Production and characterization of monoclonal antibodies against binary ethylenimine inactivated Nipah virus

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1 Journal of Virological Methods 132 (2006) Production and characterization of monoclonal antibodies against binary ethylenimine inactivated Nipah virus Yohannes Berhane a,, Jody D. Berry a, Charlene Ranadheera b, Peter Marszal a, Brigitte Nicolas a, Xin Yuan a, Markus Czub b, Hana Weingartl a a National Centre for Foreign Animal Disease, Canadian Food Inspection Agency, Canadian Science Center for Human and Animal Health, 1015 Arlington Street, Winnipeg, MB, Canada R3E 3M4 b National Microbiology Laboratory, Public Health Agency of Canada, Canadian Science Center for Human and Animal Health, Winnipeg, MB, Canada Received 24 April 2005; received in revised form 1 September 2005; accepted 6 September 2005 Available online 13 October 2005 Abstract Nipah virus, a zoonotic paramyxovirus which emerged recently was chemically inactivated using binary ethylenimine (BEI). The inactivated virus was concentrated and purified by sucrose gradient centrifugation. The gradient fractions were examined by electron microscopy and Western immunoblot, and gradient fraction containing mainly Nipah matrix (M) and nucleocapsid (N) proteins was used for immunizing BALB/c mice to generate hybridomas. Screening of the resultant hybridoma clones identified five strongly positive clones producing IgG monoclonal antibodies (mabs) reactive to the Nipah virus antigen. The protein specificity of these mabs was determined by Western immunoblot using Nipah virus and recombinant Nipah virus proteins expressed in mammalian cells. Four mabs reacted with Nipah N protein and one reacted with Nipah M protein. None of the mabs neutralized Nipah virus infectivity in vitro. However, all mabs recognized Nipah virus in ELISA and immunofluorescence assay. F45G2 mab was most suitable for immunohistochemistry on long term formalin-fixed Nipah virus infected swine tissues. Three of the anti-nucleocapsid mabs (F45G2, F45G3 and F45G6) showed cross-reactivity with closely related Hendra virus N protein in both immunofluorescence and Western Immunoblot assays. Two of the mabs were specific for the Nipah virus only, F45G4 (anti-n) and F45G5 (anti-m), and could be used in the primary identification of Nipah virus. The use of these immunoreagents to develop new diagnostic assays is discussed. Crown Copyright 2005 Published by Elsevier B.V. All rights reserved. Keywords: Nipah virus; Hendra virus; Monoclonal antibody; Nucleocapsid protein; Matrix protein; Western immunoblot; Immunofluorescence 1. Introduction Nipah virus is a zoonotic pathogen belonging to the family of Parmyxoviridae, genus Henipavirus and is listed as one of the potential biological agents for bio-terrorism in the Northern Atlantic Treaty Organization handbook (Lam and Chua, 2002). Nipah virus is classified as biosafety level 4 (BSL-4) agent and laboratory personnel needs to take appropriate precautions when working with this pathogen. In humans, Nipah virus causes severe febrile encephalitis with 40% mortality (Chua et al., 1999; Paton et al., 1999; Chua et al., 2000 and Chua, 2003). In pigs, it causes mainly a respiratory Corresponding author. Tel.: ; fax: address: berhaney@inspection.gc.ca (Y. Berhane). disease with lesser involvement of the central nervous system (CNS) and infrequent mortality (Goh et al., 2000 and Middleton et al., 2002). The virus can be transmitted to humans through close contact with infected pigs and their body secretions (Mohd Nor et al., 2000; Lam and Chua, 2002 and Middleton et al., 2002). Nipah virus is related antigenically and genetically to another zoonotic pathogen from the genus Henipavirus called Hendra virus (Murray et al., 1995; Harcourt et al., 2000; Daniels et al., 2001 and Wang et al., 2001). Hendra virus is the etiological agent of a fatal respiratory and CNS disease of horses and humans (Hooper et al., 1997a; Hooper et al., 1997b; Barclay and Paton, 2000 and Hooper et al., 2001). The genome of Henipaviruses consists of a single-stranded, linear molecule of RNA of negative sense that contains an attachment glycoprotein (G), fusion (F), matrix (M), polymerase (L), nucleocapsid (N), and P/V/C genes /$ see front matter. Crown Copyright 2005 Published by Elsevier B.V. All rights reserved. doi: /j.jviromet

2 60 Y. Berhane et al. / Journal of Virological Methods 132 (2006) Nipah virus has the largest genome among paramyxoviruses (18,246 nucleotides), and has a high degree of nucleotide homology in open reading frames (ORF) of various genes with Hendra virus (Harcourt et al., 2000). The development of mabs to Nipah virus is critical for studying the pathogenesis of the disease and for the development of diagnostic techniques. Production of quality Nipah virus antigen for mab development requires propagation of high titer virus in tissue culture; clarification by centrifugation, inactivation, concentration and purification of the virus. In addition, when working with BSL-4 pathogens such as Nipah virus, extensive safety testing of the inactivated virus is necessary prior to immunizing mice for hybridoma production under BSL-2 conditions. BEI, an aziridine compound, has been used for inactivation of adventitious viruses in biological preparations. BEI reacts with viral nucleic acids while preserving conformation and accessibility of epitopes to a much greater extent than formalin and -propriolactone (Bahnemann, 1975; Bahnemann, 1990; Kyvsgaard et al., 1997 and Blackburn and Besselaar, 1991). BEI has been used to inactivate a number of DNA viruses (African swine fever, porcine parvovirus, bovine rhinotracheitis and pseudorabies) and RNA viruses (foot and mouth disease virus, Newcastle disease virus and Rabies virus) for vaccine and antigen production (Bahnemann, 1976 and Bahnemann, 1990). This paper describes the development and characterization of murine mabs raised against BEI inactivated Nipah virus, identification of mabs to N and M proteins of Nipah virus, and their application for different types of assays. 2. Materials and methods 2.1. Virus Human isolates of Nipah and Hendra virus were kindly provided by Drs Thomas Ksiazek and Pierre Rollin, CDC, Atlanta. Nipah and Hendra virus stocks were prepared in Vero- 76 monolayers in T75 flasks (Costar, Corning Inc., Corning, NY). Confluent monolayers of Vero-76 cells were infected with Nipah/Hendra virus at 0.1 multiplicity of infection (moi). Cells were incubated at 33 C in the presence of 5% CO 2 until 80% cytopathic effect (CPE) was observed. Virus stocks were frozen at 70 C until used Cell culture African green monkey kidney (Vero-76) and porcine turbinate (PT-K 75) cells obtained from American Type Culture Collection (ATCC) were grown in T-175 flasks (Costar, Coring Inc., Coring, NY). The growth media included Dulbecco s minimum essential medium (DMEM, Sigma, St Louis, MO) supplemented with 10% fetal bovine serum (FBS) (Winset, St. Bruno, QC), 100 IU Penicillin and 100 g/ml of Streptomycin (Wisent, St. Bruno, QC), 10 mm HEPES (Sigma, St Louis, MO) and 1 mm sodium pyruvate (Gibco, Invitrogen, Grand Island, NY). The maintenance media was the same, except it contained 2% FBS Preparation of Nipah virus stocks for BEI inactivation Nipah virus stocks for BEI inactivation were prepared in PT-K 75 cells grown to 80 90% confluence and then inoculated with Nipah virus to give a multiplicity of infection (moi) of approximately 0.1. The virus was incubated for 1 h at 33 C in the presence of 5% CO 2 and cells were re-fed with 50 ml of maintenance media per flask. Cells were incubated for up to 72 h or until 70 80% cytopathic effect (CPE) was observed. Virus was harvested after freeze/thaw cycle, and clarified by centrifugation at 2000 g for 20 min at 4 C. The titer of the pooled Nipah virus was TCID 50 /ml Virus titration Virus titration was performed in 96-well plates (Costar, Coring, NY) with PT-K75 confluent cell monolayers. Ten-fold dilutions of virus inoculum were prepared in DMEM media and 50 l/well of diluted inoculum was applied to each well with cell monolayer. Following incubation for 1 h at 33 C, 5% CO 2,50 l per well of maintenance media with 4% FBS was added. The cells were incubated for 5 days and 50% tissue culture-infective dose (TCID 50 ) titers were calculated based on the presence of CPE Preparation of BEI for the inactivation of Nipah virus BEI was prepared as described elsewhere (Buonavoglia et al., 1988 and Bahnemann, 1990). For this purpose, g of 2- bromoethylamine (Sigma, St Louis, MO) was dissolved in 50 ml of 0.2N NaOH solution to produce 0.1 M BEI. The mixture was filtered through 0.22 m filter (Millipore, Corporation, Bedford, MA) and cyclization was allowed to take place at 37 C for 1 h with agitation. The BEI preparation was added to the virus suspension for a final concentration of 3 mm and the mixture was incubated at room temperature for 24 h. One-millilitre samples were collected at different intervals of time to determine virus kill curve. To each time point aliquot 10 l of ice-cold 1 M sodium thiosulphate was added to inactivate BEI Safety testing For safety testing, 10 ml of undiluted inactivated Nipah virus after 24 h of BEI inactivation was added to confluent monolayer of PT-K 75 cells grown in T-75 tissue culture flasks (Costar, Coring, NY). The cells were left to incubate for 1 h at 33 C in the presence of 5% CO 2 and were supplemented with 10 ml maintenance media with 4% FBS and were left to incubate for 5 days. Cells were monitored daily for the presence of CPE. After 5 days, the tissue culture flasks were frozen, thawed and culture supernatant was used for consecutive passages. After three passages, if no CPE was observed, the inactivated virus was considered safe for use under BSL-2 conditions.

3 Y. Berhane et al. / Journal of Virological Methods 132 (2006) Concentration and purification of viruses Initially the inactivated virus was concentrated by ultracentrifugation in Beckman 14 mm 89 mm polyallomer centrifuge tubes (Beckman Instruments Inc., Palo Alto, CA) under 30% sucrose cushion at 112,700 g for 2 h at 4 C using Beckman Optima XL-100 ultracentrifuge (Beckman Instruments Inc., Palo Alto, CA). Then, the virus pellet was resuspended in icecold TE buffer and was layered on to 20 50% (w/v) sucrose density gradient that was made in a Beckman 13 mm 51 mm Polyallomer centrifuge tubes (Beckman Instruments Inc., Palo Alto, CA). The gradient was centrifuged at 247,600 g for 2 h at 4 C. The bands from sucrose gradient were collected individually using a sterile Pasteur pipette and were transferred to a Beckman 3 mm 51 mm polyallomer tube (Beckman Instruments Inc., Palo Alto, California). The collected bands were diluted in ice-cold TE buffer and the gradient fractions were collected by centrifugation at 113,000 g for 2 h 4 C. Finally, the virus pellet was resuspended in 0.5 ml of icecold TE buffer. The viral antigen was stored at 70 C until used. Hendra virus was concentrated by ultracentrifugation in Beckman 14 mm 89 mm polyallomer centrifuge tubes (Beckman Instruments Inc., Palo Alto, CA) under 30% sucrose cushion at 112,700 g for 2 h at 4 C using Beckman Optima XL- 100 ultracentrifuge (Beckman Instruments Inc., Palo Alto, CA). Then, the virus pellet was resuspended in ice-cold TE buffer and was frozen at 70 C until used for Western immunoblot assay Transmission electron microscopy (TEM) The bands from sucrose gradient centrifugation were applied to formavar coated grids; air dried and were stained with 2% phosphotungstic acid. The grids were screened for the presence of Nipah virus structures at magnifications at 25,000 and 50,000 using TEM (Philips electron microscope, Holland). Photographs were also taken at the same magnifications Immunization of mice Five to six-week-old female BALB/c mice (Charles River, Wilmington, MA) were injected subcutaneously (SC) with 50 g of BEI inactivated, nucleoprotein enriched fraction of Nipah virus antigen from the lower band with an equal part of complete Freund s adjuvant (H37-Ra, CFA) from Difco (BD, Oakville, ON) on day 1. On day 30, the mice received 50 g of same virus SC in incomplete Freund s adjuvant in a total volume of 100 l. On days 48 and 63, the mice received 5 gof the same antigen in a total volume of 100 l SC with incomplete Freund s adjuvant. The mice received a final booster injection with 5 g of the same antigen in 200 l PBS intraperitoneally 3 days prior to hybridoma fusion. Mice were euthanized by anaesthesia overdose and exsanguinated by cardiac puncture. The spleens were excised subsequently under aseptic conditions Generation and isotyping of mabs Removal of mouse spleens, preparation of spleen and myeloma cells, and the fusion for hybridoma production were performed according to National Science Centre for Human and Animal Health (NCFAD) standard operating procedures under ISO17025 and as described previously (Berry et al., 2004). Isotyping was performed using a commercial murine isotyping dipstick test (Roche, Basel, SW) according to the manufacturer s instructions. Roller bottles (Coring Inc., Coring, NY) were seeded at equal to or greater than viable hybridoma cells per ml and allowed to grow for 7 days. Culture supernatants were concentrated 5 10-fold using Amicon stirred cell nitrogen concentrators with 30 kda cut-off Millipore (YM-30) membranes (both from Millipore, Billerica, MA) Enzyme linked immunosorbent assay (ELISA) Hybridoma culture supernatants were screened for binding to BEI inactivated Nipah virus antigen from the lower band in an ELISA assay. The Costar well 1/2 well ELISA plates (Corning, NY) were coated with either bovine serum albumin (BSA) or Nipah virus nucleoprotein enriched antigen (100 ng/well) in PBS overnight at 4 C and then blocked with 0.4% BSA in PBS, for 2 h at 37 C. The hybridoma supernatants (60 l/well) were incubated neat for 1 h at 37 C. The ELISA plates were washed eight times using an auto-washer (Tecan) with distilled water and patted dry on a paper towel. A pan-goat anti-mouse IgG-HRP antibody (Southern Biotechnology Associates, Birmingham, Alabama) was diluted to 1:2000 in 0.2% BSA in PBS, applied to the ELISA plates for 45 min at 37 C, and then washed as described above. Positive binding was detected with commercial ABTS (2,2 -azino-bis(3-ethylbenzothiazoline- 6-sulphonic acid) ELISA peroxidase substrate used according to the manufacturer s instructions (Roche, Basel, SW). The OD 405 nm was recorded at 15 and 60 min intervals after addition of the developing reagent. Mouse immune and preimmune sera were diluted 1:2000 with 2%-BSA in PBS for use as positive and negative controls, respectively, and for the establishment of the hybridoma screening assay Western immunoblot with BEI inactivated Nipah virus BEI inactivated, nucleoprotein enriched fraction of Nipah virus antigen from the lower band at a final total protein concentration of 1 g per lane were boiled in 2 SDS-loading buffers with 5% betamercaptoethanol for 10 min. Concentrated Hendra virus was also boiled in 2 SDS-loading buffer for 10 min. The samples were loaded in Criterion pre-cast gels (BioRad, Mississauga, ON) and electrophoresed at 200 V for 30 min. The proteins were transferred to Immobilon nylon membranes (Millipore, Billerica, MA) for 2 h at room temperature at 100 V, or overnight at 40 V at 4 C. Blots were blocked with 5% skim milk in TBS, rinsed three times with TBS, and reacted with mab and polyclonal guinea pig sera (Weingartl et al., 2005) overnight at 4 C. The antibody supernatants were reacted neat and the concentrated supernatants were diluted 1:50 in blocking buffer.

4 62 Y. Berhane et al. / Journal of Virological Methods 132 (2006) Blots were washed three times with TBS-Tween-20 (0.05%) for 5 min before being incubated with secondary antibody (same as above) at 1:1000 in blocking buffer for 1 h. The blots were washed three times, 5 min each and were developed using DAB insoluble substrate (Pierce, Rockford, IL) Western immunoblot using recombinant proteins Open reading frames for NiV-N, M, P, G and F were cloned into eukaryotic expression vectors pbk-cmv (cytomegalovirus) Phagemid Vector (Strategene, San Diego, CA). Recombinat plasmids were provided by Dr M. Czub, (Public Health Agency of Canada, National Microbiology laboratory, Winnipeg, MB). To enhance expression of NiV-N, the internal ribosome entry sites (IRES) gene was also cloned infront of the Nipah N gene. For recombinant protein production, 293T cells grown in 6- well plates were transfected with 4 g of Nipah N-IRES-CMV, Nipah M-CMV, Nipah P-CMV, Nipah G-CMV and Nipah F- CMV plasmids using lipofectamine (Invitrogen, Grand Island, NY). Cells were harvested in 1 ml of 2 SDS gel loading buffer 24 h after transfection. Twenty microliters of each cell lysate sample were loaded in Criterion pre-cast gels (BioRad, Mississauga, ON) and electrophoresed at 200 V for 30 min. Gel transfer and Western immunoblot procedures were the same as above Plaque reduction neutralization test (PRNT) Virus neutralization assay was carried out in 24-well cell culture plates. Nipah virus was diluted in DMEM to obtain 300 PFU/ml. Three hundred microliters of the virus dilutions and 300 l of two-fold dilution of mabs supernatants/well were added to 24-well plate. Nipah polyclonal guinea pig serum was added to positive control and DMEM alone to negative controls wells. The plates were incubated for 1 h at 37 C. Following incubation, the mixtures were added in duplicate to Vero-76 cells grown in the 24-well plates, and allowed to adsorb for 1 h at 37 C. After an hour of incubation, the inoculum was replaced with 2 ml of 2% carboxymethylcellulose, sodium salt, medium viscosity/dmem (Sigma Chemical, St. Louis, MO)/2% FBS overlay, and incubated at 37 C in the presence of 5% CO 2. After 5 days of incubation, the cells were fixed with 4% formaldehyde, stained with 0.5% of crystal violet/80% methanol/pbs and plaques were counted Immunfluorescence For the immunofluorescence assay, Vero-76 and BHK-21 cells were seeded on microscope slides (Fisher Biotech, Fisher Scientific, USA) that were set in Petri dishes. The cells were grown to 70% confluence and were infected either with Hendra or Nipah virus at 0.1 moi. After 24 h, cell monolayers were immersed in 3.7% paraformaldehyde solution (with 0.6% Triton-X. Fisher Biotech, Fisher Scientific, USA) and were incubated at 37 C for 30 min. Then, the cells were immersed in blocking buffer (1% bovine serum albumin/bsa/in PBS) for 30 min at 37 C to minimize non-specific absorption of the antibodies. After blocking, the cells were incubated with mabs against Nipah virus N (F45G1, F45G2, F45G3 and F45G4, F45G6) and M (F45G5) for 1 h at room temperature. After washing with PBS three times for 10 min each, cells were incubated with goat anti-mouse immunoglobulin G conjugated with Alexa Fluor 488 or 594 (Molecular Probes, Eugene, OR, USA). Finally, the microscope slides were washed with PBS three times and were mounted with antifade reagent in glycerol buffer (Molecular Probes, Eugene, OR, USA). The fluorescence was visualized using an Olympus FluoView laser scanning confocal microscope (Olympus, Melville, NY) Immunohistochemistry Tissues collected from pigs experimentally infected with Nipah virus were fixed in 10% formalin for minimum of 7 days as a safety precaution. Formalin fixed tissues were trimmed and embedded in paraffin, sectioned and mounted on microscopic slides. Preparation and staining of tissues for immunohistochemistry was conducted using the protocols described by Weingartl et al. (2005). Control samples included Nipahinfected tissues incubated with secondary antibody only, tissues from non-infected control pigs and tissues from classical swine fever infected pigs. 3. Results 3.1. Inactivation, concentration and purification of Nipah virus Complete BEI inactivation of Nipah virus with initial stock virus titer of TCID 50 /ml was achieved within 6 h based on virus titration assay from samples taken at different time points. During safety testing CPE was not observed in PT-K75 cells that were propagated with undiluted aliquots of BEI inactivated virus from the final time point (24 h). The inactivated virus was concentrated and partially purified in BSL-2 using sucrose gradient centrifugation. Two distinct bands were observed following the gradient centrifugation. The bands were collected separately and examined by TEM and Western immunoblot. The lower band revealed mainly herring bone structures that are characteristic of nucleocapsid proteins (Fig. 1a) of paramyxoviruses. The upper sucrose fraction mainly contained intact and partially disrupted Nipah virus particles (Fig. 1b). To confirm the protein profile of the immunogen before immunizing the mice, fractions from upper and lower bands of the sucrose gradient were analysed by Western immunoblot using guinea pig polyclonal sera as antibody. The lower fraction contained two bands: N protein at 53.2 kda, and M protein at 35.2 kda (Fig. 3). The upper band contained most Nipah virus proteins. The lower fraction was used for immunizing mice and as antigen for screening of immune serum from immunized mice as well as hybridoma supernatant.

5 Y. Berhane et al. / Journal of Virological Methods 132 (2006) Fig. 2. Enzyme linked immunosorbent assay showing IgG reactivity of mabs in hybridoma culture supernatants on BEI inactivated Nipah virus (black bars) and bovine serum albumin control (white bars). Supernatant was tested at a 1/8 dilution and polyclonal serum (from guinea pigs inoculated with Nipah virus) at 1/500 dilution in PBS. Shown is the serum IgG levels as an Fc gamma specific secondary antibody is used for all assays used to initially identify binding of clones in this study. The OD 405 nm was recorded at 1 h following the addition of substrate. Positive binding of clones were chosen as being at least five-fold above background BSA reactivity. These data are representative of one of three identical assays. The positive clones were then screened differentially for binding to a control antigen (bovine serum albumin) (Fig. 2) and from this secondary screen, six clones were identified. The clones were named F45G1, F45G2, F45G3, F45G4, F45G5 and F45G6 and their specificity and isotype are listed in Table Western immunoblot Fig. 1. (a) Electron micrograph shows presence of tubular structures with a herringbone morphology characteristic of paramyxovirus nucleocapsid structures from the lower band of the sucrose gradient. These structures are frequently observed as a result of disruption of particles followed by subsequent release of nucleocapsid structures. (b) Electron micrograph of partially disrupted Nipah virus particle with herringbone like structures in its core from the upper band of the sucrose gradient Hybridoma development and ELISA Test bleeds from immune mice showed a high titre of Nipah virus specific antibodies by indirect ELISA compared to serum from control mice (Fig. 2). A total of 708 colonies were picked and screened by ELISA, producing the first set of positive clones. Western immunoblot was carried out to identify the protein specificity of mabs to Nipah virus. Specific reactions were detected with all six mabs. Monoclonal antibodies F45G1, F45G2, F45G3, F45G4 and F45G6 reacted with Nipah N at 53.2 kda, and F45G5 reacts with M protein at 35.2 kda (Fig. 3). Reactivity of the antibodies with Nipah N protein was confirmed for F45G2, F45G3, F45G6 and F45G4 (Fig. 4a), and for M protein with F45G5 (Fig. 4b) using recombinant Nipah protein expressed transiently in 293T cells. F45G1 was not tested further as it reacted weakly with N protein on Western immunoblot compared to the other mabs. None of the mabs reacted with other recombinant Nipah proteins (P, G and F) or negative control cell lysate. The mabs were also tested for cross-reactivity with Hendra virus proteins by Western immunoblot. Three of the anti-nipah mabs specific for the N (F45G2, F45G3, F45G6) reacted positively with Hendra N protein (Fig. 5). Two of the anti-nipah virus mabs (F45G4, anti-n; F45G5, anti-m) did not react with Hendra virus proteins based on the results of Western immunoblot Immunofluorescence Monoclonal antibodies specific to the Nipah N and M proteins were tested for their ability to recognize Nipah virus epitopes on BHK-21 and Vero-76 cells infected with Nipah virus using immunofluorescence. All of the mabs that reacted with Nipah

6 64 Y. Berhane et al. / Journal of Virological Methods 132 (2006) Table 1 Characterization of mabs to Nipah virus mabs Isotype Target protein Neutral antibody Western blot Immunofluorescence Hendra Nipah Hendra Nipah F45G2 IgG1/k N F45G3 IgG1/k N F45G4 IgG1/k N + + F45G5 IgG2a/k M + + F45G6 IgG2a/k N N and M proteins in Western immunoblot assay gave specific positive fluorescence with Nipah virus infected but not uninfected cells (Fig. 6d). Using mabs that react with N protein, an intense and specific fluorescence was observed mainly in the perinuclear regions as well as in the cytoplasm of Nipah virus infected cells (Fig. 6a) in the form of focal aggregates. Anti-Nipah M mabs also reacted with cells infected with Nipah virus and permeabilized (0.6% of Triton X 100). According to our immunofluorescence assay data, the M protein was present at the cytoplasmic side of the plasma membrane diffusely and in the form of tiny focal aggregates (Fig. 6b). The pattern of fluorescence for both M and N proteins was similar in both BHK-21 and Vero-76 cells. The mabs were also tested for their ability to cross-react with Hendra virus infected Vero-76 and BHK-21 cells. Nipah N mabs (F45G2 and F45G6) cross-reacted with Hendra virus infected cells and the pattern of fluorescence was similar to Nipah virus infected cells (Fig. 6c). However, mabs F45G4 (Nipah N) and F45G5 (Nipah M) did not cross-react with Hendra virus infected cells as assayed by immunofluorescence. F45G3 was also reactive with Hendra virus infected cells, but the fluorescence was not as intensive as for F45G2 and F45G Plaque reduction neutralization test and immunohistochemistry A plaque reduction neutralization assay with Vero cells was performed to determine which mabs possess neutralizing properties. Under the conditions performed in this study, none of the mabs neutralized Nipah virus. A study was also undertaken to determine if the mabs could be used for routine immunohistochemical diagnosis using formalin fixed swine tissues. Results from the immunohistochemistry test demonstrated (Fig. 7a) that one N protein specific mab (F45G2) gave a strong positive reaction with a variety of Nipah virus infected swine tissues following long term formalin fixation. In addition, Nipah mabs F45G4 (N) and F45G5 (M) gave weak positive staining under the same conditions. 4. Discussion This study describes the development of murine mabs using nucleprotein enriched fraction of sucrose gradient from BEI inactivated Nipah virus as immunogen. These mabs recognize Nipah virus antigen in ELISA, immunofluorescence assay, Western immunoblot and immunohistochemistry. Target identification of the mabs in Western immunoblot with BEI inactivated Nipah virus and recombinant Nipah virus proteins collectively indicated that five mabs reacted with the N protein and one with M protein. Electron microscopy analysis of the immunogen used revealed mainly intact and partially disrupted herringbone structures of Nipah virus and Western immunoblot analysis showed presence of mainly N and M proteins. Upon immunizing mice only anti-nipah N and M mabs were developed and no mabs were developed to Nipah fusion and attachment glycoproteins. Neither the N nor the M mabs possess neutralizing effect on Nipah virus infectivity in vitro and all the mabs tested reacted by immunofluorescence assay with Vero-76 and BHK-21 cells infected with Nipah virus. Fig. 3. Western blot analysis of anti Nipah mabs using BEI inactivated Nipah virus as antigen. 1: prestained kaleidoscope marker and molecular weights are indicated in the middle; 2: BEI inactivated and Nipah virus nucleoprotein enriched fraction of the sucrose gradient (lower band) without betamercaptoethanol; 3: BEI inactivated and Nipah virus nucleoprotein enriched fraction of the sucrose gradient (lower band) with betamercaptoethanol; 4: control negative control hybridoma supernatant.

7 Y. Berhane et al. / Journal of Virological Methods 132 (2006) Fig. 4. (a) Specificity of anti Nipah N mab (F45G4) towards recombinant Nipah virus proteins as shown by Western immunoblot. Lane 1: prestained kaleidoscope marker (molecular weights are indicated); Lane 2: Nipah P; Lane 3: Nipah G; Lane 4: Nipah N; Lane 5: Nipah F; Lane 6: Nipah M; Lanes 7, 8 and 9: mock transfected 293T cell lysates. Lane 10: Nipah virus. (b) Specificity of Nipah M mab (F45G5) towards recombinant Nipah virus proteins in Western immunoblot. Lane 1: prestained kaleidoscope marker (molecular weights are indicated); Lane 2: Nipah P; Lane 3: Nipah G; Lane 4: Nipah N; Lane 5: Nipah F; Lane 6: Nipah M; Lanes 7, 8 and 9: mock transfected 293T cell lysates; Lane 10: Nipah virus. Furthermore, three mabs to Nipah N protein reacted with cells infected with Hendra virus with variable intensity in immunofluorescence as well as Western immunoblot assays. These findings are consistent with previously published reports that demonstrated the ability of polyclonal sera and mabs to Hendra and Nipah viruses to cross-react/neutralise to a limited degree (Goh et al., 2000; Imada et al., 2004). Genetically, there is also a high degree of homology between the coding sequences of the N protein of Nipah and Hendra which is 78.4% (Harcourt et al., 2000). Hence, this close serological relationship between them ensures that immunologic assays such as ELISA using Hendra or Nipah virus antigens can be used to detect antibodies to both viruses. Some of the anti-nipah N protein mabs such as F45G2, F45G3 and F45G6 could also be used for the laboratory diagnosis and in studying the pathogenesis of Hendra virus.the immunohistochemical study demonstrated that not all the mabs recognize long-term formalin fixed swine tissues as compared to results from immunofluorecence assay studies using Nipah infected cells after short term formalin fixation. Only one N protein mabs (F45G2) and to a lesser extent anti M protein (F45G5) and anti N protein (F45G4) mabs were able to react with Nipah virus infected porcine tissues. This could be as a result of deleterious effects on the epitopes recognized in immunohistochemistry by several of these mabs due to the long-term formalin fixation as has been seen previously described with porcine reproductive and respiratory syndrome (PRRS) viral antigen (Van Alstine et al., 2002). Protracted delays in the shipping or processing of formalin fixed tissues could thus reduce or prevent the detection of viral antigens via immunohistochemistry. This study demonstrates that BEI inactivated Nipah virus can be successfully used as an immunogen for mab development. BEI has also been widely used to inactivate a number of viruses for vaccine and mab production (Hulskotte et al., 1997; Bahnemann, 1975 and Buonavoglia et al., 1988). Indeed, BEI was shown to be nearly twice as efficient as formalin for the inac- Fig. 5. Cross reactivity of Nipah N mabs with Hendra virus in Western blot. Lane 1: prestained precision ladder (BioRAD) and molecular weights are indicated in the middle; Lane 2: BEI inactivated and Nipah virus nucleoprotein enriched fraction of the sucrose gradient (lower band); Lane 3: Hendra virus; Lane 4: uninfected control cell lysate.

8 66 Y. Berhane et al. / Journal of Virological Methods 132 (2006) Fig. 6. Immunofluorescence of Vero-76 cells infected with Nipah virus using mabs F45G4 (a) recognizing Nipah N protein and using mab F45G5 (b) recognizing Nipah M protein. (c) Demonstrates cross-reactivity of the mab F45G2 with Hendra virus N protein in Vero-76 cells infected with Hendra virus. (d) Uninfected control cells using mab F45G4, as an example. The green fluorescence in (b) and (d) is from cells costained with human autoimmune serum specific for fibrillarin provided by Marvin Fritzler (University of Calgary, Calgary, Alberta, Canada), followed by staining with goat anti-human antibody conjugated with FITC. tivation of paramyxoviruses such as Newcastle disease virus. It also preserves conformation and accessibility of the epitopes compared to formalin and -propriolactone (Buonavoglia et al., 1988; Blackburn and Besselaar, 1991). Currently, confirmation of a Nipah virus infection is provided only after isolation of the virus from clinical specimens, a confirmed positive RT-PCR or detection of antibody seroconversion. Virus isolation is time-consuming, and RT-PCR requires technical equipment, which is not available in every laboratory. Clearly a quality mab based immunoassay would be faster and cheaper laboratory test. Experiments with Nipah virus infected pigs demonstrate that antibodies to N protein are produced early during experimental infection as assayed by Western immunoblot using BEI inactivated Nipah virus as an antigen (manuscript

9 Y. Berhane et al. / Journal of Virological Methods 132 (2006) The authors are grateful to Mrs Lynn Burton for her expertise on transmission electron microscopy. The authors also would like to thank Greg Smith and Jason Gren for their laboratory support, Dr Stefanie Czub, Ms Lisa Manning and Ms Shelley Ganske for their expertise on the Nipah mabs immunohistochemistry. This research was supported by CFIA TD funds and the CBRN Research and Technology Initiative (CRTI) under projects 0196 (HW) and 0091 (JDB). References Fig. 7. Immunohistochemistry of submandibular lymph node from pigs experimentally infected with Nipah virus using F45G2 mabs (a) and using Nipah polyclonal serum developed in guinea pigs (b). The antigen is localized mainly in the vascular/lymphatic endothelial cells with few positively stained dendritic cells. in preparation). Therefore, the mabs to N protein may allow the development of highly sensitive and specific Nipah virus antigen-capture ELISAs for the rapid identification of Nipah virus or could also be used for the development of competitive ELISA that will enable detection of antibodies in diseased animals. Acknowledgements Bahnemann, H.G., Binary ethylenimine as an inactivant for foot-andmouth disease virus and its application for vaccine production. Arch. Virol. 47, Bahnemann, H.G., Inactivation of viruses in serum with binary ethyleneimine. J. Clin. Microbiol. 3, Bahnemann, H.G., Inactivation of viral antigens for vaccine preparation with particular reference to the application of binary ethylenimine. Vaccine 8, Barclay, A.J., Paton, D.J., Hendra (equine morbillivirus). Vet. J. 160, Berry, J.D., Jones, S., Drebot, M.A., Andonov, A., Sabara, M., Yuan, X.Y., Weingartl, H., Fernando, L., Marszal, P., Gren, J., Nicolas, B., Andonova, M., Ranada, F., Gubbins, M.J., Ball, T.B., Kitching, P., Li, Y., Kabani, A., Plummer, F., Development and characterisation of neutralising monoclonal antibody to the SARS-coronavirus. J. Virol. Methods 120, Blackburn, N.K., Besselaar, T.G., A study of the effect of chemical inactivants on the epitopes of Rift Valley fever virus glycoproteins using monoclonal antibodies. J. Virol. Methods 33, Buonavoglia, C., Fioretti, A., Tollis, M., Menna, F., Papparella, V., A preliminary vaccine potency trial of a Newcastle disease virus inactivated with binary ethylenimine. Vet. Res. Commun. 12, Chua, K.B., Goh, K.J., Wong, K.T., Kamarulzaman, A., Tan, P.S., Ksiazek, T.G., Zaki, S.R., Paul, G., Lam, S.K., Tan, C.T., Fatal encephalitis due to Nipah virus among pig-farmers in Malaysia. Lancet 354, Chua, K.B., Bellini, W.J., Rota, P.A., Harcourt, B.H., Tamin, A., Lam, S.K., Ksiazek, T.G., Rollin, P.E., Zaki, S.R., Shieh, W., Goldsmith, C.S., Gubler, D.J., Roehrig, J.T., Eaton, B., Gould, A.R., Olson, J., Field, H., Daniels, P., Lin, A.E., Peter, C.J., Anderso, L.J., Mahy, B.W., Nipah virus: a recently emergent deadly Paramyxovirus. Science 288, Chua, K.B., Nipah virus outbreak in Malaysia. J. Clin. Virol. 26, Daniels, P., Ksiazek, T., Eaton, B.T., Laboratory diagnosis of Nipah and Hendra virus infections. Microbes Infect. 3, Goh, K.J., Tan, C.T., Chew, N.K., Tan, P.S.K., Kamarulzaman, A., Sarji, S.A., Wong, K.T., Abdullah, B.J., Chua, K.B., Lam, S.K., Clinical features of Nipah virus encephalitis among pig farmers in Malaysia. N. Engl. J. Med. 342, Harcourt, B.H., Tamin, A., Ksiazek, T.G., Rollin, P.E., Anderson, L.J., Bellini, W.J., Rota, P.A., Molecular characterization of Nipah virus, newly emergent paramyxovirus. Virology 271, Hooper, P.T., Ketterer, P.J., Hyatt, A.D., Russell, G.M., 1997a. Lesions of experimental equine morbillivirus pneumonia in horses. Vet. Pathol. 34, Hooper, P.T., Westbury, H.A., Russell, G.M., 1997b. The lesions of experimental equine morbillivirus disease in cats and guinea pigs. Vet. Pathol. 34, Hooper, P., Zaki, S., Daniels, P., Middleton, D., Comparative pathology of the diseases caused by Hendra and Nipah viruses. Microbes Infect. 3, Hulskotte, E.G., Dings, M.E., Norley, S.G., Osterhaus, A.D., Chemical inactivation of recombinant vaccinia viruses and the effects on antigenicity and immunogenicity of recombinant simian immunodeficiency virus envelope glycoproteins. Vaccine 15, Imada, T., Abdul Rahman, M.A., Kashiwazaki, Y., Tanimura, N., Syed Hassan, S.S., Jamaluddin, A., Production and characterization of monoclonal antibodies against formalin inactivated Nipah virus isolated from the lung of pigs. J. Vet. Med. Sci. 66, Kyvsgaard, N.C., Hoier, R., Bruck, I., Nansen, P., Effect of two virus inactivation methods: electron beam irradiation and binary ethylenimine

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