Presence of broadly reactive and group-specific neutralizing epitopes on newly described isolates of Crimean-Congo hemorrhagic fever virus

Asim A. Ahmed1, Jeanne M. McFalls1, Christian Hoffmann1, Claire Marie Filone1, Shaun M. Stewart1, Jason Paragas2, Shabot Khodjaev3, Dilbar Shermukhamedova3, Connie S. Schmaljohn2, Robert W. Doms1 and Andrea Bertolotti-Ciarlet1

1 Department of Microbiology, University of Pennsylvania, Philadelphia, PA 19104, USA
2 Virology Division, United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, Frederick, MD 21702, USA
3 Institute of Virology, Ministry of Health, Tashkent, Uzbekistan

Correspondence
Andrea Bertolotti-Ciarlet
aciarlet{at}mail.med.upenn.edu


   ABSTRACT
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Crimean-Congo hemorrhagic fever virus (CCHFV), a member of the genus Nairovirus of the family Bunyaviridae, causes severe disease in humans with high rates of mortality. The virus has a tripartite genome composed of a small (S), a medium (M) and a large (L) RNA segment; the M segment encodes the two viral glycoproteins, GN and GC. Whilst relatively few full-length M segment sequences are available, it is apparent that both GN and GC may exhibit significant sequence diversity. It is unknown whether considerable antigenic differences exist between divergent CCHFV strains, or whether there are conserved neutralizing epitopes. The M segments derived from viral isolates of a human case of CCHF in South Africa (SPU 41/84), an infected tick (Hyalomma marginatum) in South Africa (SPU 128/81), a human case in Congo (UG 3010), an infected individual in Uzbekistan (U2-2-002) and an infected tick (Hyalomma asiaticum) in China (Hy13) were sequenced fully, and the glycoproteins were expressed. These novel sequences showed high variability in the N-terminal region of GN and more modest differences in the remainder of GN and in GC. Phylogenetic analyses placed these newly identified strains in three of the four previously described M segment groups. Studies with a panel of mAbs specific to GN and GC indicated that there were significant antigenic differences between the M segment groups, although several neutralizing epitopes in both GN and GC were conserved among all strains examined. Thus, the genetic diversity exhibited by CCHFV strains results in significant antigenic differences that will need to be taken into consideration for vaccine development.

The GenBank/EMBL/DDBJ accession numbers for the sequences described in this paper are AY900141–AY900145.

Supplementary figures are available in JGV Online.


   INTRODUCTION
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Crimean-Congo hemorrhagic fever virus (CCHFV) causes a haemorrhagic disease in humans with mortality rates that range from 10 to 80 % (Whitehouse, 2004). CCHFV can be isolated from ticks, livestock and humans (Whitehouse, 2004). Infection can occur through the bite of an infected tick, exposure to tissue and fluids from an infected animal or through contact with infected human bodily fluids. CCHFV infection was first described during an outbreak in Russia during the 1940s, when more than 200 cases of severe haemorrhagic fever were reported among agricultural workers and soldiers in the Crimean peninsula (Chumakov et al., 1968, 1970). Since then, the virus has spread or has been recognized throughout many regions of the world, including sub-Saharan Africa (Williams et al., 2000; Wood et al., 1978), Bulgaria, the Arabian Peninsula, Iraq, Pakistan, the former Yugoslavia, northern Greece and north-west China (Chumakov et al., 1970; Hoogstraal, 1979; Olaleye et al., 1996; Onishchenko et al., 2000, 2001a, b).

CCHFV is a member of the genus Nairovirus within the family Bunyaviridae (Schmaljohn, 1996). Members of this enveloped virus family have a tripartite, single-stranded RNA genome of negative polarity. The small segment (S) encodes the viral nucleocapsid, the medium segment (M) encodes the two glycoproteins, GN and GC, and the large segment (L) encodes an RNA-dependent RNA polymerase. The viral glycoproteins, like those of other members of the family Bunyaviridae, are synthesized as a polyprotein precursor (Schmaljohn, 1996) that undergoes proteolytic cleavage events to yield the mature glycoproteins (Vincent et al., 2003). The GN precursor protein (Pre-GN) contains an N-terminal domain with a high proportion of Ser, Thr and Pro residues. This region resembles the mucin-like domain present in the glycoproteins of other viruses, most notably the Ebola virus glycoprotein (Simmons et al., 2002).

The GN and GC glycoproteins of CCHFV probably influence the host range, cell tropism and pathogenicity of this vertebrate and tick virus, and are the targets for neutralizing antibodies. Studies thus far indicate that portions of GN are highly variable compared with other regions of GN and with GC (Chinikar et al., 2004; Hewson et al., 2004a, b; Morikawa et al., 2002; Papa et al., 2002). However, there is limited sequence information available on CCHFV isolates from regions outside China and the former Soviet Union (Chinikar et al., 2004; Hewson et al., 2004a, b; Morikawa et al., 2002; Papa et al., 2002). We previously described the first neutralizing mAbs to CCHFV (Bertolotti-Ciarlet et al., 2005). In addition, some of these antibodies were shown to be protective in a suckling mouse animal model (Bertolotti-Ciarlet et al., 2005). However, it is not clear whether significant antigenic differences exist between divergent CCHFV isolates or whether conserved neutralizing epitopes are present. This information is important for vaccine development, as the identification of conserved neutralizing epitopes may lead to the development of vaccines and entry inhibitors.

To further characterize the genetic diversity of the CCHFV M segment, we cloned and expressed glycoproteins from divergent CCHFV strains that were passaged a limited number of times. Additionally, to assess antigenic differences between CCHFV isolates, we cloned and fully sequenced the open reading frames from five CCHFV isolates obtained from humans or ticks in South Africa, Congo, Uzbekistan and China. Phylogenetic analyses indicated that one or more of these new strains segregated with three of the four previously described M segment groups (Hewson et al., 2004b). The glycoproteins from each strain were expressed transiently in cell lines and their ability to be recognized by a panel of mAbs to GN and GC was determined. The genetic proximity of strains and their antigenic similarity were imperfectly correlated. Whilst some epitopes were conserved, others were not, indicating that CCHFV vaccines designed to induce neutralizing antibodies may have to include immunogens derived from several CCHFV strains, or in some way focus the immune response on conserved neutralizing epitopes.


   METHODS
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Virus strains and cells.
African green monkey kidney fibroblast (CV-1), Vero, Vero E6, human cervix carcinoma (HeLa) and human embryonic kidney (HEK-293T) cells, obtained from the ATCC (Manassas, VA, USA), were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10 % fetal bovine serum (FBS; Invitrogen). Similarly, the human tumour cell line SW-13 (adrenocortical carcinoma) was grown in DMEM supplemented with 2·5 % FBS. CCHFV strains Hy13, U2-2-002, SPU 41/84, SPU 128/81, SPU 94/87 and UG 3010 were used in this study. All of the viruses were passaged by intracerebral inoculation of 1-day-old mice with each CCHFV isolate, using a dose resulting in the death of 50 % of the mice. The mice were killed 24 h post-infection and the brains were harvested. Brains were homogenized to 10 % (w/v) with Hanks' salt solution and clarified by centrifugation at 10 000 r.p.m. in an SW41 rotor for 30 min. CCHFV prototype strain IbAr10200, first isolated in 1976 from ticks (Hyalomma excavatum) from Sokoto, Nigeria, was grown in African green monkey kidney Vero or Vero E6 cells (Sanchez et al., 2002). Republic of South Africa CCHFV strain SPU 41/84 was isolated from an infected human in 1984 and passaged in suckling mice four times. Republic of South Africa CCHFV strain SPU 128/81 was isolated in 1981 from infected ticks (Hyalomma marginatum rufipes) and passaged in suckling mice three times. Congolese strain UG 3010 was isolated in 1956. This was one of the first ‘Congo’ strains isolated (Simpson et al., 1967; Woodall et al., 1967). Chinese strain Hy13 was isolated from infected ticks (H. asiaticum) in 1968 and was passaged in suckling mice three times. CCHFV strain U2-2-002/U-6415 from Uzbekistan was isolated from an infected human and passaged in suckling mice four times. All work with replication-competent CCHFV was conducted in a biosafety level 4 facility at the United States Army Medical Research Institute of Infectious Diseases (USAMRIID).

RNA purification, RT-PCR and sequencing.
Consensus primers were designed based on an alignment of known full-length M segment sequences available in GenBank. In order to amplify the 5' half of the M segment from each strain, primers CCHF 5' (5'-TCTCAAAGAAACACGTGCCGC-3') and CCHF 3519 R (5'-GTACTCRAAGACAGGRGARTACAT-3') were designed. CCHF 2325 F (5'-AATGCAATAGAYGCTGARATGCA-3') and CCHF 3'R (5'-TCTCAAAGAWATAGTGGCGGCACGCAGTC-3') were designed to amplify the 3' half of the M segment for each strain. Wobble code includes R=A or G, Y=C or T and W=A or T. The two designed amplicons share 1 kb overlapping sequence at the centre of the M segment. This strategy of amplification of the M segment in two halves was utilized for most of the strains. Total RNA was isolated from lysates of SW-13 cells infected with the different CCHFV strains by utilizing TRIzol LS (Invitrogen) and removed from biocontainment. The samples were chloroform-extracted, followed by high-speed centrifugation and isolation of the resulting aqueous layer. RNA was precipitated by using propan-2-ol and pellets were resuspended in RNase-free distilled water. RNA was further purified through the RNeasy system (Qiagen) according to the manufacturer's directions.

Reverse transcription of the entire M RNA segment was performed by using 5 µl RNA from above, CCHF 3'R (300 ng) and 1 µl of a mixture of the four dNTPs (at 10 µM each) in 12 µl. This mixture was heated to 65 °C for 5 min and chilled rapidly on ice. Four microlitres of 5x RT buffer, 2 µl 0·1 M dithiothreitol and RNasin (40 U) were added to the mixture and heated to 42 °C for 2 min. Then, 1 µl Superscript II (Invitrogen) reverse transcriptase (RT, 200 U) was added to the reaction mixture and incubated at 42 °C for 1 h. The resultant cDNA generated from this reaction was used as a template in subsequent PCRs. PCR was performed by using 2 µl cDNA generated from the RT reaction, 5 µl CCHF primers (10 µM each), 5 µl 10x PCR buffer, 1·5 µl dNTP mixture, 2 µl MgSO4 (50 µM) and 0·6 µl Hi-Fidelity polymerase (5 U) in a 50 µl reaction. PCR thermocycler conditions were used as recommended by the manufacturer with an annealing temperature of 45 °C. When the consensus primer set was unable to generate a PCR product for one half of the M segment, a gene-specific internal primer was designed based on sequences from the half of the M segment that did yield a product. This was the case with UG 3010; the 3' half of the UG 3010 M segment was amplified by using a gene-specific internal primer, 3370F (5'-TGAACACAGGGGCAACAAAATC-3'), in combination with the 3' external consensus primer CCHF 3'R. Resultant PCR products were TA-cloned into pCR4-TOPO using the TOPO cloning for sequencing system (Invitrogen) according to the manufacturer's instructions. Recombinant clones were confirmed by sequencing in both directions. On average, three clones from two PCRs were sequenced in both directions to generate a sequence for each M segment half. By using the data from the 5' and 3' ends of each M segment that shared a 1 kb overlap, the sequence of each strain's M segment was resolved. These two overlapping fragments were utilized for cloning a full-length M segment into the expression vector pCAGGS (Niwa et al., 1991). The sequences have been deposited in GenBank (accession numbers AY900141–AY900145).

Mapping of mAb 11E7.
In order to map the epitope recognized by mAb 11E7, we constructed expression plasmids that represent fragments of the GC ectodomain. Primers were synthesized according to the published sequence for strain IbAr10200 (Sanchez et al., 2002) and standard PCR technology was performed to clone the amplicons into the pcDNA3.1D/V5-His-TOPO vector (Invitrogen). The 5' primers included the CACC sequence at the 5' end and the start codon to allow for directional cloning. The 3' primers did not possess a stop codon to allow the inclusion of the V5 cassette and polyhistidine epitope tags at the C terminus of the protein. Cloning was performed as described by the manufacturer (Invitrogen) and all constructs were sequenced. All primer sequences are available upon request.

Protein analysis.
To analyse protein expression, HEK-293T cells were infected with recombinant vaccinia virus vTF1.1 expressing T7 polymerase (Alexander et al., 1992) and transfected 40 min later by using Lipofectamine 2000 (Invitrogen). At 24 h post-transfection, cell extracts were prepared in 50 mM Tris/HCl (pH 7·4), 5 mM EDTA, 1 % Triton X-100 and Complete Protease Inhibitor cocktail (Roche Applied Sciences). Cell lysates were incubated at 4 °C for 3 min and then centrifuged at 10 000 g for 10 min. The supernatant was mixed with sample buffer [0·08 M Tris/HCl (pH 6·8), 2 % SDS, 10 % glycerol, 5 % {beta}-mercaptoethanol, 0·005 % bromophenol blue] and incubated at 56 °C for 10 min before electrophoresis in a Criterion SDS-PAGE 4–15 % Tris/HCl gel (Bio-Rad). Western blot analysis was performed by using mouse anti-V5 (Invitrogen) or mAb 11E7 as primary antibodies and sheep anti-mouse horseradish peroxidase-conjugated secondary antibody (Amersham Biosciences) followed by visualization with ECL-Plus Western blotting detection reagents (Bioscience). In the case of Western blotting developed with mAb 11E7, samples were not treated with {beta}-mercaptoethanol.

Immunofluorescence (IF) microscopy.
To determine whether there were antigenic differences among glycoproteins from different CCHFV strains and to characterize their localization within cells, we performed indirect IF microscopy as described previously (Morais et al., 2003). HeLa cells grown to 50 % confluence on glass coverslips were transfected with the different pCAGGS plasmids containing the CCHFV M segments. At 24 h post-transfection, the cells were fixed with 2 % (w/v) formaldehyde in PBS, permeabilized with 0·5 % Triton X-100 and stained with ascites containing a GN- or GC-specific mAb, diluted 1 : 250 in PBS containing 0·5 mM MgCl2 and 4 % FBS. Then, cells were washed with PBS and incubated for 1 h with the secondary antibody conjugated to Alexa Fluor 488 (goat anti-mouse) (Molecular Probes) diluted 1 : 500 in PBS containing 4 % FBS. Finally, cells were washed in PBS, mounted with Fluoromount-G (Southern Biotechnology Associates) and examined on a Nikon E600 microscope at x60 magnification utilizing UV illumination.

Sequence analysis.
We studied the relationships between the newly sequenced CCHFV M segments and previously published full-length isolates. The sequence alignments were produced by using CLUSTAL_X (Thompson et al., 1997) and checked manually for accuracy. The phylogenetic trees were drawn by using the PHYLIP package version 3.57c (Felsenstein, 1997). Briefly, the trees were obtained by using distance methods; SEQBOOT was used to obtain 1000 bootstrap replications of the original sequence alignment. The bootstrapped alignments were used for construction of a consensus tree with NEIGHBOR and CONSENSE as described in the package documentation. Distance between species shown in Fig. 1 was obtained from the original alignment. Consensus trees were rooted with the Dugbe strain, using TREEVIEW version 1.6.1 (Page, 1996).



View larger version (19K):
[in this window]
[in a new window]
 
Fig. 1. Phylogenetic trees showing the relationships between CCHFV M segments. The bootstrap values shown are percentages of 1000 replications of the original dataset. All sequences were retrieved from GenBank. Strains marked in bold were sequenced as part of this study (GenBank accession numbers AY900141–AY900145). (a) Phylogenetic tree constructed by utilizing the full-length M segment sequence. The branch length for the Dugbe sequence (outgroup) was cropped for presentation purposes. The small tree at the bottom left of the figure shows the correct branch-length relationship between Dugbe and the remaining sequences. (b) Phylogenetic tree constructed with only the mucin-like domain sequence. Bars, 0·1 substitution per base position.

 

   RESULTS AND DISCUSSION
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Cloning and expression of M segments from diverse regions of the world
There is limited sequence information on CCHFV isolates from regions outside China and the former Soviet Union, with only one full-length M segment from an African strain described previously (Chinikar et al., 2004; Hewson et al., 2004a, b; Morikawa et al., 2002; Papa et al., 2002). It is not known whether divergent CCHFV strains exhibit significant antigenic variability or share neutralizing epitopes – information that is important for vaccine development. In addition, only the glycoproteins of the extensively passaged IbAr10200 and Matin strains have been well characterized with regard to processing and cellular localization (Sanchez et al., 2002). To define the genetic and antigenic diversity of geographically diverse CCHFV strains, we cloned, sequenced and expressed the M segments from five isolates. Congolese strain UG 3010 was isolated in 1956 from a physician who became ill after handling blood taken from an infected boy at the Kisangani Hospital (Simpson et al., 1967; Woodall et al., 1967). Republic of South Africa CCHFV strain SPU 41/84 was isolated from a patient in South Africa in 1984 (Blackburn et al., 1987), whilst Republic of South Africa strain SPU 128/81 was isolated from H. marginatum ticks (Shepherd et al., 1985). Chinese strain Hy13 was isolated from H. asiaticum ticks in XinJiang, China, and Uzbekistan strain U2-2-002/U-6415 was isolated from an infected human. The viruses were passaged in suckling mice for between three and 11 times, as described in Methods.

M segment phylogeny
Hewson et al. (2004b) thoroughly described CCHFV phylogeny, revealing the existence of four M segment groups termed M1, M2, M3 and M4. We found that Chinese strain Hy13 clustered with group M1, along with several other Chinese strains and Pakistan strain Matin (Fig. 1a). South African strains SPU 41/84 and SPU 128/81 and Uzbekistan strain U2-2-002 clustered with group M2, along with previously described strains from China, Uzbekistan, Pakistan, Iraq and Nigeria (Hewson et al., 2004b; Morikawa et al., 2002; Sanchez et al., 2002). Congo strain UG 3010 clustered with group M3, which contains two previously described Chinese strains (Fig. 1a) (Morikawa et al., 2002). As noted previously, whilst there is some geographical clustering of CCHFV strains, there are also examples of geographically distant but genetically closely related virus isolates, perhaps reflecting trade in livestock or dispersal of infected ticks by migratory birds (Hewson et al., 2004b).

We repeated the phylogenetic analysis of the strains using different regions of the M segment (the mucin-like domain or P35 domains of GN, GN lacking these domains, and GC). The same phylogenetic tree was obtained in all cases (data not shown), even when only the highly variable mucin-like domain was used (Fig. 1b). This indicates that sequencing only a small portion of the M segment should make it possible to categorize new CCHFV isolates accurately.

Pairwise analysis of M segments sequences
The five completed M segment sequences had lengths ranging from 1684 to 1699 aa. The CCHFV glycoprotein precursor has been described to contain 78–80 cysteine residues on average, suggesting the presence of an exceptionally large number of disulfide bonds and a complex secondary structure. Cysteine residues were highly conserved, as were the sequences at the predicted proteolytic cleavage sites that have been described previously (Vincent et al., 2003). The number of potential N-linked glycosylation sites ranged from nine to 14. The M segments of the newly described strains were aligned with published sequences by using the CLUSTAL_X program (Jeanmougin et al., 1998; Thompson et al., 1997), and an identity matrix was constructed by using the program BioEdit (Tippmann, 2004). The GN precursor protein (Pre-GN) contains a highly variable domain at its N terminus that contains a high proportion of serine, threonine and proline residues, and it is predicted to be heavily O-glycosylated, thus resembling a mucin-like domain (Table 1) (Hewson et al., 2004a, b; Morikawa et al., 2002; Sanchez et al., 2002). When the identity values for the M segments were calculated based only on the mucin domain, the M1, M2, M3 and M4 strains were clearly distinct, consistent with the phylogenetic analyses (Table 1). When the same type of comparison was performed by using the full-length sequences or other portions of GN or GC, distinctions between the subgroups were not as obvious (data not shown), although the M3 group was the best defined and differentiated of the four subgroups (Table 1).


View this table:
[in this window]
[in a new window]
 
Table 1. Complete M segment deduced amino acid identities of CCHFV virus strains

 
Antigenic analysis of GN and GC
Antigenic variation of arboviruses is of relevance because it may provide clues on the possible directions of epidemics or endemic spread. Little is known about antigenic relationships among CCHFV strains, in part because of the lack of adequate reagents. Early studies have shown that strains from diverse parts of the world have close antigenic relationships (Tignor et al., 1980). However, these studies were performed by utilizing polyclonal serum obtained from animals inoculated with infected mouse-brain tissue, which usually results mainly in antibodies directed against the nucleocapsid (Blackburn et al., 1987). Indeed, with the exception of a recent report from our laboratory (Bertolotti-Ciarlet et al., 2005), the CCHFV mAbs described thus far are directed against the nucleocapsid (Blackburn et al., 1987). The viral glycoproteins might exhibit a degree of higher antigenic variability than the nucleocapsid protein as a result of immune selection and the adaptation needed to efficiently bind to and enter diverse cell types. Therefore, we determined antigenic differences between GN and GC from different strains, utilizing a panel of eight mAbs to GN and nine mAbs to GC (Bertolotti-Ciarlet et al., 2005). These mAbs bind to conformation-dependent epitopes and so were characterized for their ability to recognize the different GN and GC proteins by IF microscopy utilizing constructs expressing only one of the glycoproteins (Bertolotti-Ciarlet et al., 2005). The GN and GC proteins from each of the five strains were recognized by a subset of the mAbs and were localized to both the endoplasmic reticulum and the Golgi, consistent with correct processing and transport (Table 2 and Fig. 2) (Andersson & Pettersson, 1998; Andersson et al., 1997a, b; Chen & Compans, 1991; Chen et al., 1991; Gerrard & Nichol, 2002). The Golgi localization was confirmed by IF microscopy using a marker for TGN46 (Serotec), a sheep antibody specific for a heavily glycosylated protein localized primarily in the trans-Golgi network (data not shown). The M segments from each of the five virus strains appeared to be expressed at similar levels, as they were all recognized well by mAb 11E7 (see Supplementary Fig. S1, available in JGV Online). In addition, by using a rabbit polyclonal serum, we were able to show that the GN glycoproteins from each of the five CCHFV strains were expressed and processed properly (see Supplementary Fig. S2, available in JGV Online). With regards to mAb reactivity, two of the M2 group strains (SPU 128/81 and U2-2-002) were virtually identical to IbAr10200, which itself is an M2 group strain. However, the closely related SPU 41/84 M2 strain was not recognized by two of the GC mAbs or by two of the GN mAbs (Table 2). On the other hand, the M1 group strain Hy13 was recognized by seven of the eight GN mAbs, but by only three of nine GC mAbs. The M3 strain UG 3010, which was genetically the most distantly related to IbAr10200, shared a high degree of antigenic similarity with this prototype CCHFV strain. Altogether, the mAbs exhibited eight different reactivity patterns, including some mAbs that recognized only M2 virus strains and others that recognized all strains tested. Of the seven mAbs known to neutralize IbAr10200 potently in vitro (Bertolotti-Ciarlet et al., 2005), only 11E7 bound to all six virus strains. Of the five mAbs described previously to be able to protect at least 70 % of suckling mice challenged with IbAr10200 (Bertolotti-Ciarlet et al., 2005), 11E7 and 8F10 could bind to all six virus strains. It is important to note that, in each experiment, we used the parental IbAr10200 strain as a positive control (as it was recognized by all of the mAbs) and mock-transfected cells as a negative control (see Supplementary Fig. S3, available in JGV Online). These results suggest that there are significant antigenic differences between CCHFV strains that may not correlate well with genotypic or geographical characteristics. In addition, a number of epitopes to which neutralizing or protective mAbs can be directed are not highly conserved. However, at least one broadly cross-reactive, potently neutralizing mAb that can protect mice from a lethal CCHFV challenge (11E7) was identified.


View this table:
[in this window]
[in a new window]
 
Table 2. Reactivity of IbAr10200 mAbs with different CCHFV strains

The characterization of neutralization and protection has been described previously (Bertolotti-Ciarlet et al., 2005). Neutralization is shown as the plaque-reduction neutralization titre (PRNT 80 %) and protection data as the number of surviving mice compared with the total number of mice treated. +, Positive signal by IF; –, negative result. The identity of the antibodies was determined by IF analysis using constructs that contain GN or GC alone (Bertolotti-Ciarlet et al., 2005).

 


View larger version (129K):
[in this window]
[in a new window]
 
Fig. 2. IF analysis of CCHFV GN and GC glycoproteins. Transfected HeLa cells expressing CCHFV glycoproteins from different virus strains were processed for IF microscopy and stained with each of 17 different mouse anti-CCHFV mAbs (red) as described previously (Bertolotti-Ciarlet et al., 2005). Nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI; blue). Representative examples are shown in the following panels: (a) 11E7 anti-GC mAb, from left to right: CCHFV strains M1 Hy13, M2 SPU 128/81, M3 UG 3010; (b) 8A1 anti-GC mAb, from left to right: M1 Hy13, M2 U2-2-002, M3 UG 3010; (c) 7F5 anti-GN mAb, from left to right: M1 Hy13, M2 SPU 41/84, M3 UG 3010. HeLa cells were transfected with CCHFV M segments by using Lipofectamine 2000 (Invitrogen) and processed 24 h later.

 
Mapping of the 11E7 mAb epitope
As the neutralizing mAb 11E7 was able to recognize GC by Western blot under non-reducing conditions, we were able to partially map its epitope by testing its ability to recognize fragments of GC produced in HEK-293T cells. This is of relevance because mAb 11E7 protects mice in vivo from challenge with CCHFV strain IbAr10200 (Bertolotti-Ciarlet et al., 2005). Passive immunization can be effective for the treatment of CCHFV infection in humans, emphasizing the importance of identification of neutralizing antibodies and the epitopes to which they bind (Vassilenko et al., 1990).

We found that a GC construct lacking the transmembrane and cytoplasmic domains was recognized by mAb 11E7 (Fig. 3). Therefore, we constructed three fragments that covered the length of the GC ectodomain (C1, C2 and C3). All fragments contained a V5 epitope tag at the C terminus to allow detection of the fragment and to confirm their expression (Fig. 4). Most of the constructs, when expressed, formed SDS-resistant oligomers to some extent (Fig. 4). However, the relevance of this oligomerization is not clear, as the fragments represent only small portions of the protein and may therefore aggregate. Nonetheless, of these three fragments, only construct C3, located at the C terminus of the GC ectodomain, was recognized by 11E7. Therefore, we focused our attention on this area, further dividing it into three new fragments (C3A, C3B and C3C). The antibody recognized none of these fragments. Next, we decided to divide the C3 fragment into two overlapping regions (C3.1 and C3.2); however, this resulted in disruption of the 11E7 epitope (Figs 3 and 4). Therefore, we performed a small deletion within the C3 C terminus (C3-T1). The antibody recognized this construct. Additionally, a small deletion of the N terminus of the C3 region also yielded a fragment recognized by mAb 11E7 (C3-T2) (Figs 3 and 4). Therefore, we conclude that the neutralizing epitope of mAb 11E7 is contained between aa 1443 and 1566 of the M segment of IbAr10200 strain, a highly conserved region of the protein (Figs 3 and 4).



View larger version (15K):
[in this window]
[in a new window]
 
Fig. 3. Mapping of the epitope recognized by neutralizing mAb 11E7. A schematic representation of the different IbAr10200 GC fragments utilized to map the 11E7 epitope is shown. All of the constructs were expressed in mammalian cells and included a V5 epitope tag at the C terminus to control for expression. The numbers at the end of each construct represent the amino acid numbers based on the full-length IbAr10200 M segment.

 


View larger version (50K):
[in this window]
[in a new window]
 
Fig. 4. Western blot analyses for mapping of the mAb 11E7 epitope. Western blotting was performed by using lysates of HEK-293T cells transfected with some of the constructs shown in Fig. 3 and developed by using mAb 11E7 in parallel with a mAb for the V5 tag (Invitrogen). Some of the smaller fragments ran as both monomers and oligomers in SDS-PAGE. Molecular markers are shown in kDa (Prestained SDS-PAGE standards, broad range; Bio-Rad). GFP, Green fluorescent protein.

 
Conclusion
In summary, we report the first description of CCHFV glycoprotein antigenic structure and relatedness, as well as initial mapping of a cross-reactive neutralizing epitope present on divergent CCHFV strains. CCHFV strains can exhibit considerable genetic variability, with the mucin-like domain in GN in particular being highly divergent. We also found a considerable amount of antigenic variability, which may not follow phylogenetic groupings of CCHFV strains. Even the highly conserved GC protein exhibited antigenic variability, suggesting that CCHFV glycoproteins are subject to immune selection. Nonetheless, at least some epitopes to which neutralizing and/or protective antibodies bind are conserved between divergent CCHFV strains, and definition of these antibody-binding sites may be useful for vaccine design.


   ACKNOWLEDGEMENTS
 
We thank Aura Garrison, Louis Altamura and Donald Pijak for expert technical assistance. This work was supported in part by training grant NIH T32 AI055400, Department of Defense Peer Reviewed Medical Research Program grant PRMRP PR033269 and R21-AI-063308.


   REFERENCES
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Alexander, W. A., Moss, B. & Fuerst, T. R. (1992). Regulated expression of foreign genes in vaccinia virus under the control of bacteriophage T7 RNA polymerase and the Escherichia coli lac repressor. J Virol 66, 2934–2942.[Abstract]

Andersson, A. M. & Pettersson, R. F. (1998). Targeting of a short peptide derived from the cytoplasmic tail of the G1 membrane glycoprotein of Uukuniemi virus (Bunyaviridae) to the Golgi complex. J Virol 72, 9585–9596.[Abstract/Free Full Text]

Andersson, A. M., Melin, L., Bean, A. & Pettersson, R. F. (1997a). A retention signal necessary and sufficient for Golgi localization maps to the cytoplasmic tail of a Bunyaviridae (Uukuniemi virus) membrane glycoprotein. J Virol 71, 4717–4727.[Abstract]

Andersson, A. M., Melin, L., Persson, R., Raschperger, E., Wikström, L. & Pettersson, R. F. (1997b). Processing and membrane topology of the spike proteins G1 and G2 of Uukuniemi virus. J Virol 71, 218–225.[Abstract]

Bertolotti-Ciarlet, A., Smith, J., Strecker, K. & 7 other authors (2005). Cellular localization and antigenic characterization of Crimean-Congo hemorrhagic fever virus glycoproteins. J Virol 79, 6152–6161.[Abstract/Free Full Text]

Blackburn, N. K., Besselaar, T. G., Shepherd, A. J. & Swanepoel, R. (1987). Preparation and use of monoclonal antibodies for identifying Crimean-Congo hemorrhagic fever virus. Am J Trop Med Hyg 37, 392–397.[Medline]

Chen, S.-Y. & Compans, R. W. (1991). Oligomerization, transport, and Golgi retention of Punta Toro virus glycoproteins. J Virol 65, 5902–5909.[Medline]

Chen, S.-Y., Matsuoka, Y. & Compans, R. W. (1991). Golgi complex localization of the Punta Toro virus G2 protein requires its association with the G1 protein. Virology 183, 351–365.[CrossRef][Medline]

Chinikar, S., Persson, S.-M., Johansson, M. & 7 other authors (2004). Genetic analysis of Crimean-Congo hemorrhagic fever virus in Iran. J Med Virol 73, 404–411.[CrossRef][Medline]

Chumakov, M. P., Butenko, A. M., Shalunova, N. V. & 10 other authors (1968). New data on the viral agent of Crimean hemorrhagic fever. Vopr Virusol 13, 377 (in Russian).[Medline]

Chumakov, M. P., Smirnova, S. E. & Tkachenko, E. A. (1970). Relationship between strains of Crimean haemorrhagic fever and Congo viruses. Acta Virol 14, 82–85.[Medline]

Felsenstein, J. (1997). An alternating least squares approach to inferring phylogenies from pairwise distances. Syst Biol 46, 101–111.[Medline]

Gerrard, S. R. & Nichol, S. T. (2002). Characterization of the Golgi retention motif of Rift Valley fever virus GN glycoprotein. J Virol 76, 12200–12210.[Abstract/Free Full Text]

Hewson, R., Chamberlain, J., Mioulet, V. & 9 other authors (2004a). Crimean-Congo haemorrhagic fever virus: sequence analysis of the small RNA segments from a collection of viruses world wide. Virus Res 102, 185–189.[CrossRef][Medline]

Hewson, R., Gmyl, A., Gmyl, L., Smirnova, S. E., Karganova, G., Jamil, B., Hasan, R., Chamberlain, J. & Clegg, C. (2004b). Evidence of segment reassortment in Crimean-Congo haemorrhagic fever virus. J Gen Virol 85, 3059–3070.[Abstract/Free Full Text]

Hoogstraal, H. (1979). The epidemiology of tick-borne Crimean-Congo hemorrhagic fever in Asia, Europe, and Africa. J Med Entomol 15, 307–417.[Medline]

Jeanmougin, F., Thompson, J. D., Gouy, M., Higgins, D. G. & Gibson, T. J. (1998). Multiple sequence alignment with Clustal X. Trends Biochem Sci 23, 403–405.[CrossRef][Medline]

Morais, V. A., Crystal, A. S., Pijak, D. S., Carlin, D., Costa, J., Lee, V. M.-Y. & Doms, R. W. (2003). The transmembrane domain region of nicastrin mediates direct interactions with APH-1 and the {gamma}-secretase complex. J Biol Chem 278, 43284–43291.[Abstract/Free Full Text]

Morikawa, S., Qing, T., Xinqin, Z., Saijo, M. & Kurane, I. (2002). Genetic diversity of the M RNA segment among Crimean-Congo hemorrhagic fever virus isolates in China. Virology 296, 159–164.[CrossRef][Medline]

Niwa, H., Yamamura, K. & Miyazaki, J. (1991). Efficient selection for high-expression transfectants with a novel eukaryotic vector. Gene 108, 193–199.[CrossRef][Medline]

Olaleye, O. D., Tomori, O. & Schmitz, H. (1996). Rift Valley fever in Nigeria: infections in domestic animals. Rev Sci Tech 15, 937–946.[Medline]

Onishchenko, G. G., Lomov, Iu. M., Markov, V. I. & 12 other authors (2000). The laboratory diagnosis of an outbreak of hemorrhagic fever at Oblivskaya village, Rostov Province: proof of the etiological role of the Crimean-Congo hemorrhagic fever virus. Zh Mikrobiol Epidemiol Immunobiol 32–36 (in Russian).

Onishchenko, G. G., Efremenko, V. I., Kovalev, N. G. & 12 other authors (2001a). Specific epidemiologic features of Crimean haemorrhagic fever in Stavropol' region in 1999–2000. Zh Mikrobiol Epidemiol Immunobiol 86–89 (in Russian).

Onishchenko, G. G., Markov, V. I., Merkulov, V. A., Vasil'ev, N. T., Berezhnoi, A. M., Androshchuk, I. A. & Maksimov, V. A. (2001b). Isolation and identification of Crimean-Congo hemorrhagic fever virus in the Stavropol territory. Zh Mikrobiol Epidemiol Immunobiol 7–11 (in Russian).

Page, R. D. M. (1996). TREEVIEW: an application to display phylogenetic trees on personal computers. Comput Appl Biosci 12, 357–358.[Medline]

Papa, A., Ma, B.-J., Kouidou, S., Tang, Q., Hang, C.-S. & Antoniadis, A. (2002). Genetic characterization of the M RNA segment of Crimean Congo hemorrhagic fever virus strains, China. Emerg Infect Dis 8, 50–53.[Medline]

Sanchez, A. J., Vincent, M. J. & Nichol, S. T. (2002). Characterization of the glycoproteins of Crimean-Congo hemorrhagic fever virus. J Virol 76, 7263–7275.[Abstract/Free Full Text]

Schmaljohn, C. S. (1996). Bunyaviridae: the viruses and their replication. In Fileds Virology, 3rd edn, pp. 1447–1471. Edited by B. N. Fields, D. M. Knipe & P. M. Howley. Philadelphia, PA: Lippincott-Raven.

Shepherd, A. J., Swanepoel, R., Shepherd, S. P., Leman, P. A., Blackburn, N. K. & Hallett, A. F. (1985). A nosocomial outbreak of Crimean-Congo haemorrhagic fever at Tygerberg Hospital. Part V. Virological and serological observations. S Afr Med J 68, 733–736.[Medline]

Simmons, G., Wool-Lewis, R. J., Baribaud, F., Netter, R. C. & Bates, P. (2002). Ebola virus glycoproteins induce global surface protein down-modulation and loss of cell adherence. J Virol 76, 2518–2528.[Abstract/Free Full Text]

Simpson, D. I., Knight, E. M., Courtois, G., Williams, M. C., Weinbren, M. P. & Kibukamusoke, J. W. (1967). Congo virus: a hitherto undescribed virus occurring in Africa. I. Human isolations – clinical notes. East Afr Med J 44, 86–92.[Medline]

Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F. & Higgins, D. G. (1997). The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25, 4876–4882.[Abstract/Free Full Text]

Tignor, G. H., Smith, A. L., Casals, J., Ezeokoli, C. D. & Okoli, J. (1980). Close relationship of Crimean hemorrhagic fever-Congo (CHF-C) virus strains by neutralizing antibody assays. Am J Trop Med Hyg 29, 676–685.[Medline]

Tippmann, H.-F. (2004). Analysis for free: comparing programs for sequence analysis. Brief Bioinform 5, 82–87.[CrossRef][Medline]

Vassilenko, S. M., Vassilev, T. L., Bozadjiev, L. G., Bineva, I. L. & Kazarov, G. Z. (1990). Specific intravenous immunoglobulin for Crimean-Congo haemorrhagic fever. Lancet 335, 791–792.

Vincent, M. J., Sanchez, A. J., Erickson, B. R., Basak, A., Chretien, M., Seidah, N. G. & Nichol, S. T. (2003). Crimean-Congo hemorrhagic fever virus glycoprotein proteolytic processing by subtilase SKI-1. J Virol 77, 8640–8649.[Abstract/Free Full Text]

Whitehouse, C. A. (2004). Crimean–Congo hemorrhagic fever. Antiviral Res 64, 145–160.[CrossRef][Medline]

Williams, R. J., Al-Busaidy, S., Mehta, F. R. & 7 other authors (2000). Crimean-Congo haemorrhagic fever: a seroepidemiological and tick survey in the Sultanate of Oman. Trop Med Int Health 5, 99–106.[CrossRef][Medline]

Wood, O. L., Lee, V. H., Ash, J. S. & Casals, J. (1978). Crimean-Congo hemorrhagic fever, Thogoto, Dugbe, and Jos viruses isolated from ixodid ticks in Ethiopia. Am J Trop Med Hyg 27, 600–604.[Medline]

Woodall, J. P., Williams, M. C. & Simpson, D. I. (1967). Congo virus: a hitherto undescribed virus occurring in Africa. II. Identification studies. East Afr Med J 44, 93–98.[Medline]

Received 10 May 2005; accepted 22 August 2005.



This Article
Abstract
Full Text (PDF)
Supplementary figures
Alert me when this article is cited
Alert me if a correction is posted
Citation Map
Services
Email this article to a friend
Similar articles in this journal
Similar articles in PubMed
Alert me to new issues of the journal
Download to citation manager
Google Scholar
Articles by Ahmed, A. A.
Articles by Bertolotti-Ciarlet, A.
PubMed
PubMed Citation
Articles by Ahmed, A. A.
Articles by Bertolotti-Ciarlet, A.
Agricola
Articles by Ahmed, A. A.
Articles by Bertolotti-Ciarlet, A.


HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
INT J SYST EVOL MICROBIOL MICROBIOLOGY J GEN VIROL
J MED MICROBIOL ALL SGM JOURNALS