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
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession numbers for the sequences described in this paper are AY900141AY900145.
Supplementary figures are available in JGV Online.
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INTRODUCTION |
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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.
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METHODS |
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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 AY900141AY900145).
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 %
-mercaptoethanol, 0·005 % bromophenol blue] and incubated at 56 °C for 10 min before electrophoresis in a Criterion SDS-PAGE 415 % 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
-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
).
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RESULTS AND DISCUSSION |
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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 7880 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
).
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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
).
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ACKNOWLEDGEMENTS |
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Received 10 May 2005;
accepted 22 August 2005.
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