Virus stocks were prepared by infecting vero cells at low m.o.i RVFV antigen preparation for immunization

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1 Journal of Virological Methods 131 (2006) Production of monoclonal antibodies against Rift Valley fever virus Application for rapid diagnosis tests (virus detection and ELISA) in human sera A. Zaki a, D. Coudrier b, A.I. Yousef a, M. Fakeeh a, M. Bouloy b, A. Billecocq b, a Virology Laboratory, Dr. Fakeeh Hospital, P.O. Box 2537, Jeddah 21461, Saudi Arabia b Unité degénétique moléculaire des Bunyavirides, Institut Pasteur, Paris Cedex 15, France Received 1 April 2005; received in revised form 29 June 2005; accepted 11 July 2005 Available online 15 August 2005 Abstract This paper describes the production and characterization of RVFV monoclonal antibodies. The characteristics of 32 out of 55 ELISA and/or IFA positive monoclonal antibodies were determined, including the RVFV components against which they are directed. One monoclonal antibody recognized the nucleoprotein, 15 the Gc and 16 the Gn. Among the latter ones, five monoclonal antibodies possess another specificity and recognized both Gn and either the nucleoprotein (four of them) or the NSs protein (one). To validate the use of these monoclonal antibodies for diagnosis tests, a pool of monoclonal antibodies reacting with the structural proteins was prepared and used successfully to detect RVFV from cell culture as well as viral antigen antibody complex in ELISA Elsevier B.V. All rights reserved. Keywords: Plebovirus; Bunyaviridae; Hybridoma; Immunofluorescence; Serological tests 1. Introduction Rift Valley fever (RVF) is an emerging epidemic disease of humans and livestock which is caused by Rift Valley fever virus (RVFV). This virus belongs to the family Bunyaviridae, and the plebovirus genus (Elliott, 1996). The virus is transmitted to livestock and humans by bites of infected mosquitoes or exposure to tissues or blood of infected animals. Massive epizootics are typically observed in livestock during times of unusually high and sustained rainfall because of the presence of breeding sites and overabundance of adult competent mosquito vectors (Linthicum et al., 1999). Infections caused by RVFV are characterized by severe disease and abortion in livestock, particularly sheep and cattle. In the epidemic region, populations are at high risk for RVFV infection, potentially leading to thousands of human cases. Humans infected with RVFV have self-limited febrile illness, Corresponding author. Tel.: ; fax: address: abilleco@pasteur.fr (A. Billecocq). but retinal degeneration (5 10%), hemorrhagic fever (<1%), or encephalitis (<1%) may also develop (Meegan and Bailey, 1989; Laughlin et al., 1979). The virus used to be restricted to sub-saharan Africa, but in 1977 it emerged for the first time in Egypt causing severe epizootics and epidemics with increased morbidity and mortality (Meegan, 1979; Laughlin et al., 1979). In 2000, the virus spread beyond Africa causing severe epidemics in humans in Saudi Arabia and Yemen (CDC, 2000a, 2000b). RVFV has a trisegmented RNA genome of negative (L and M segments) or ambisense (S segment) polarity. The L and M segments code, respectively, for the RNA-dependent RNA polymerase and for a polypeptide precursor which is cleaved during translation, thus generating the envelope glycoproteins Gn, Gc and the nonstructural proteins 14K and 78K. The S segment utilizes an ambisense strategy: the 5 halves of the antigenomic and genomic strands code, respectively, for the nucleoprotein N and the nonstructural protein NSs (Giorgi, 1996; Schmaljohn, 1996). Diagnosis of RVF can be achieved by different methods, namely virus isolation, RT-PCR or detection of viral /$ see front matter 2005 Elsevier B.V. All rights reserved. doi: /j.jviromet

2 A. Zaki et al. / Journal of Virological Methods 131 (2006) antigens. Virus isolation is considered as the gold standard but it requires biosafety type three laboratory. RT-PCR is highly sensitive and reproducible (Garcia et al., 2001; Sall et al., 1999, 2002; Drosten et al., 2002). However, it is still a research tool which is not available in every laboratory. The viral antigens can also be detected in patient blood during viremia, but the assays may lack sensitivity. In parallel with virological methods which detect the virus or its components, IgM capture and IgG sandwich ELISAs (Niklasson et al., 1984; Martin et al., 2000; Johnson et al., 2000; Paweska et al., 2005), are widely utilized in clinical laboratories. These techniques are rapid, sensitive, specific and useful to reveal infected animal in endemic areas or during an epizootic (Paweska et al., 2003a, 2003b). However, they require RVFV specific antigens and antibodies to monitor the presence of antibody-antigen complexes. RVFV specific polyclonal antibodies are usually produced in mice but the antibody titer varies with each batch. On the other hand, the production of monoclonal antibodies specific for RVFV insures a constant source. These antibodies are revealed with anti-mouse IgG conjugated to peroxidase or are themselves conjugated to this enzyme. Not only, the RVFV monoclonal antibodies are necessary for serology but also they can be helpful for a rapid identification of a RVFV infected cell culture by indirect immunofluorescent assay (IFA). This paper describes the production of monoclonal antibodies that can be used to identify RVFV in cell cultures or to detect RVFV antibodies by IgM or IgG capture ELISA using a monoclonal antibody-based capture ELISA was reported. These monoclonal antibodies were pooled and, for IgM capture ELISA, they were conjugated with peroxidase. 2. Materials and methods 2.1. Cells C6/36 cells were grown in Eagle minimal essential medium supplemented with 10% foetal calf serum (FCS), 10% tryptose phosphate; BHK-21 in Glasgow minimal essential medium (MEM) supplemented with 5% FCS, 10% tryptose phosphate and 10 mm HEPES; BSR cells (a clone of BHK-21cells) in Glasgow MEM supplemented with 10% FCS; and Vero cells in Dulbecco modified Eagle medium supplemented with 5% FCS. Penicillin (5 U/ml) and streptomycin (5 g/ml) were added. The cells were incubated at 37 Cina5%CO 2 atmosphere except for C6/36 which were cultured at 28 C in closed flasks Virus The virus Gizan 02 strain was isolated in Saudi Arabia during the 2000 epidemic and passaged in suckling mice twice. The Kenyan strain (Ken97) was kindly provided by Swanepoel. The ZH548 strain was isolated from a human infection during the 1977 outbreak in Egypt. Virus stocks were prepared by infecting vero cells at low m.o.i RVFV antigen preparation for immunization Suckling mice were injected with the Gizan 02 strain by intracerebral route. When symptoms appeared, mice were killed and brains were harvested and homogenized as 10% (w/v) suspension in phosphate buffered saline which was formalin inactivated when used for mice immunization Animal immunization Groups of five BALB/c female mice of 8 weeks age were first inoculated subcutaneously with two doses of formalininactivated RVFV infected brain suspension at 15 days interval followed by three doses of infectious virus at 10-day interval. The first two injections were combined with Freund s complete adjuvant, while the rest of injections were combined with Freund s incomplete adjuvant. The mice were killed 4 days after the last injection, their sera analyzed for the presence of antibodies and their spleens were used for production of hybridoma Hybridoma production The splenocytes from antibody-positive mice were fused with SP2/0-Ag14 myeloma cells by using 50% (w/v) polyethylene glycol (molecular weight, ; Sigma Chemical Co.) according to the procedure described (Xu et al., 1997). The fused cells were grown in hybridoma selective medium (Iscove s modified Dulbeco medium; Sigma Chemicals Co.) containing 20% fetal bovine serum (Gibco BRL) and hypoxanthine aminopterin thymidine (HAT) selective medium (Sigma Chemical Co.). After 2 weeks, HAT was replaced by hypoxanthine thymidine (HT) medium (Sigma Chemical Co.). After 5 days of incubation, the supernatants from viable hybridoma clones were screened for antibodies against RVFV by IFA. Positive hybridoma cells were subcloned two to three times by limit dilution. Supernatants from positive hybridoma were further analyzed by ELISA, Western blotting and plaque reduction neutralization tests to determine their specificity Immunofluorescence assay IFA was used to screen hybridoma clones and determine the specificities of the monoclonal antibodies. RVFV infected Vero cells were mixed with non infected Vero cells and deposited on Teflon coated 10-well slides. The slides were air dried inside a biosafety cabinet and fixed in chilled acetone or acetone/methanol for 20 min. The assay was modified from a previously described procedure (Xu et al., 1997). Briefly, the wells were overlaid with 30 l of supernatants from hybridoma clones, and the plates were incubated in a moist chamber at 37 C for 30 min before they were

3 36 A. Zaki et al. / Journal of Virological Methods 131 (2006) washed three times in PBS. The bound antibody was detected with fluorescein isothiocyanate-conjugated goat anti-mouse immunoglobulin G (IgG) (Sigma Chemicals Co.) diluted 1:80 in PBS containing 3% nonfat dry milk and 0.2% Evans blue (Sigma Chemicals Co.). The slides were washed, mounted with Fluoprep (BioMerieux) and examined under a Leitz fluorescence microscope Purification of monoclonal antibodies and peroxidase conjugation Supernatants positive for RVFV antibodies were pooled and IgG monoclonal antibodies were separated using Protein A kit (Sigma Chemicals Co.), according to the manufacturer instructions. Purified IgGs from pooled monoclonals were conjugated to peroxidase using Peroxidase conjugation kit (Alpha Diagnostic International Co.), according to the manufacturer instructions Virus isolation Blood from 70 patients suspected of RVF was inoculated into Vero cells grown in 25 ml tissue culture flasks. Cells maintained with minimal essential Eagle medium containing 2% fetal bovine serum were observed daily for 7 days. When they exhibited CPE, they were harvested and spot slides made for identification of RVFV by IFA using the monoclonal antibody pool RT-PCR RT-PCR was carried out from patient blood and from cells inoculated with patient specimen (Ibrahim et al., 1997). RNA was extracted from blood and from infected cell culture using the Qiagen whole blood extraction kit (Qiagen) as described by the manufacturer. RT-PCR was done using the Qiagen one-step RT-PCR and specific primers in a 50 l volume. Nested PCR was performed using the Qiagen hot start PCR kit (Qiagen), using 3 l of the RT-PCR product and specifics primers. Amplified products were analyzed by agarose gel electrophoresis followed by ethidium bromide staining RVFV antigen for ELISAs Gizan 02 infected C6/36 cells were recovered at 6 days p.i. and cell extract treated by -propiolactone to inactivate infectious virus (Groen and Elliot, 1982). Kenyan or ZH548-infected Vero cells were recovered at 3 days p.i., the cell pellets were lysed in 10 volumes of borate buffer ph 9 containing 1% of Triton X100 and ultrasonicated. Control antigen was prepared similarly using non infected Vero cells IgM capture ELISA IgM antibody-capture ELISA (MAC-ELISA) was performed using 96-well plates (costar) coated with goat antihuman IgM chain specific (KPL), as described elsewhere (Martin et al., 2000). Peroxidase-labeled monoclonal pool was used for detection. Briefly, plates were coated overnight with 100 l of 1/500 dilution of goat anti-human IgM in carbonated buffer ph 9. Human sera from suspected cases were diluted 1/100 using phosphate-buffered saline with 0.5% Tween 20 and 5% FCS incubated at 37 C for 1 h, and unfixed material was eliminated by washing. Positive (Gizan O2 infected C6/36 cell extract) and control (C6/36 cell extract) antigens were added to adjacent wells, incubated for 1 h at 37 C and washed. The pool of peroxidase-conjugated monoclonal antibodies was added at a dilution 1/2000 (dilution determined from previous titration) and incubated at 37 C for 1 h. The plates were washed and the TMB substrate (Sigma Chemicals Co.) added for 10 min, followed by the stopping solution. The optical densities were measured at 450 nm (OD 450 ) corrected for the absorption due to the plastic of the wells, given by OD 620. Cut-off was calculated as the mean plus 3 standard deviations of optical densities from 10 negative samples IgG ELISA RVFV IgG antibodies detection by ELISA was performed as described (Billecocq et al., 2003), the plates being directly coated with the positive (Kenyan or ZH548 infected Vero cell extract) and control (Vero cell extract) antigens Recombinant antigen Recombinant RVFV proteins were expressed via SFV replicon. psfv-n and psfv-nss are reported in Billecocq et al. (2000). For the construction of psfv- Gn, the Gn coding region was amplified by RT-PCR using the primers (StartG: 5 -GAACGGATCCGAGAG- AAACCATGGCAGGGATTGCA-3 [BamH1 site is underlined] and 3G2: 5 -GGCCAGATCTCTATGCTGATGCATA- TGAGACAATC 3 [BglII site is underlined]) and introduced into psfv-1 in the BamHI site. For Gp expression, the full-length M segment was amplified by RT-PCR (primer sequences available on request) and cloned into psfv-1 at the SmaI site. The production of the recombinant suicide viruses was carried out in BHK-21 using recombinant psfv-1 and helper plasmid psfv-h2 according to Liljeström and Garoff (1991) (Berglund et al., 1983). Each antigen was produced in BSR cells (Billecocq et al., 2000, 2003) and at 24 h p.i., the rsfv-infected cells were resuspended in 10 volumes of lysis buffer (Tris 25 mm ph 7.4, NaCl 50 mm, EDTA 2 mm, NP40 0.6%) and the nuclei discarded by centrifugation. In the case of the nuclear NSs protein, whole cell extracts were prepared by lysing the cells in the lysis buffer containing 0.2% SDS. When cells were used for IFA, they were recovered using trypsine/edta, washed in PBS, deposited on Teflon coated 10-well slides and fixed as described above. The specificity of the recombinant antigens was checked in IFA and Western blotting using specific antibodies.

4 A. Zaki et al. / Journal of Virological Methods 131 (2006) Plaque reduction neutralisation test (PRNT) For PRNT, serial dilutions of the hybridoma culture supernatant were preincubated with a suspension of 50 pfu of RVFV ZH548 for 1 h at 37 C before incubation with Vero cells in 6-well plates. After 1 h incubation at 37 C, the inoculum was discarded and cells were incubated for 6 days under an overlay consisting of DMEM, 2% FCS, antibiotics and 1% of agarose (Indubiose, Biosepra) at 37 C. The lytic plaque were counted after staining with a solution of crystal violet (0.2% in 10% formaldehyde and 20% ethanol). The titer is the dilution giving 80% reduction of the number of plaques observed in the control (Pifat et al., 1988) Western blot analysis Proteins from rsfv-infected cell extracts were separated by electrophoresis in SDS 10% polyacrylamide and transferred onto an Hybond C extra membrane (Amersham). The immunological reaction was performed as described (Billecocq et al., 2003). Monoclonal antibodies were used at 1/100 dilution and peroxidase-labeled goat anti mouse (Sigma Chemicals Co.) at 1/5000. The reaction was revealed by chemiluminescence (Super Signal, Pierce). The expression of recombinant protein (Gn, Gc, N and NSs) was checked by Western blotting using an hyperimmune ascitic fluid anti RVFV (dilution 1/500) (Fig. 1). Fig. 1. Analysis by PAGE and Western blot of recombinant RVFV antigen expressed via SFV replicon using an hyperimmune ascite anti RVFV. 3. Results A total of 55 monoclones tested for their reactivity against RVFV were found positive by ELISA or/and IFA. The tests were performed using RVFV strains representative of two of the three phylogenic lineages: the Kenyan (KEN97) and the ZH548 strains for Eastern/Central Africa and Egyptian lineages, respectively. For most of the monoclonal antibodies, a similar response was observed with the two strains tested in parallel (Table 1). Moreover, they were specific since all of them were ELISA negative to closely related viruses in the plebovirus genus (Arumowot, Gabek, Toscana ISS, Sandfly fever Naples, Sandfly fever Sicilian, Corfou, Salehabad and Belterra) (data not shown). To characterize these monoclones, the viral proteins against which they were directed were determined. Thus the nucleoprotein N, the nonstructural NSs or the glycoprotein Gn as well as them full length glycoprotein precursor (Gp) which generates Gn and Gc and the nonstructural proteins 14K and 78K were expressed individually, via the SFV replicon. This system allows the expression of large amounts of recombinant proteins, making the test more sensitive. The reactivity of the monoclonal antibodies against cells expressing either Gn alone or the glycoprotein precursor should allow to distinguish monoclonal antibodies reacting with Gn from those recognizing Gc or the nonstructural proteins. Moreover, IFA and Western blot allowed to determine the characteristic of the epitopes recognized by the monoclonal antibodies: monoclonal antibodies positive by Western blot detect linear epitopes since the proteins are denatured and monoclonal antibodies positive by IFA, could recognize linear or conformational epitopes localized at the surface of the proteins. For 23 hybridoma cultures reacting by ELISA or IFA, neither ELISA or Western blot using the rsfv allowed to determine the protein against which they are directed. Thus, these monoclonal antibodies were not further studied. The specificity of the remaining 32 cloned hybridoma was determined (Table 1): 27 were monospecific and five supernatants displayed reactivities against two viral proteins, the glycoprotein Gn and the nucleoprotein (four monoclonal antibodies) or the NSs protein (one monoclonal antibody). Most probably, these five hybridoma cultures would require further subcloning. Among the 27 monospecific monoclonal antibodies, one was directed against the nucleoprotein but was faintly reactive in IFA and in ELISA and 26 monoclonal antibodies recognized one glycoprotein: 11 reacted with SFV-Gn and SFV-Gp infected cells and 15 were positive using SFV-Gp but not SFV-Gn infected cells, suggesting that these latter monoclonal antibodies reacted with Gc or with the nonstructural proteins 14K or 78K. Among the monoclonal antibodies reacting by IFA with SFV-Gp but not SFV-Gn, one monoclonal (SP1-P2 W18) recognized the glycoprotein Gc by Western blot and the others were directed against a conformational epitope. For the latter ones, the protein (Gc, 14K or 78K) against which they are directed could not be identified. So, they are referred as Gc/NSm (see Table 1). According to

5 38 A. Zaki et al. / Journal of Virological Methods 131 (2006) Table 1 Characterization of the RVFV monoclonal antibodies Hybridoma ELISA a IFA WB b PRNT c ZH548 Ken 97 ZH548 d Ken 97 d Targeted protein e Targeted protein e ZH548 Included in the pool reagent SP1-P2 W Gc/Nsm +Gc 160 SP1-P1 W Gc/Nsm 20 SP2-P2 W Gc/Nsm 80 SP1-P2 W Gc/Nsm 40 SP2-P1 W Gc/Nsm 40 SP2-P2 W Gc/Nsm 40 SP2-P2 W Gc/Nsm 40 SP1-P1 W Gc/Nsm 40 SP1-P2 W Gc/Nsm 20 SP2-P1 W Gc/Nsm 20 SP1-P1 W Gc/Nsm 40 SP2-P2 W Gc/Nsm 20 SP1-P2 W Gn +Gn 40 SP1-P1 W Gn 160 SP1-P2 W Gn 20 SP2-P1 W Gn 20 SP1-P1 W Gn 20 SP2-P2 W Gn +Gn, +N 0 SP1-P1 W Gn +Gn, +N 160 SP1-P1 W Gn, N +Gn 20 SP2-P2 W Gn, NSs +Gn 160 SP1-P1 W Gn, N 20 Not included SP2-P2 W Gc 0 SP2-P1 W Gc 0 SP1-P2 W Gn +Gn 0 SP1-P2 W Gn +Gn 0 SP1-P1 W Gn +Gn 0 SP1-P1 W Gn 0 SP2-P2 W Gn 0 SP1-P2 W Gn 0 SP1-P1 W Gn 0 SP2-P1 W N 0 a ELISA: ++, OD > 1000; +, OD: ;, negative. b WB = Western blotting: +, positive;, negative. c PRNT = Plaque reduction neutralization test: reciprocal of monoclonal antibody dilution giving 80% virus neutralization. d ++ and +, indicates relative fluorescence intensity;, negative. e Recombinant viral protein(s) expressed via SFV replicon targeted by the specific monoclonal antibody. another report (Saluzzo et al., 1989), the nonstructural protein 14K and 78K is a poor inducer, suggesting that these monoclonal antibodies are likely directed against Gc rather than against 14K or the 78K protein. Finally, the neutralizing ability of the monoclonal antibodies was tested by plaque reduction assay: 21 appeared to have neutralizing activities among which some were highly reactive by ELISA and IFA. To validate the use of these monoclonal antibodies for diagnosis, two types of tests were performed, virus isolation in tissue culture and IgM detection by ELISA, using sera from patients suspected of RVF disease. Because of the epitope variability among RVFV strains, 22 different monoclonal antibodies were pooled, of which 21 possessed neutralizing activity and one did not neutralize but was highly responsive in ELISA. The 22 monoclonal antibodies comprised those which displayed two specificities, allowing the detection of the N, Gn and Gc proteins. The characteristics of the selected monoclonal antibodies is indicated in Table 1. Furthermore, to determine the RVFV positive samples by a method different from virus isolation, RT-PCR in parallel experiments were carried out. Out of 70 blood samples analyzed, 21 were positive by RT-PCR and for infectious virus when inoculated to Vero cells for virus isolation and assayed by IFA using the pool of monoclonal antibodies. In addition, none of the RT-PCR negative sera reacted positively by IFA in cell culture when tested for virus isolation. The second type of test involving the utilization of monoclonal antibodies was the IgM capture ELISA. It usually consists in a five-step procedure: coating anti chain on the plate, incubating with the patient serum, followed by the antigen and detecting the IgMantigen complexes by antigen specific antibodies revealed by

6 A. Zaki et al. / Journal of Virological Methods 131 (2006) Table 2 IgM capture ELISA results, showing P/N ratio, OD readings, and final IgM titer Serum number P/N ratio OD reading IgM titer peroxidase-labeled secondary antibodies. The conjugation of peroxidase directly to the monoclonal antibody pool, saves one step and thus 1 h in the procedure and no secondary antibodies are necessary (Martin et al., 2000). To validate the use of peroxidase-labeled monoclonal antibodies, the 70 serum samples screened for RT-PCR were evaluated by this fourstep IgM ELISA in comparison with the classical five-step ELISA. The results obtained with both methods were similar: IgMs were detected in 11 sera of the 70 cases tested. The IgM titers, the intensity of the reaction (OD at serum dilution of 1/100) as well as the ratio between ODs obtained with the sample and negative control (P/N ratio) are given in Table 2 showing the specificity and sensibility of the pool of peroxidase conjugated monoclonal antibodies. These monoclonals could also be used successfully in sandwich ELISA for IgGs (data not shown). In conclusion, the monoclonal antibody pool gave a specific response and represents a sensitive reagent in the immunological tests used to detect RVFV infectious virus in cells or RVFV antibodies in IgM capture and IgG sandwich ELISA. 4. Discussion During an outbreak, one of the most effective way of prophylaxis against RVFV infection is mosquito control. However, implementation of this measure requires an efficient and reliable diagnosis. For the diagnosis of RVFV infections, virus isolation or reverse transcriptase polymerase reaction (RT-PCR) from patient s blood are the currently used methods. These methods require well equipped virological laboratories, and although very sensitive and specific, they have limitations because of the short duration of viremia. In addition, manipulation of RVFV generates biohazards. The second group of diagnostic tests are the serologic techniques for the detection of IgM, IgG or neutralizing RVFV specific antibodies. Usually IgM can be detected after 5 8 days of symptoms. These serologic techniques, unlike virus isolation can be used in any clinical laboratory with minimal hazards for the workers and the environment, except for the detection of neutralizing RVFV specific antibodies. Both virus isolation and serologic techniques require immune reagents, like RVFV specific immune sera or monoclonal antibodies. Monoclonal antibodies compared to immune sera have many advantages including the unlimited and easy production by the hybridomas. This paper reports the production of RVFV monoclonal antibodies and their characterization. The monoclonal antibodies were evaluated first using IFA and ELISA with the Egyptian and Kenyan RVFV strains. Fifty-five monoclonal antibodies were found positive with both strains but negative when tested by ELISA with several closely related phleboviruses. Using recombinant antigen in ELISA and Western blotting, it has been possible to determine the specificities of 32 hybridoma among which 5 possessed a double activity and should be subcloned further. Five monoclonal antibodies are directed against the nucleoprotein (13%), 15 against the Gc/NSm (41%), 16 against the Gn (43%) and only 1 against the NSs (3%). Of note, a high percentage of the monoclonal antibodies directed against Gn or Gc possesses neutralizing activities. Since the nucleoprotein is considered as the major antigen, we were surprised by the low number of clones directed against this protein. Production of RVFV specific monoclones has been reported by Saluzzo et al. (1989) and a high number of anti nucleoprotein antibodies (31%) has been found, although the majority of the monoclones were directed against the Gn (25%) or the Gc (42%) and few against the NSs (3%). However, the immunization protocole was different since they utilized a live mouse-attenuated strain (Saluzzo et al., 1989). Another study addressing topological mapping of the two glycoproteins did not indicate if monoclonal antibodies directed against the nucleoprotein or the NSs were also selected (Besselaar and Blackburn, 1991). In these studies, neutralizing epitopes have been observed in Gn as well as Gc (Keegan and Collett, 1986; Besselaar and Blackburn, 1991). Similar results were obtained in this study. As a validation procedure these monoclonal antibodies were used to detect RVFV infected cells by IFA as well as IgM in ELISA. All the cultures inoculated with patient sera which were positive by RT-PCR were also positive by IFA, indicating that the pool of monoclonal antibodies is reliable for detection of RVF viruses. Moreover, these monoclonal antibodies were used successfully in ELISA. The use of a pool of several specific virus group-reactive Mab conjugates reacting with a combination of virus-specific antigens represents a system to detect various arboviruses which can be screened concurrently by using a single virusadaptable procedure. Such a procedure has been described by Martin et al. (2000). Our set of monoclonal antibodies could be useful for this kind of diagnosis kit. In conclusion, these RVFV specific monoclonal antibodies are valuable tools for use in different aspect of diagnosis of RVFV infections, including virus identification in cell cultures, IgM capture and IgG sandwich ELISAs.

7 40 A. Zaki et al. / Journal of Virological Methods 131 (2006) References Berglund, P., Sjöberg, M., Garoff, H., Atkins, G.J., Sheaham, B.J., Liljeström, P., Semliki forest virus expression system: production of conditionally infectious recombinant particles. Biotechnology 11, Besselaar, T.G., Blackburn, N.K., Topological mapping of antigenic sites on the Rift Valley fever virus envelope glycoproteins using monoclonal antibodies. Arch. Virol. 121, Billecocq, A., Coudrier, D., Boue, F., Combes, B., Zeller, H., Artois, M., Bouloy, M., Expression of the nucleoprotein of the Puumala virus from the recombinant Semliki Forest virus replicon: characterization and use as a potential diagnostic tool. Clin. Diagn. Lab. Immunol. 10, Billecocq, A., Vazeille-Falcoz, M., Rodhain, F., Bouloy, M., Pathogen-specific resistance to Rift Valley fever virus infection is induced in mosquito cells by expression of the recombinant nucleoprotein but not NSs non-structural protein sequences. J. Gen. Virol. 81, CDC, 2000a. Outbreak of Rift Valley fever Yemen, August October MMWR Morb. Mortal Wkly. Rep. 49, CDC, 2000b. Update: outbreak of Rift Valley Fever Saudi Arabia, August November MMWR Morb. Mortal Wkly. Rep. 49, Drosten, C., Gottig, S., Schilling, S., Asper, M., Panning, M., Schmitz, H., Gunther, S., Rapid detection and quantification of RNA of Ebola and Marburg viruses, Lassa virus, Crimean-Congo hemorrhagic fever virus, Rift Valley fever virus, dengue virus, and yellow fever virus by real-time reverse transcription-pcr. J. Clin. Microbiol. 40, Elliott, R.M., The Bunyaviridae. Plenum Press, New York, London. Garcia, S., Crance, J.M., Billecocq, A., Peinnequin, A., Jouan, A., Bouloy, M., Garin, D., Quantitative real-time PCR detection of Rift Valley fever virus and its application to evaluation of antiviral compounds. J. Clin. Microbiol. 39, Giorgi, C., Molecular Biology of Phlebovirus. Plenum Press, New York, NY. Groen, G.V.D., Elliot, L.H., Use of betapropionolactone inactivated Ebola, Marburg and Lassa intracellular antigens in immunofluorescence antibody assay. Ann. Soc. Belge Méd. Trop. 62, Ibrahim, M.S., Turell, M.J., Knauert, F.K., Lofts, R.S., Detection of Rift Valley fever virus in mosquitoes by RT-PCR. Mol. Cell. Probes 11, Johnson, A.J., Martin, D.A., Karabatsos, N., Roehrig, J.T., Detection of anti-arboviral immunoglobulin G by using a monoclonal antibody-based capture enzyme-linked immunosorbent assay. J. Clin. Microbiol. 38, Keegan, K., Collett, M.S., Use of bacterial expression cloning to define the amino acid sequences of antigenic determinants on the G2 glycoprotein of Rift Valley fever virus. J. Virol. 58, Laughlin, L.W., Meegan, J.M., Strausbaugh, L.J., Morens, D.M., Watten, R.H., Epidemic Rift Valley fever in Egypt: observations of the spectrum of human illness. Trans. R. Soc. Trop. Med. Hyg. 73, Liljeström, P., Garoff, H., A new generation of animal cell expression vectors based on the Semliki Forest virus replicon. Biotechnology (NY) 9, Linthicum, K.J., Anyamba, A., Tucker, C.J., Kelley, P.W., Myers, M.F., Peters, C.J., Climate and satellite indicators to forecast Rift Valley fever epidemics in Kenya. Science 285, Martin, D.A., Muth, D.A., Brown, T., Johnson, A.J., Karabatsos, N., Roehrig, J.T., Standardization of immunoglobulin M capture enzyme-linked immunosorbent assays for routine diagnosis of arboviral infections. J. Clin. Microbiol. 38, Meegan, J.M., Bailey, C.J., Rift Valley Fever, vol. IV. CRC Press Inc., Boca Raton, FL. Meegan, J.M., The Rift Valley fever epizootic in Egypt Description of the epizzotic and virological studies. Trans. R. Soc. Trop. Med. Hyg. 73, Niklasson, B., Peters, C.J., Grandien, M., Wood, O., Detection of human immunoglobulins G and M antibodies to Rift Valley fever virus by enzyme-linked immunosorbent assay. J. Clin. Microbiol. 19, Paweska, J.T., Burt, F.J., Anthony, F., Smith, S.J., Grobbelaar, A.A., Croft, J.E., Ksiazek, T.G., Swanepoel, R., 2003a. IgG-sandwich and IgM-capture enzyme-linked immunosorbent assay for the detection of antibody to Rift Valley fever virus in domestic ruminants. J. Virol. Methods 113, Paweska, J.T., Burt, F.J., Swanepoel, R., Validation of IgG-sandwich and IgM-capture ELISA for the detection of antibody to Rift Valley fever virus in humans. J. Virol. Methods 124, Paweska, J.T., Smith, S.J., Wright, I.M., Williams, R., Cohen, A.S., Van Dijk, A.A., Grobbelaar, A.A., Croft, J.E., Swanepoel, R., Gerdes, G.H., 2003b. Indirect enzyme-linked immunosorbent assay for the detection of antibody against Rift Valley fever virus in domestic and wild ruminant sera. Onderstepoort J. Vet. Res. 70, Pifat, D.Y., Osterling, M.C., Smith, J.F., Antigenic analysis of Punta Toro virus and identification of protective determinants with monoclonal antibodies. Virology 167, Sall, A.A., Macondo, E.A., Sene, O.K., Diagne, M., Sylla, R., Mondo, M., Girault, L., Marrama, L., Spiegel, A., Diallo, M., Bouloy, M., Mathiot, C., Use of reverse transcriptase PCR in early diagnosis of Rift Valley fever. Clin. Diagn. Lab. Immunol. 9, Sall, A.A., Zanotto, P.M., Sene, O.K., Zeller, H.G., Digoutte, J.P., Thiongane, Y., Bouloy, M., Genetic reassortment of Rift Valley fever virus in nature. J. Virol. 73, Saluzzo, J.F., Anderson Jr., G.W., Hodgson, L.A., Digoutte, J.P., Smith, J.F., Antigenic and biological properties of Rift Valley fever virus isolated during the 1987 Mauritanian epidemic. Res. Virol. 140, Schmaljohn, C., Bunyaviridae the Viruses and Their Replication. Raven Press, New York, NY. Xu, W., Beati, L., Raoult, D., Characterization of and application of monoclonal antibodies against Rickettsia africae, a newly recognized species of spotted fever group rickettsia. J. Clin. Microbiol. 35,