In vitro Epstein-Barr Virus Infection Model of Rabbit Lymphocytes from Peripheral Blood or Spleen

Most humans become lifelong carriers of Epstein-Barr virus (EBV) by adulthood. Primary EBV infection in adolescents causes infectious mononucleosis. EBV infection is associated with various diseases, neoplasms and hematological disorders. Recently, we reported that EBV can infect rabbits by intravenous, intranasal and/or peroral inoculation, which caused primary EBV infection in rabbits with heterogeneous host reactions. Some rabbits showed chronic and lifelong EBV infection with hemophagocytosis. In this study, to reveal detailed mechanisms in rabbit EBV infection, an in vitro investigation was performed. We elucidated that: (1) EBV can infect rabbit peripheral blood mononuclear cells and splenic lymphocytes in vitro, because EBV gene expressions were confirmed. (2) It is highly likely that the B cell is the main target cell of rabbit EBV infection and is immortalized similar to humans. (3) CD8+ T cells increased in the rabbit in vivo model after EBV inoculation, whereas an increase of B cells occurred after their transient decrease. These data suggest that EBV-infected B cells were proliferated, while CD8+ T cells increased to recognize and kill them. This system may explain Received: December 28, 2009 Accepted after revision: March 9, 2010 Published online: August 6, 2010 Kyosuke Kanai, PhD, and Kazuhiko Hayashi, MD Division of Molecular Pathology, Faculty of Medicine, Tottori University 86 Nishi-cho, Yonago, Tottori 683-8503 (Japan) Tel. +81 859 38 6063, Fax +81 859 38 6060 E-Mail kyosuke @ gmail.com or hayashik @ med.tottori-u.ac.jp © 2010 S. Karger AG, Basel 0300–5526/11/0541–0017$38.00/0 Accessible online at: www.karger.com/int D ow nl oa de d by : 54 .7 0. 40 .1 1 11 /1 /2 01 7 12 :2 7: 27 A M Kanai et al. Intervirology 2011;54:17–24 18 thorax-associated lymphoma, opportunistic lymphoma including post-transplant lymphoproliferative disorders, some cases of gastric carcinoma, nasopharyngeal carcinoma and other neoplasms [2, 8–10] . Therefore, it is important to clarify the mechanism and pathogenesis of these tumors for successful treatment. Investigation of the pathogenesis of EBV has been described in some animal models. EBV infects not only humans but also certain types of New World monkeys. So far, animal models of the common marmoset, cottontop tamarin, and douroucouli have been described [11–13] . Some New World monkeys developed post-transplant lymphoproliferative disorder-like B cell lymphoma, infectious mononucleosis-like disease, or latent infection. A rhesus monkey model using a rhesus lymphocryptovirus similar to EBV has been useful for elucidating primary and persistent infection of EBV [14] . However, these monkeys are precious natural resources. Although EBV does not infect mice and severe combined immunodeficiency (SCID) mice in general, it can infect humanized SCID or mice with immunodeficiency such as NOD/SCID, NOD/Shiscid /IL-2R null (NOG), and Rag2 –/– c –/– , and which were transplanted with human hematopoietic stem cells [15–18] . It has been reported previously that rabbits infected with herpesvirus papio (HVP), a type of lymphocryptovirus in baboons, developed virus-associated lymphoproliferative disorders and hemophagocytic syndrome, and are a possible animal model for studying the EBV-associated hemophagocytic syndrome [19–21] . In our previous reports, we recently revealed that EBV could frequently infect rabbit lymphocytes in vivo by intravenous inoculation, which caused a heterogeneous host reaction in the rabbits [22] ; some rabbits were also infected via the intranasal or peroral route [23] . In addition, we confirmed that in some of the rabbits inoculated intravenously, EBV-DNA were detected continuously or intermittently lifelong over 1,000 days. Autopsy of rabbits with lifetime EBV infection revealed the infiltration of lymphocytes expressing EBVencoded small RNA 1 (EBER1) or latent membrane protein 1 (LMP1) in the spleen and lymph nodes. Additionally, mild or moderate hemophagocytosis and some large atypical lymphocyte infiltrations were also observed in the autopsied spleens or lymph nodes [unpubl. data]. These results mean that EBV inoculated into rabbits causes transient viral infection and/or is maintained and survives in the rabbits for life without loss of viral function. An EBV-infected rabbit model may be appropriate to investigate in vivo EBV infection and the mechanisms for maintaining EBV. However, details of the cell or molecular biology of EBV-infected rabbits are still unclear. Therefore, in vitro investigations are needed to reveal the detailed mechanism of EBV infection. In this study, we investigated whether EBV can infect and immortalize rabbit peripheral blood mononuclear cells (PBMC) or splenic lymphocytes in vitro, and tried to determine the subsets of in vitro EBV-infected lymphocytes. Therefore, each abundance and transition of lymphocyte subsets in the rabbit after in vivo EBV inoculation were also analyzed by flow cytometry. Materials and Methods EBV-Producing Cells and Culture B95-8, which is an EBV-producing marmoset lymphoid cell line established by infection with a virus derived from a patient with infectious mononucleosis, was cultured in RPMI1640 medium (Nissui, Tokyo, Japan) with 5% fetal bovine serum (FBS), glutamine, and antibiotics (penicillin-G and streptomycin). B95-8 cells were incubated at 37 ° for 1 week and then incubated at 33 ° for 2 weeks; cell growth was accelerated at 37 ° , while EBV production was increased at 33 ° . Supernatants obtained from the B95-8 culture were filtrated using 0.45m filters to eliminate the cell debris and were centrifuged at 16,000 g for 90 min to remove supernatants by a Compact High Speed Refrigerated Centrifuge 7780 (Kubota, Tokyo, Japan). After centrifugation, the pellets were diluted with the fresh RPMI 1640 medium and stored at –80 ° until use. Preparations of Rabbit PBMC and Spleen Lymphocytes, and Exposure to EBV The collected peripheral bloods in the tubes with EDTA-2K were diluted with phosphate-buffered saline (PBS) into three times volumes and spleens were dissected out and suspended in PBS. These suspensions were placed on the mixture of Lymphosepar I and Lymphosepar II (Lymphosepar I and Lymphosepar II = 2:1) (Immuno-Biological Laboratories, Takasaki, Japan), and centrifuged at 490 g for 30 min. The layers of PBMC and/or lymphocytes were collected in the new tube and washed. Rabbit PBMC and/or spleen lymphocytes were exposed for infection by EBV. For exposure to EBV, washed 1 ! 10 6 cells were suspended in 1 ml of EBV suspension as described previously in a 15-ml tube and incubated for 2 h at 37 ° with tapping. Subsequently, cells were washed in PBS, and cultured in fresh 10% FBS, RPMI1640 medium in 37 ° until the optional days. Detection of EBV-Related Genes mRNA in PBMC by RT-PCR EBV-exposed rabbit PBMC were cultured and sampled every 2 days until 10, 20 and 30 days, and EBNA1 and ZEBRA mRNA expression were determined. EBV-exposed spleen lymphocytes were cultured and sampled every 2 days until 10 days, and EBNA1, EBNA2 and ZEBRA mRNA expression was analyzed. The RNA was extracted with the RNeasy Micro Kit (Qiagen, Hilden, Germany) and QIA shredder (Qiagen) in accordance with the manufacturer’s protocol. RNA from PBMC were quantified by NanoDrop (Thermo Scientific, Wilmington, Mass., USA) with each RNA sample measuring 500 ng. RT-PCR was performed with TaKaRa RNA PCR TM Kit (AMV) version 3.0 (TaKaRa Bio., D ow nl oa de d by : 54 .7 0. 40 .1 1 11 /1 /2 01 7 12 :2 7: 27 A M In vitro EBV Infection of Rabbit Lymphocytes Intervirology 2011;54:17–24 19 Otsu, Japan) in accordance with its protocol. The mRNA expression of EA, EBNA1, EBNA2, and ZEBRA was investigated by PCR or nested PCR using the described primers [23] . The mRNA of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the internal control. The PCR mixture comprised 2  l of 10 ! Ex Taq buffer, 1.6  l of dNTP Mixture, 0.1  l of TaKaRa Ex Taq HS, 12.3  l of H 2 O, 1 l of sense primer, 1  l of antisense primer, and 2 l of the sample. BJAB cells exposed to EBV and incubated for 2 days were used as positive control, and nuclease free H 2 O used for mixture dilution was used as negative control. Immunofluorescence Staining of Rabbit PBMC or Splenic Lymphocytes For discrimination of EBV-infected cells, EAD expression in rabbit PBMC and/or spleen lymphocytes were examined by an immunofluorescence staining method. Rabbit spleen lymphocytes were exposed to EBV and cultured for 4 days. Lymphocytes were washed in PBS, smeared on sides, and fixed (acetone:methanol = 1: 2). 1 g/ml sheep IgG, PBS was used for blocking. Then, 1: 600 diluted mouse anti-EAD antibody (Millipore, Billerica, Mass., USA) was used as the 1st antibody after washing, and 0.1% diluted fluorescein isothiocyanate (FITC)-conjugated anti-mouse IgG sheep antibody (Cappel, West Chester, Pa., USA) was applied as the second antibody. The slides were incubated at 37 ° for 30 min after the addition of each antibody, respectively. Finally, PI or DAPI were added for staining of nuclei and incubated at room temperature for 15 min. The slides were then observed under a fluorescence microscope (Olympus, Tokyo, Japan). To determine the cell type of expressed EAD, double-immunofluorescence staining was performed. 1: 200-diluted mouse anti-rabbit CD3 and CD79a (Spring Valley Laboratories, San Diego, Calif., USA) were also used as first antibody and cell type markers, and incubated. A rabbit polyclonal to mouse IgG-F(ab) 2 fragment (rhodamine) (Abcam, Cambridge, UK) was used as the second antibody and incubated. After 3 times washing in PBS, antiEAD antibody (Millipore) conjugated with biotin by Biotin Labeling Kit-NH 2 (Dojindo Laboratories, Kumamoto, Japan) was used as the first antibody for EAD detection, and rabbit polyclonal to biotin (FITC) (Abcam) was used as the second antibody. After each addition of antibody, the slides were incubated at 37 ° for 30 min. Finally, after DAPI staining, the slides were observed as before. Colony Formation Assay of EBV-Exposed Rabbit PBMC Methylcellulose medium culture was performed to reveal the immortalization potential of EBV-exposed rabbit PBMC. PBMC obtained from 2 individual rabbits were exposed to EBV for 2 h and 3 ! 10 6 PBMC were suspended in 3 ml of RPMI1640 medium containing glutamine, antibiotics, 30% FBS, 100  M 2-mercaptoethanol, and 1.2% methylcellulose: each 1 ! 10 6 cells plated on 3 dishes and incubated at 37 ° for 3 weeks. EBV-exposed human PBMC were plated on 3 dishes as the positive control, and EBV-unexposed PBMC were also plated to use as the negative control. The colonies that appeared were observed under a stereomicroscope (Leica mzfliii R-LS, Leica Microsystems, Wetzlar, Germany). Intravenous Inoculation with EBV into the Rabbit Rabbits were specific-pathogen-free normal male Japanese White Rabbits (weight, 2–3 kg) obtained from Shimizu Laboratory Supplies (Kyoto, Japan). The viral solutions of 9.2 ! 10 5 copies of EBV-DNA were inoculated into the rabbit intravenously. The day of viral inoculation was defined as ‘week 0’, and blood was collected every week. The animal experiment was carried out under the control of the committee in accordance with ‘The Guidelines for Animal Experimentation in the Faculty of Medicine, Tottori University’, ‘The Law Concerning the Human Care and Control of Animals’ (Law No. 105; October 1, 1973) and ‘The Japanese Government Notification on Feeding and Safekeeping of Animals’ (Notification No. 6, March 27, 1980). Flow Cytometry Analysis for Lymphocyte Subsets of PBMC from EBV-Inoculated Rabbits To determine the abundance of rabbit lymphocyte cell types, B cells, CD4+ and CD8+ T cells, and flow cytometry analysis were carried out in PBMC from EBV-inoculated rabbits. The following antibodies for B cells, CD4+ T cells or CD8+ T cells were used: goat anti-rabbit IgM ( heavy chain specific) with R-phycoerythrin (R-PE) conjugated with B cells (SouthernBiotech, Birmingham, Ala., USA), mouse anti-rabbit CD4 conjugated with FITC (AbD Serotec, Oxford, UK) and mouse anti-rabbit CD8 conjugated with FITC (AbD Serotec). PBMC were separated by the method described previously, 5 ! 10 5 cells were suspended in 10% rabbit serum, 0.02% NaN 3, Hanks’ BSS and incubated at 4 ° for 10 min for blocking. After centrifugation (490 g ) for 5 min, the supernatants were aspirated. Antibodies were diluted by 2% rabbit serum, 0.02% NaN 3 , Hanks’ BSS to a final concentration of 0.5 g in 100  l, and added to cells in the tube, suspended and incubated at 4 ° for 30 min. Cells were centrifuged and the supernatants were aspirated and 0.2 ng/200  l PI was added, suspended and incubated at room temperature for 15 min. Prepared cells were analyzed using EPICS XL (Beckman Coulter, Calif., USA) and obtained data were analyzed by WinMDI version 2.9. The sums of CD4 and CD8 values were considered as value of total T cell in this experiment. The incremental values of each lymphocyte subset that was calculated by data obtained by flow cytometry were shown by relative values which were considered on the principle that the value of day 0 was 100 (fig. 4a). Real-Time Quantitative PCR of EBV-DNA The PCR primers selected for this assay were specific for the BALF5 gene, which encodes the viral DNA polymerase [24] . The upstream and downstream primer sequences were 5 -CGGAAGCCCTCTGGACTTC-3 and 5 -CCCTGTTTATCCGATG-3 , respectively. A fluorogenic probe (5 -TGTACACGCACGAGAAATGCGCC-3 ) with a sequence located between the PCR primer sequences was synthesized by PE Applied Biosystems (Foster City, Calif., USA). PCR was performed using the TaqMan PCR kit (PE Applied Biosystems) [25] . Either 250 ng of DNA from peripheral blood mononuclear cells (PBMC), plasma, or culture supernatant was added to the PCR mixture containing 10 m M Tris (pH 8.3); 50 m M KCl; 10 m M EDTA; 5 m M MgCl 2 ; 400 m M each of dNTP; 0.2 m M each primer; 0.1 m M fluorogenic probe, and 1.25 U AmpliTaq Gold (PE Applied Biosystems). Following a 10min activation of AmpliTaq Gold at 95 ° , 45–50 cycles of 15 s at 95 ° and 1 min at 62 ° were carried out by a 7700 Sequence Detector (PE Applied Biosystems). A plasmid containing the BALF5 gene (pGEM-BALF5) was used as the positive control. Each sample was tested in duplicate, and the mean of the two values was considered as the copy number of the samples. Samples were deD ow nl oa de d by : 54 .7 0. 40 .1 1 11 /1 /2 01 7 12 :2 7: 27 A M Kanai et al. Intervirology 2011;54:17–24 20 fined as negative if the threshold cycle values exceeded 50. When EBV-DNA copies were lower than the minimal sensitivity (20 copies/10 6 WBCs), they were considered negative and indicated as ! 2 ! 10 1 (fig. 4).