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Short Communication |
1 Laboratoire de Virologie, Unité des Arbovirus et Fièvres Hémorragiques Virales, Institut Pasteur de Bangui, Central African Republic
2 GEVSM-SRSMC, UMR 7565 CNRS Nancy-University, France
3 CHU de Nancy Brabois, Nancy-University, France
4 INSERM U525, Nancy-University, France
5 Unité de Biologie Moléculaire chez les Extremophiles (BMGE), Institut Pasteur, Paris, France
Correspondence
B. H. Rihn
b.rihn{at}chu-nancy.fr
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ABSTRACT |
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). One of the largest is the Bunyamwera serogroup, named after the prototype member of the family Bunyaviridae, isolated from mosquitoes collected in Uganda in 1943 (Smithburn et al., 1946). Most viruses belonging to this serogroup have been isolated from arthropods, such as mosquitoes and culicoid midges, as well as from vertebrate hosts. The classification of the Bunyamwera serogroup is based upon antigenic relationships, determined by plaque-reduction neutralization, haemagglutination-inhibition, complement-fixation and radial-immunodiffusion tests (Shope & Causey, 1962; Calisher & Karabatsos, 1988). Although serological immunoassays are available for antigen detection of a few viruses, cross-reactions are common and may impair their identification (Artsob et al., 1984; Hildreth et al., 1982). Molecular diagnosis of orthobunyavirus species would facilitate surveillance of vectors and reservoirs, as well as laboratory diagnosis. Positive samples can be submitted to further investigations to identify species and subtypes.
Until now, orthobunyaviruses from the Central African Republic (CAR) have been exclusively classified serologically, and molecular characterization of circulating viruses is lacking. Developing specific molecular tools for African viruses is thus essential for improving virological surveillance in arthropod populations in endemic areas, as well as for clinical diagnosis. For this purpose, it would be of interest to have primers that allow detection of all circulating viruses, using as few separate reaction mixtures as possible. Here, we have isolated 133 strains of orthobunyavirus that have been classified by using serological assays: complement-fixation, haemagglutination-inhibition and neutralization tests in the Centre de Référence OMS de Recherche pour les Arbovirus (Dakar, Senegal) from samples obtained in CAR from mosquitoes and/or blood of symptomatic forest workers (Shope & Causey, 1962; Calisher & Karabatsos, 1988). Twelve of these strains were selected for determination of their complete S segment sequences. The purpose of this study was to determine whether it is possible to design specific primers for the detection of most African orthobunyavirus strains from samples or after cell culture.
Twelve strains of orthobunyavirus from the 133 strains of the collection of the Institut Pasteur de Bangui (CAR), classified as belonging to the Bunyamwera, Bakau, Turlock, Simbu and Nyando serogroups, were selected. They were grown on VeroE6 (green monkey kidney) cells in Eagle's minimum essential medium (Sigma) supplemented with 2 % fetal bovine serum (Gibco-BRL) and antibiotics (penicillin/streptomycin; Sigma-Aldrich). To prevent any cross-contamination, each virus was grown individually. Cell layers and supernatants were harvested when approximately 75 % of cells were exhibiting cytopathic effect (CPE) and centrifuged at 880 g (Heraeus Megafuge 1.0R, rotor BS4402/A). RNA was extracted from the supernatant by using a QIAamp viral RNA mini kit (Qiagen) according to the manufacturer's instructions.
Using the obtained purified RNA extracts, the S segment was amplified by using primers specific for the Bunyamwera and California virus serogroups: BUNYA1, 5'-GTCACAGTAGTGTACTCCAC-3', and BUNYA2, 5'-CTGACAGTAGTGTGCTCCAC-3'; and primers corresponding to the highly conserved terminal sequences of the S segment of Bunyamwera virus (GenBank accession no. NC_001927
[GenBank]
): BUNS274C, 5'-CTTAACYTTGGGGGCTGGA-3', and BUNS957R, 5'-CCCCIACCACCCACCC-3' (Dunn et al., 1994; Bowen et al., 2001).
As only partial sequences of the S segment could be obtained with the above sets of primers, four new set of primers (A, B, C and D) were designed on the basis of the GenBank NC_001927 [GenBank] S RNA segment, as follows: set A, BUNS1 (5'-AGTAGTGTACTCCACACTACAAACT-3') and BUNS3 (5'-TCGTCAGGAACTGGGTTGTTCCGG-3'); set B, BUNS1 (5'-AGTAGTGTACTCCACACTACAAACT-3') and BUNS9 (5'-AGGAATCCACTGAGGCGGTGGAGG-3'; set C, BUNS4 (5'-CTGGCAACCGGAACAACCCAGTT-3') and BUNS5 (5'-GAGACAACTGTCAGTGCAGACTGAA-3'); set D, BUNS10 (5'-TCAGTCTGCACTGACAGTTGTCTC-3') and BUNS2 (5'-AGTAGTGTGCTCCACCTAAAACTTA-3').
Complete S segment sequencing was performed by genome walking. Reverse transcription was performed by incubation (65 °C, 10 min) of the RNA extract (1.0 µl) in the presence of 25 pmol of the appropriate forward primers (BUNS274C and BUNYA1) in RNA-free H2O in a total volume of 10.5 µl. The mixture was quenched immediately in an ice–water bath to prevent the reannealing of the RNA with the forward primer. Reverse transcription mix (9.5 µl), composed of 5x First-Strand buffer, 10 mM dithiothreitol, 10 mM each dNTP, 20 U RNasin and 50 U SuperScript II reverse transcriptase (Invitrogen), was added to the first components. cDNA was synthesized at 25 °C for 10 min, 42 °C for 50 min and 72 °C for 15 min. The amplification mix was composed of 2.0 µl cDNA, 5.0 µl 10x Expand Long Template buffer (1.75 mM MgCl2), 0.2 mM each dNTP, 25 pmol each primer and 2.5 U Expand Long Template buffer (Expand Long Template PCR System; Roche) in a total volume of 50.0 µl in autoclaved distilled water. Amplification was carried out by using a cycle of melting at 95 °C for 15 min, followed by 35 cycles of melting at 95 °C for 1 min, annealing at 55 °C for 1 min and extension at 72 °C for 2 min. The thermal cycling was terminated by a final extension step at 72 °C for 10 min.
Amplified products were detected by staining with ethidium bromide following electrophoresis in a 1 % agarose gel in Tris/borate/EDTA buffer (pH 8.0). DNA bands of the expected size were located by UV transillumination, sliced from the gel and purified by using a QIAquick gel extraction kit (Qiagen), following the manufacturer's instructions. The sequences of amplified products were determined by using ABI PRISM BigDye Terminator v1.1 cycle sequencing ready reaction kits with AmpliTaq DNA Polymerase FS (Applied Biosystems), 3.2 pmol each primer and 30 ng amplified cDNA. Nucleic acid sequences were obtained by using an ABI PRISM 3100 Avant Genetic Analyzer (Applied Biosystems). Each sequence was determined twice to demonstrate reproducibility.
To examine the phylogeny of orthobunyaviruses, N open reading frame (ORF) nucleotide sequences of CAR strains were aligned with the corresponding ones from the Bunyamwera strain (GenBank accession no. NC_001927
[GenBank]
) and from 18 additional viruses of the Bunyamwera, California and Simbu serogroups that were retrieved from GenBank by using CLUSTAL_W at the European Bioinformatics Institute (http://www.ebi.ac.uk/clustalw). Unfortunately, sequences from the Bakau, Turlock and Nyando serogroups were not available at the time of testing. From the resulting alignment, 689 unambiguously aligned positions were selected and a maximum-likelihood (ML) tree was constructed by using PHYML (Guindon & Gascuel, 2003), using the general time-reversible model including an estimation of base frequencies and a correction for rate heterogeneity among sites (four rate categories and an estimation of the alpha parameter describing the gamma distribution of rates). Statistical support at nodes was calculated by bootstrap from 100 resamplings of the original dataset.
CPE was observed at 3 days after inoculation on average with orthobunyavirus strains, except for M’Poko virus, for which 100 % CPE was observed after 24 h. The primers designed for the California and Bunyamwera serogroups (Bowen et al., 2001) allowed amplification of the genome of 10 of the 12 strains selected. These primers allowed the amplification of part of the S RNA sequences corresponding to approximately nt 320–800 of the Bunyamwera prototype genome (GenBank accession no. NC_001927
[GenBank]
). Combination of the published and designed primers allowed determination of complete S segment sequences of 10 of the 12 selected orthobunyaviruses (Table 1), but was unsuccessful for Pongola (Bwamba) and Batama (Tete) viruses. Moreover, attempted amplification using the terminal conserved 11 nt sequence generated only faint bands of approximately 250 bp as seen in agarose gel (data not shown).
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). Their organization was identical to that of viruses of the Bunyamwera, California and Simbu serogroups of the genus Orthobunyavirus (Elliott, 1989, 1990) with two overlapping ORFs, predicted to encode the N and NSs proteins, respectively (Dunn et al., 1994). The larger ORFs, encoding the N protein, consisted of 648–723 nt (nt 80/86–785/808) and were initiated by a methionine codon, whilst the smaller ORFs, encoding the NSs protein, consisted of 252–330 nt (nt 105–357/435) and began with two methionine codons, except for the Birao strain, which had only one. The coding regions were flanked by terminal non-coding regions (designated 5' and 3' NCRs) of 79–85 nt at the 5' end and of 107–227 nt at the 3' end (Table 1) with complementary sequences. The S segment showed 5–15 % difference at the nucleotide level from the Bunyamwera virus sequence (GenBank accession no. NC_001927
[GenBank]
).
Alignment of the amino acid sequences of the N protein (213–240 aa) from 10 CAR viruses (Fig. 1a) showed sequence divergence of 4–27 % with respect to the Bunyamwera virus prototype. However, a number of conservative amino acid substitutions were observed that replace an amino acid with another amino acid of similar properties and structure, and may have no effect on protein function (Fig. 1a). Several regions were highly conserved and may contain the complement-fixation site that cross-reacted in serological tests within viruses of the genus Orthobunyavirus. The N protein of the M’Poko ArB365 strain was the most divergent when compared with the Bunyamwera strain (GenBank accession no. NC_001927
[GenBank]
; 26 % amino acid variation). Similarly, the NSs sequence of the M’Poko ArB365 strain appeared highly divergent (37–43 % dissimilarity) compared with other analysed viruses, but some conservative substitutions could be seen.
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). The ten CAR viruses (indicated by 
) grouped within the Bunyamwera cluster with high statistical confidence (100 % bootstrap value). The two other groups, corresponding to the Simbu and California serogroups, were also each supported by a 100 % bootstrap value. Although not all nodes within the Bunyamwera group are supported strongly, the ten CAR strains clustered at a bootstrap-support level of 97 % with the Bunyamwera and Mboke viruses (GenBank accession nos NC_001927
[GenBank]
and AY593727
[GenBank]
, respectively). Surprisingly, the ArB2633 Ingwavuma strain, which was classified in the Simbu serogroup by serological methods, clustered confidently with two Bunyamwera serogroup viruses and stood far from the other Simbu serogroup sequences. Alignment of the amino acid sequence of the N protein from the Ingwavuma ArB2633 strain showed sequence divergence of 42 % with respect to the published Ingwavuma strain An4165 (GenBank accession no. AF362395
[GenBank]
). Similarly, the strains assigned to the Turlock (ArB365), Bakau (ArB2882) and Nyando (ArB16055) serogroups clustered confidently within the Bunyamwera group (Fig. 2).
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) confirms the fact that they belong to the genus Orthobunyavirus. However, it remains to be determined which method (serological or molecular) is more appropriate for definition of the different groups of orthobunyaviruses. In fact, serogrouping based on serological analysis and genome sequencing produced very different results, making their comparison difficult. In addition, no S RNA segment nucleotide sequences of the Turlock (M’Poko virus), Bakau (Nola virus) and Nyando (Nyando virus) serogroups were available in GenBank and their exact taxonomic positions remain to be determined. Regarding the amino acid sequence variation between the two Ingwavuma strains, it would be interesting to determine whether the Ingwavuma ArB2633 strain from CAR was initially misclassified in the Simbu serogroup.
The presence of two successive AUG codons at the beginning of the NSs ORF is a characteristic of viruses belonging to the California and Bunyamwera serogroups (Bowen et al., 1995; Huang et al., 1996). The presence of a unique AUG codon in the NSs ORF solely in the Birao strain is not surprising, as this characteristic is present in other strains (Germiston, GenBank accession no. M19420
[GenBank]
; Buttonwillow, accession no. AF362398
[GenBank]
; Oropouche, accession no. AY993912
[GenBank]
; and Tinaroo, accession no. AB000819
[GenBank]
). Although it retains the main characteristics of Bunyamwera group viruses, the M’Poko strain appears as an outlier by its greater sequence divergence. M’Poko also showed a higher divergence in G2 protein sequence. Moreover, it emerged at the base of the Bunyamwera serogroup in a phylogenetic tree constructed with G2 protein sequences that was inconsistent with the tree built by using the N sequences (data not shown). However, this placement may be the product of an artefact of phylogenetic reconstruction induced by the presence of very long branches. Furthermore, in this tree, the Bunyamwera serogroup was not monophyletic, as it also included the Simbu serogroup.
Our study allowed the identification of primer sets suitable for detection of complete S RNA fragment sequences of most Bunyamwera serogroup viruses isolated from CAR in a single RT-PCR step. These primer sets have been shown to be effective in detecting the most serologically distinct viruses in the complex (M’Poko, Nyando, Ingwavuma and Nola viruses). Further studies are necessary to better characterize the two viruses (Pongola and Batama) that were not amplified by using the primer sets described above.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTMAIN TEXTREFERENCES |
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Bowen, M. D., Jackson, A. O., Bruns, T. D., Hacker, D. L. & Hardy, J. L. (1995). Determination and comparative analysis of the small RNA genomic sequences of California encephalitis, Jamestown Canyon, Jerry Slough, Melao, Keystone and Trivittatus viruses (Bunyaviridae, genus Bunyavirus, California serogroup). J Gen Virol 76, 559–572.
Bowen, M. D., Trappier, S. G., Sanchez, A. J., Meyer, R. F., Goldsmith, C. S., Zaki, S. R., Dunster, L. M., Peters, C. J., Ksiazek, T. G. & Nichol, S. T. (2001). A reassortant bunyavirus isolated from acute hemorrhagic fever cases in Kenya and Somalia. . RVF Task Force. Virology 291, 185–190.[CrossRef][Medline]
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Dunn, E. F., Pritlove, D. C. & Elliott, R. M. (1994). The S RNA genome segments of Batai, Cache Valley, Guaroa, Kairi, Lumbo, Main Drain and Northway bunyaviruses: sequence determination and analysis. J Gen Virol 75, 597–608.
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Received 13 October 2006;
accepted 12 February 2007.
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