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Short Communication |
1 Unité des Virus Emergents (EA3292, IFR48, IRD UR0178), Faculté de Médecine La Timone, 27 boulevard Jean Moulin, 13005 Marseille, France
2 IRD – UR0178, Conditions et Territoires d'Emergence des Maladies, BP 1386, 18524 Dakar, Senegal
3 IRD – UR0178, Mahidol University, Research Center for Emerging Viral Diseases/Center for Vaccine Development Institute of Sciences, Salaya, 25/25 Phutthamonthon 4, Nakhonpathom 73170, Thailand
4 Department of Zoology, University of Oxford, Oxford, UK
5 Centre International de Recherches Médicales de Franceville, BP 769 Franceville, Gabon
6 Department of Entomology, University of Minnesota, St Paul, MN 55108, USA
7 Department of Biology, The Pennsylvania State University, University Park, PA 16802, USA
Correspondence
Gilda Grard
gilda.grard{at}gulliver.fr
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession number for the sequence reported in this paper is DQ400858.
A supplementary figure showing electron micrographs of Ngoye virus is available in JGV Online.
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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), West Nile virus has recently spread across northern America, causing several hundred human and many thousands of animal and avian deaths (Komar, 2003; http://www.cdc.gov/ncidod/dvbid/westnile/) and a range of tick-borne flaviviruses are responsible for human encephalitis or haemorrhagic fevers in Europe and Asia (Gritsun et al., 2003). In addition to these major pathogens, more than 20 other flaviviruses have been reported to cause diseases in humans (Calisher & Gould, 2003).
The flaviviruses are also notable for their diversity, with more than 70 different viruses described to date within the genus Flavivirus. The majority belong to a single, antigenically related phylogenetic group within which viruses are distributed in different branches according to the nature of their natural arthropod vector (ticks or Aedes or Culex mosquitoes) (Gaunt et al., 2001). However, even among these ‘classical’ flaviviruses, some can be regarded as atypical in that they have no arthropod vector. These latter viruses form two genetic subgroups: the first encompasses viruses isolated from bats and rodents that are related distantly to tick-borne viruses (Billoir et al., 2000; Cook & Holmes, 2006), whilst the second contains viruses related to Aedes mosquito-borne flaviviruses that have probably lost the capacity for vector transmission secondarily (Kuno et al., 1998).
A number of other viruses are related more distantly to the classical flaviviruses. This group includes Cell fusing agent virus (CFAV) (Cammisa-Parks et al., 1992) and Kamiti River virus (KRV) (Crabtree et al., 2003). Although magnetofection-forced entry into mammalian cells results in active replication, these viruses do not infect mammalian cells spontaneously (X. de Lamballerie, unpublished data) and are therefore considered to be insect viruses. Recently, it was shown that long genomic fragments of viruses related to CFAV and KRV exist in DNA form in the genome of Aedes mosquitoes (Crochu et al., 2004) and that infection of mosquito cells by both CFAV and KRV is followed by the synthesis of genomic DNA forms (Cook et al., 2006).
Finally, a single tentative species named Tamana bat virus (TABV) occupies a unique position within the flavivirus phylogeny. This virus was isolated in 1973 from an insectivorous bat in Trinidad (Price, 1978) and for 25 years it remained a taxonomic enigma. It was shown recently to constitute a highly divergent evolutionary lineage within the flaviviruses, constituting a sister group to all other known taxa (de Lamballerie et al., 2002). In contrast to CFAV and KRV, TABV infects only vertebrates and does not replicate in arthropod cells.
Herein, we report the description and analysis of a novel virus that similarly occupies a unique phylogenetic position within the genus Flavivirus. Field collection of African ticks was organized in order to investigate the presence and distribution of tick species possibly associated with arboviruses. Following the collection of 110 ticks in October 2002 in the village of Ngoye (14° 37' N 16° 25' W), in the Fatick region of Senegal, this novel virus was identified in three Rhipicephalus evertsi evertsi ticks (family Ixodidae) sampled from two caprines (Capra hircus, subfamily Caprinae, family Bovidae) and one ovine (Ovis sp., subfamily Caprinae, family Bovidae). The virus was named Ngoye virus (NGOV) after the location from which it was isolated. It was subsequently identified from another R. evertsi evertsi tick (out of 46 ticks tested) sampled from a bovine (Bos taurus, subfamily Bovinae, family Bovidae) in December 2004. The sequence of the latter isolate (reported under our reference NGOV-strain JJL-329) is described in the current study. The NGOV genome was first identified in RNA extracts from ticks by using a highly degenerate primer set designed from the sequences of the polymerase domain of the flavivirus NS5 gene (PF1S/PF2R; Crochu et al., 2004) and a standard RT-PCR protocol (Access RT-PCR system; Promega). Although cell culture of NGOV was attempted, tests using specific primers [JJL3S, 5'-GAGCCGGTTTCTTGAGTTTG-3' (forward), and JJL4R, 5'-GCCACGTATCCGACTCCTG-3' (reverse)] for the presence of the virus genome in these cultures were negative. Aedes albopictus C6/36, green monkey Vero, human SW13 and Xenopus XTC cells were used and none resulted in viral amplification. Results from cerebral injection into newborn mice were also negative, as were cell cultures using Rhipicephalus appendiculatus (RAE25) and Ixodes scapularis (ISE6) cells. A third field collection was organized in the same village in February 2006. Out of 67 ticks analysed as described above, two tested positive for NGOV RNA. Both of them were Rhipicephalus guilhoni ticks sampled from one caprine and one ovine. New attempts to isolate the virus on Vero cells were made. Faint amounts of viral RNA could be detected in cell pellets after three passages, but the viral titre could not be increased to a significant level. The low replicative activity of NGOV in mammalian cells and mice appears unusual with respect to other arboviruses in general and to the flaviviruses in particular. With the previously noted exceptions of CFAV and KRV, all flaviviruses can be propagated in such systems. The failure to isolate NGOV may suggest that it is an arthropod virus with a low ability to infect vertebrates. Nonetheless, because replication was not detected in tick cell lines, the possibility that the absence of replication is due to non-adapted experimental procedures cannot be excluded. Hence, new attempts of propagation of NGOV by intracerebral inoculation of newborn mice and experimental infection of ticks merit further investigation.
The viral nature of NGOV was investigated by molecular biology and electron microscopy. A total nucleic acid extract (DNA+RNA) from the original JJL-329 tick sample was submitted to specific PCR amplification with or without a reverse-transcription step. RT-PCR provided a positive result, whilst direct amplification remained negative, demonstrating that the genome is an RNA molecule. The clarified suspension obtained from tick JJL-355 (2006 field collection) was submitted to nuclease digestion using benzonase (Novagen), an enzyme with strong DNase and RNase activity. This was performed following the manufacturer's recommendations before and after ultracentrifugation purification (the suspension was applied to a 4 ml 25 % sucrose cushion; the sucrose gradient was then centrifuged at 32 000 r.p.m. for 2.5 h at 4 °C in an MLS 50 rotor). Subsequent RNA extraction and RT-PCR amplification provided positive results, strongly supporting the encapsidated nature of the NGOV RNA. Electron microscopy was also performed before and after ultracentrifugation, leading to the identification of viral particles of 40–50 nm in diameter and spherical in shape, compatible with the morphology of flaviviruses (see Supplementary Fig. S1, available in JGV Online).
The only tick-borne flavivirus previously identified in Africa, Kadam virus (KADV), was isolated from the tick Rhipicephalus appendiculatus. Accordingly, we decided to determine whether NGOV was related to KADV or comprised a phylogenetic lineage distinct from other members of the genus Flavivirus. By using the same protocol as described above and a degenerate set of primers designed from the NS3 gene of the flaviviruses (X1, box 4 of the protease; X2, motif V of the helicase; Crochu et al., 2004), a 750 bp sequence of NGOV was characterized. The remaining sequence between the NS3 and NS5 genes was then determined by using specific primers deduced from the original sequences and long-range RT-PCR (cMaster RTplusPCR system kit; Eppendorf). The final contiguous sequence subjected to phylogenetic analyses was 4176 bp in length (GenBank accession no. DQ400858
[GenBank]
). The corresponding amino acid sequence was then aligned with those of other flaviviruses by using the MUSCLE algorithm (Edgar, 2004). The highly divergent TABV was excluded from this analysis, as a previous study failed to identify its precise phylogenetic position (de Lamballerie et al., 2002). Phylogenetic trees were estimated by using the maximum-likelihood (ML) method available in TREE-PUZZLE (Strimmer & von Haesler, 1996), with 10 000 puzzling steps. To choose the model of amino acid replacement that best fitted the empirical data, the likelihood scores of trees produced by all six models of amino acid replacement available in TREE-PUZZLE were compared, both with equal rates of substitution and with a gamma (
) distribution of rate heterogeneity with a shape parameter (
) of 1.0. The model that produced the phylogeny with the highest likelihood score for this dataset was then used for further analyses, with the ML value of the shape parameter () then estimated from the empirical data. By using our dataset, the Whelan–Goldman (WAG) model of amino acid replacement, with a distribution of rate heterogeneity (with eight rate categories), gave the phylogeny with the highest likelihood. The ML tree of these data is shown in Fig. 1(a). This confirms that NGOV is indeed a member of the genus Flavivirus, but that it forms a distinct phylogenetic lineage related distantly to other viruses, including KADV and other tick-borne arboviruses. However, NGOV is also clearly related more closely to the classical flaviviruses than to CFAV and KRV or TABV. This is confirmed by an analysis of pairwise amino acid distances measured after the readdition of the TABV sequence to our original alignment using CLUSTAL_W (Fig. 1b).
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). As expected, the NS3/NS4A, NS4A/2K and NS4B/NS5 cleavage sites for the NGOV polyprotein are clearly mediated by the viral protease, occurring after two basic residues. In common with the flaviviruses, the 2K/NS4B cleavage is presumably mediated by a host signalase. This cleavage site is predicted by SignalP 3.0 (Bendtsen et al., 2004) and the amino acids located downstream are similar to those of the flaviviruses. The lengths of the NS4A, 2K and NS4B proteins resulting from the polyprotein processing are 126, 23 and 254 aa, respectively, similar to those of the flaviviruses (for example, 126, 23 and 255 aa, respectively, in the case of JEV).
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) and can also be identified in the NGOV polyprotein (Fig. 2b
), suggesting that NGOV NS3 supports the same enzymic activity. Similarly, motifs that have been implicated in the methyltransferase activity of the NS5 protein could be mapped within the NGOV sequence (Fig. 2b). Moreover, the analysis of hydropathy profiles along non-structural proteins of flaviviruses shows that some hydrophilic patterns can be associated with enzymic proteins (NS3, NS5), whilst proteins of the replication complex associated with cellular membranes (NS4A, 2K and NS4B) are more hydrophobic. Comparative hydropathy plots of YFV and NGOV non-structural proteins (Fig. 3) show a similar distribution of hydrophilic and hydrophobic patterns. Taken together, these data suggest that the processing of the NGOV polyprotein and the organization of its replication complex are similar to those of the flaviviruses.
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). The recent discovery of novel arboviruses transmitted by Culex mosquitoes (Bakonyi et al., 2005; Kono et al., 2000; Nisbet et al., 2005) supports this hypothesis. However, the identification of KRV and cell silent agents 1 and 2 and the characterization of TABV, as well as the discovery of Ngoye virus, suggest that a large number of viruses related distantly to the group of classical arthropod-borne flaviviruses also remain to be discovered. Crucial in the identification of novel flaviviruses has been the use of molecular amplification systems employing highly conserved enzymic motifs of the NS3 and NS5 genes. These have allowed the characterization of cell silent agents 1 and 2 (Crochu et al., 2004), TABV (de Lamballerie et al., 2002), new American isolates of CFAV (Cook et al., 2006) and Ngoye virus, and it is evident that they will continue to constitute valuable tools for the discovery of classical and atypical flaviviruses.
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ACKNOWLEDGEMENTS |
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Received 27 March 2006;
accepted 23 June 2006.
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