited system to measure changes in gene expression by factors passing through the membrane and reaching the target cells. To analyze the role of innate immune response in the establishment of interference against VHSV, interferon (ifn) and Mx protein (mx) gene expression was measured in EPC cells exposed to different stimuli by quantitative real-time RTPCR (Fig. 6). Direct exposure to conditioned medium from EPCIPNV cells appears to be the best ifn and mx inducer on EPC cells. Treatment with poly I:C as well as co-cultivation with poly I:C treated cells led to the up-regulation of the mx gene, but not the ifn gene. One additional control sample (EPC cells exposed to 100 TCID50 IPN virus) was included to show that small amounts of IPN virus produced by the IPNV-carrier cells do not exert any effect on the target cells. This issue had to be checked to rule out that any effect we were seeing was due to the passage of IPNV particles through the membrane pores. EPCIPNV cells showed an upregulation of both ifn and mx gene expression relative to control EPC cells,
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Fig. 3. Rectangular chambers co-culture system. A. Image of a 60 mm diameter culture plate with the three-chamber plastic grid showing the culture of target and donor cells. The size of each chamber is 1 2 cm. Once the plastic grid is removed, the two types of cells can be incubated under the same medium for the desired amount of time before testing the antiviral state by VHSV challenge. 3B. Response to VHSV challenge by fresh EPC cells co-cultured with poly I:C treated (20 mg/ml poly I:C for 24 h) EPC or with EPCIPNV cells. Cells were incubated sharing the culture medium for 24 h before infection with VHSV (moi ¼ 0.01). In the rightmost image, EPC and EPCIPNV cells were kept separated by the plastic grid during growth and virus infection. Plates were incubated 8 days at 14 C and VHSV-induced cytopathic effect was revealed by crystal violet staining. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 4. Transwell® co-culture experimental setup. Transwell membrane inserts (0.4 mm pore size) were used to separate the donor cells (inner chamber) from naïve target cells (outer chamber). Cells seeded on wells with no transwell inserts were used as controls. Donor cells were usually IPNV-carrier EPC cells or EPC cells pretreated with poly I:C. Following 48 h of incubation the transwell inserts were removed and the target cells were challenged with VHSV. Once the cytopathic effect was evident in the virus-infected control, the cells were fixed and stained to determine cell survival to VHSV infection. A duplicate transwell plate of the experiment is set up to collect the cells for RNA extraction and measurement of gene expression.
as expected. Incubating EPC cells with donor EPCIPNV cells led to an increase of ifn and mx RNA levels (Fig. 6C,D). 4. Discussion Infectious pancreatic necrosis virus is capable of establishing a persistent infection both in vivo [3,4,19] as well as in vitro [5,17]. Previously we found that an IPNV carrier state could be achieved under certain conditions in the cyprinid cell line EPC [5]. We named this IPNV-carrier cell line EPCIPNV. A noteworthy feature of the EPCIPNV culture is that EPCIPNV cells display resistance to superinfection with heterologous viruses such as VHSV. Our previous studies determining the antiviral activity in EPCIPNV culture supernatants suggested that one or more soluble factors may be responsible of the establishment of the antiviral state in our EPC/ EPCIPNV cell system [6]. Our next step was to design experimental setups to study the interaction between the two cell populations: co-culture systems specifically aimed to investigate the effect of persistently infected cells over naive cells in our EPCIPNV model. We
first hypothesized that cells harbouring IPNV in the carrier culture could be producing some soluble antiviral factor. Therefore, reducing the number of IPNV-positive cells within the culture by “diluting” the EPCIPNV cells in fresh EPC cells would lead to the loss of the anti-VHSV state since the antiviral factor would not reach an effective concentration in the growth medium. Our data appear to support that hypothesis. This is in accordance with the previously reported anti-VHSV activity of the conditioned medium from EPCIPNV cells where the conditioned medium lost the antiviral activity when it was serially diluted in fresh medium [6]. The simplest co-culture system we tested was the cell culture in droplets. Here no special device is required, and the visualization and interpretation of the results is very straightforward. EPC cells should be destroyed by VHSV, whereas a drop of EPC cells placed next to a drop of EPCIPNV cells should show some sign of induced protection against subsequent VHSV infection. We proved that this was the case. Unfortunately, the cell droplet co-culture system has a number of drawbacks: the droplets can be perturbed by any movement during incubation and more important: it is not feasible
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Fig. 5. VHSV-induced cytopathic effect on EPC cells exposed to different donor cells in a transmembrane co-culture system. In brackets: donor cells in the inner chamber. Donor and target cells were co-cultured for 48 h before removing the transwell insert and infecting the cells in the outer chamber with 0.1 TCID50 VHSV/cell. At 4 days post-infection the cells were fixed with methanol and stained with Giemsa. 10 images of representative fields captured by the microscope are shown.
Fig. 6. Determination of ifn and mx gene expression by real-time RT-PCR in donor and target cells in a transwell co-culture setup. In brackets: cells seeded in the inner chamber. A and B: ifn and mx gene expression in experimental controls. IPNV (100): exposure to100 TCID50 units of IPNV; CM-EPCipnv: exposure to growth medium from EPCIPNV culture clarified by centrifugation and diluted 1:4 in fresh medium. Poly I:C: exposure to 10 mg/ml poly I:C. The normalized relative expression to untreated EPC cells is shown. C and D: ifn gene and mx gene expression in the target cells at the indicated donor/target cells matches. All data are presented as the mean ± sd from two independent cDNA samples with three replicates per sample. Statistical significance of gene expression compared to EPC control cells was determined by Dunnet's Multiple Comparison Test (*, p < 0.05).
to perform an independent harvesting of donor and target cells for RNA extraction and quantitative analysis of gene expression. The absence of a physical barrier between the two drops also precludes
to carry out any treatment (i.e: with an interferon inductor such as poly I:C) of one of the cell types apart from the other cell type. A better suited method for independent handling of two types
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of cells is the rectangular chamber grid co-culture system described in here: the two cell types are kept physically separated by a plastic grid until desired. That feature allowed us to test the effect of cocultivating EPC cells previously treated poly I:C with fresh EPC cells. This is something that could not be done with the drop culture system. Once the plastic grid is removed the two types of cells can interact just by overlaying the two cell patches with growth medium. At a later time in the experiment the plastic grid can be placed again over the plate in order to harvest the two rectangular patches of cells separately for RNA extraction and measurement of gene expression. In our hands RNA amounts of 0.5e1.5 mg could be obtained from one rectangular patch containing z6 105 cells (not shown). The limitations of the rectangular grid culture system are that it allows for a relatively small number of experimental points per assay, plus the high consumption of medium and reagents that the use of 60 mm dishes implies. The third co-culture system, the Transwell®, makes use of porous membrane inserts. The cells are grown in inserts with the permeable material at the bottom that are placed on the top (but not touching) the cell monolayer in the plate underneath. In our experimental designs the inserts contain the donor cells for convenience, since it was easier to visualize the effect of co-culture on the target cells if they were kept on the plastic bottom of the well. There are a number of examples of Transwell® system applications in the literature [20] but very few are used to analyze the interplay between virus-infected cells and naive cells. The Transwell® system is costly but it is very well adapted to our purposes due to its versatility. For instance, the cells in the inserts could be pretreated with poly I:C separately, and later on be placed on top the target cells. Likewise, time of contact between donor and target cells can be ended by simply removing the insert from the well, making the transwell a suitable system to perform time-course assays. Most important, it is the co-culture system best suited to analyze gene expression since it is easy to extract RNA from both donor and target cells independently at different time points along the experiment. Here we show how co-cultivation of EPC cells (outer chamber) with EPCIPNV (insert) leads to the protection of the EPC target cells against subsequent VHSV challenge. From our previous studies [6] we suspected the role of the interferon pathway in the establishment of the anti-VHSV state. Particularly, the production of the antiviral protein Mx (one of the interferon-stimulated genes) in response to virus infection has been observed in several fish species [21e23]. In fact, most fish viruses have some capacity to induce the interferon pathway in the diseased animal [24,25] including IPNV [26]. In the aforementioned works the role of the innate immune system in the protection of fish against viral challenge seems well proven. Here we show that following exposure to inserts containing IPNV-carrier cells the target EPC cells underwent an up-regulation of the ifn and mx gene expression, strongly supporting the role of the ifn-dependent immune response in the establishment of the antiviral state. Poly I:C is a good mx inducer on EPC cells. However we did not observe ifn induction by poly I:C treatment. Lack of ifn gene up-regulation by poly I:C has been reported before [14,27]. That may imply that an interferon-independent mx induction may be taking place in poly I:C treated cells. In summary, we have explored three different approaches to investigate the superinfection exclusion that IPNV exerts over VHSV in EPC cells. The co-culture systems employed along this work have proven useful to demonstrate the induction of an antiviral state by IPNV-carrier cells on naive cells by means of soluble factors. They will be most helpful in elucidating the molecular basis of broad range non-specific antiviral responses in fish cells, with potential implications in the design of novel strategies to control viral diseases of fish.
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