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1 Department of Zoology, University of Oxford, South Parks Road, Oxford OX1 3PS, UK
2 Asia-Pacific Institute of Tropical Medicine and Infectious Diseases, University of Hawaii at Manoa, 3675 Kilauea Avenue, Honolulu, HI 96816, USA
3 Department of Biology, The Pennsylvania State University, University Park, PA 16802, USA
4 Unité des Virus Emergents, Faculté de Médecine de Marseille, Université de la Méditerranée, 27 Boulevard Jean Moulin, 13385 Marseille Cedex 05, France
Correspondence
Shelley Cook
shelley.cook{at}balliol.oxon.org
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ABSTRACT |
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Published online ahead of print on 18 January 2006 as DOI 10.1099/vir.0.81475-0.
The GenBank/EMBL/DDBJ accession numbers for the sequences described in this paper are DQ181421–DQ181515.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Similarly, Yellow fever virus (YFV) affects 200 000 individuals annually (Monath, 2001), with a case-fatality rate of around 20 % (Monath & Heinz, 1996), and the disease caused by Japanese encephalitis virus (JEV) is believed to represent the earliest documented aetiology of viral encephalitis (Halstead & Jacobson, 2003).
A number of recent advances have been made in understanding the taxonomic distribution of the flaviviruses. First, the complete genome sequence of Tamana bat virus (TABV) was determined (de Lamballerie et al., 2002). TABV was a hitherto unclassified flavivirus originally isolated in 1973 from the insectivorous bat Pteronotus parnellii (Price, 1978). TABV was found to share many characteristics with the flaviviruses, including similar genomic organization, hydropathy plots and conserved polyprotein-cleavage sites and enzyme domains. Second, by using a phylogenetic approach to estimate the level of taxon sampling in a clade, Pybus et al. (2002) suggested the number of unsampled taxa in the mosquito-borne flavivirus clade to be approximately 2000. This was supported by the recent isolation and identification of a new flavivirus, Kamiti River virus (KRV), from Aedes macintoshi mosquitoes in Kenya (Crabtree et al., 2003; Sang et al., 2003). In terms of both nucleotide sequence and growth kinetics in culture, KRV was most similar to the only other known insect-only flavivirus, cell fusing agent virus (CFAV). Perhaps most surprising of all was the observation that sequences related to the flaviviruses persist in DNA form integrated into the genome of some Aedes sp. mosquitoes (Crochu et al., 2004). Specifically, an open reading frame (ORF) predicted to encode a protein of 1557 aa, related closely to CFAV and KRV, was observed in both laboratory-bred and wild Aedes albopictus mosquitoes and the cell line C6/36. Similarly, in both the Aedes aegypti cell line A20 and laboratory-bred and wild A. aegypti samples, a 492 aa-encoding ORF related to CFAV and KRV sequences was detected. Other flavivirus-like sequences, in which genes were truncated or contained multiple stop codons, were also found. These sequences probably resulted from two or more independent integration events, following infection of each mosquito species by a virus (or viruses) related to the CFAV group. Taken together, these findings indicate that further members of the CFAV group may exist in the wild that remain unidentified to date.
CFAV was first recognized in 1975 as the agent that caused A. albopictus cells to undergo massive syncytium formation following inoculation with medium from an A. aegypti cell line (Stollar & Thomas, 1975). Further characterization of the agent showed that it comprised a single-stranded, positive-sense RNA virus, with a number of properties suggesting that it was related to the flaviviruses. For example, virions were approximately 50 nm and, of the three structural proteins, the larger 49 and 16·5 kDa proteins were glycosylated and associated with the envelope, whereas the smaller 13 kDa protein was associated with the viral RNA (Igarashi et al., 1976; Stollar & Thomas, 1975). However, the 16·5 kDa envelope protein was much larger than that typically seen in flaviviruses, there was a lack of haemagglutination-inhibition activity when tested against several flaviviral antigens and a failure of anti-CFAV serum to react with cells infected with DENV-2, JEV or Bovine viral diarrhea virus. Therefore, at the time of its isolation, CFAV was considered to be an ungrouped member of the family Togaviridae, together with the flaviviruses and the pestiviruses (Porterfield et al., 1978). Later work focused on determination of the complete nucleotide sequence of CFAV and highlighted its relationship with viruses of the genus Flavivirus, family Flaviviridae (Cammisa-Parks et al., 1992). In the Seventh Report of the International Committee on Taxonomy of Viruses, CFAV was assigned as a tentative species in this genus (Heinz et al., 2000). In contrast to the majority of flaviviruses, CFAV does not appear to have a vertebrate host, as in culture, the virus replicates in mosquito cells, but not in mice or other vertebrate cells (Harbach, 1988; Stollar & Thomas, 1975). The CFAV genome comprises a single, long ORF that encodes three structural and seven non-structural proteins. Amino acid sequence similarity between CFAV proteins and those of other flaviviruses is highest for the non-structural proteins NS5 and NS3 and lowest for the structural proteins.
As part of a study to investigate the distribution and biodiversity of flaviviruses and their vectors, we collected mosquitoes in Puerto Rico at the end of the rainy season of 2002. Samples were screened via RT-PCR using degenerate primers designed across the entire genus Flavivirus. Initial results suggested the presence of a sequence related closely to CFAV in a number of samples. Following further RT-PCR and culture work, we determined the presence of a new strain of CFAV in a large number of samples, which we identified to mosquito species level by using molecular identification via COI and COII sequences (Cook et al., 2005). Notably, the virus was found in wild-caught mosquitoes of both sexes, from sampling sites across Puerto Rico itself and an offshore island, Culebra, and in mosquito species other than A. aegypti, including A. albopictus and Culex spp. This represents the first isolation of a novel strain of CFAV and the first discovery of this virus in a natural mosquito population.
In addition to investigating the distribution of the CFAV Culebra strain, we show here the results of a phylogenetic analysis of the virus with the other members of the genus Flavivirus. Finally, we present results from experimental-infection tests conducted using the original CFAV strain. Here, the production of DNA forms of the CFAV genome was investigated, with the aim of contributing to the recent observations of DNA forms of flaviviral sequences inserted into the genomes of Aedes sp. mosquitoes made by Crochu et al. (2004).
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METHODS |
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shows sampling locations in Puerto Rico. Standard CDC fan-augmented light traps were supplemented with 2·27 kg (5 lb) dry ice and placed in trees at various heights in and around public spaces for 8 h trapping periods (McNelly, 1989). Commercial propane-powered traps (Mosquito Magnet; American Biophysics Corp.) were also used to attract hungry females via production of CO2, heat and water vapour. In addition, manual aspiration was conducted regularly in both natural (e.g. campus bathrooms, residential basements) and artificial (e.g. box traps made from small, dark-coloured cardboard boxes with a partially open lid) roosting areas. Mosquitoes were placed on ice upon collection at the sampling site. Upon arrival at the laboratory, samples were sorted on a chill table according to trap, location and sex (including noting engorged status for females) and placed in microtitre plates before storage at –80 °C.
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Pooled RNA extraction and controls.
Three 65 µl homogenate samples from three mosquitoes were combined per pool for RNA extraction. Ten microlitres of a supernatant of enterovirus culture (echovirus 30 propagated in MRC5 human fibroblast cell culture until gross cytopathic effect) was also added to each pool to act as a positive control for RNA extraction. RNA extraction was then conducted via the MagNApure LC system using an RNA II Culture Cells kit and the standard manufacturer's protocol (Roche Diagnostics). Ten microlitres of each resultant RNA extract was used in an enterovirus-detection TaqMan assay (Applied Biosystems) to check for successful extraction and the presence of inhibitors. The TaqMan protocol followed that of Watkins-Riedel et al. (2002), with slight modification of the primers EV1S (5'-CCCTGAATGCGGCTAATCC-3'), EV1R (5'-ATTGTCACCATAAGCAGCCA-3') and probe EV_ENTTM1 (FAM-CADGGACACCCAAAGTAGTCGGTTCC-TAMRA).
Initial screening for flaviviruses via RT-PCR.
Semi-nested RT-PCR was conducted on all RNA extracts by using degenerate primers designed across the flaviviruses. First-round amplification consisted of a one-step RT-PCR using primers PF1 and PF2 (20 µM; Invitrogen) and a Qiagen One-Step RT-PCR kit, according to the standard manufacturer's protocol. Second-round PCR amplifications were performed in 25 µl volumes using primers PF2 and PF3 (50 µM; Invitrogen). Each mastermix was prepared in a flow hood in an area separate from all other DNA and RNA work in order to avoid contamination. PCR was conducted according to the standard manufacturer's protocol (ABGene). See Table 1 for all primer sequences and reaction conditions.
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). Before large-scale screening was conducted on the Puerto Rican samples, the system was tested on RNA extracted from viral cultures of both tick- and mosquito-borne members of the genus, in addition to RNA extracted from DENV-inoculated mosquitoes. Having determined the efficacy of the system, we used the DENV-inoculated mosquitoes as positive controls for large-scale screening. A number of laboratory-bred mosquitoes were then tested for infection with viral CFAV and RNA from related flaviviruses, using PF1–PF2–PF3. These were used as negative controls for large-scale screening. Both positive and negative controls were carried through all stages of the study, from initial homogenization to second-round PCR.
CFAV genome from individual mosquito samples.
For the first four pools that gave positive results for CFAV using the primer set PF1–PF2–PF3, RNA was re-extracted directly from the original individual 12 mosquito homogenates by using Promega RNAgents according to the manufacturer's instructions. One-step RT-PCRs were conducted on these new RNA extractions, using a range of primer sets designed along the CFAV genome (‘CFA’ and ‘MCFA’ primer pairs, 20 µM; Invitrogen; see Table 1
). Of these 12 mosquitoes, three particular individuals, labelled mosquitoes A, B and C, gave consistently clean, positive results for the majority of CFAV-specific primer pairs. As a consequence, their viral sequences were used for phylogenetic analyses.
SeqMan II (DNASTAR Inc.) was used to combine reverse and forward viral sequences from each sample. In total, 3618 bp of the novel CFAV strain was obtained by using the primer pairs detailed in Table 1. These sequences were then compared with those of other members of the genus Flavivirus. Table 2 lists the flaviviral sequences that were used in analyses, comprising all those that are available in public databases at present. Datasets were prepared for the NS5 gene, NS3 gene and entire genomes and compiled by using Se-Al (available at http://evolve.zoo.ox.ac.uk/software/Se-Al/main.html). Sequences were then aligned by using CLUSTAL_X version 1.8 (Thompson et al., 1997). Phylogenetic trees were inferred from deduced amino acid sequences by using the maximum-likelihood (ML) method available in TREE-PUZZLE (Strimmer & von Haeseler, 1996). In all cases the Whelan–Goldman model of amino acid replacement was used (Whelan & Goldman, 2001), with values for the 
(gamma) distribution of rate variation (with eight categories) estimated from the data. For analysis of the genome dataset, regions in which alignment was ambiguous were removed to produce a concatenated dataset. Final datasets used for analysis comprised 1147 aa and 27 sequences for the concatenated genome data, 244 aa and 32 sequences for the NS3 data and 93 aa and 75 sequences for the NS5 data. Phylogenetic trees were rooted on TBV, which was previously shown to be a viable outgroup (de Lamballerie et al., 2002).
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For each passage, detection of CFAV sequence in both RNA and DNA forms was attempted by using CFAV-specific primers designed from the complete genome sequence of the virus (MCFA_V1S and MCFA_V1R, or CFA-3R and CFA-3S; see Table 1). For the detection of RNA forms of the virus genome, extraction from the culture supernatant was performed by using a MagNApure LC System RNA II Culture Cells kit and followed by a 60 min digestion in the presence of 10 U bovine pancreas DNase I (Roche). RT-PCR was performed by using the One-Step Access RT-PCR system (Promega). For the detection of DNA forms of the virus genome, extraction from pelleted C6/36 cells was performed by using the Chelex 100 method (de Lamballerie et al., 1992) and PCR amplification was conducted by using the One-Step Access RT-PCR system (Promega) in the absence of avian myeloblastosis virus reverse transcriptase (RT).
Extraction, PCR amplification and sequencing of mosquito DNA.
The COI and COII genes for each individual CFAV-positive mosquito were sequenced. DNA was extracted from mosquitoes by using the Chelex 100 method (de Lamballerie et al., 1992) or the Biorobot EZ1 system according to the manufacturer's instructions (Qiagen). COI and COII genes were amplified by using the PCR primers UEA3 and FLY10, and SCTL2-J-3037 and TK-N-3785, respectively (10 µM; Invitrogen; Table 1; Cook et al., 2005).
Phylogenetic analysis of mosquito sequences.
SeqMan II (DNASTAR Inc.) was used to combine reverse and forward sequences from each mosquito and final datasets were compiled by using Se-Al. These sequences were combined with those currently available from GenBank as detailed in Table 3.
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+I model of nucleotide substitution, with the GTR substitution matrix, the base composition, the gamma distribution of among-site rate variation () and the proportion of invariant sites (I) all estimated from the data. All parameter values are available from the authors on request. To assess the robustness of particular phylogenetic groupings, a bootstrap-resampling analysis was undertaken by using 100 replicate neighbour-joining trees using the ML substitution matrix described above. All phylogenetic analyses were performed by using the PAUP* package (Swofford, 2000).
Detection of DNA forms of the CFAV genome during experimental infection of mosquito-cell cultures.
C6/36 and AA23 A. albopictus cells, A. aegypti (A20) and Aedes W-albus cells were grown in 12·5 cm flasks at 28 °C as reported previously (Crochu et al., 2004). Subconfluent cultures were infected with 100 TCID50 CFAV, original strain. After a 30 min incubation at room temperature, the cell layer was washed twice with PBS (pH 7) and incubated at 28 °C in the presence of fresh culture medium. Any possible contaminating DNA was removed carefully from the infecting suspension by centrifugation (30 min at 3000 g) and treatment by benzonase (Novagen) according to the manufacturer's recommendations. Infection experiments using C6/36 cells were repeated in the presence of nucleoside RT inhibitors (azydothymidine, didanosine, stavudine, lamivudine and tenofovir) and non-nucleoside RT inhibitors (nevirapin, efavirenz and abacavir). All RT inhibitors were tested in quadruplicate at 50 and 100 µg ml–1 final concentrations in the culture medium. They were added to the culture medium 1 h before infection and again in the fresh medium used at time 0 p.i. In the case of azydothymidine, an additional experiment was conducted in which the inhibitor was present in the culture medium 12 h before infection. The presence of DNA forms of the viral genome was investigated at 2, 4, 8, 12, 24, 48 and 72 h p.i. DNA was extracted from pelleted cells by using the same protocols as described above.
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RESULTS |
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In total, 1584 mosquitoes were screened in 528 pools via RT-PCR with degenerate primers PF1–PF2–PF3. Positive results for a strain of CFAV, as identified via BLAST (Altschul et al., 1997), were obtained in 67 pools. The nucleotide sequences of all 67 CFAV amplicons obtained during initial flaviviral screening using primers PF1–PF2–PF3 were virtually identical and variation in viral sequence between mosquitoes appeared to be surprisingly low.
RT-PCR of the CFAV genome by using specific primers
Fig. 2 shows results from the concatenated-genome phylogenetic analysis. In terms of general topology, the no known vector (NKV) group and the tick-borne flaviviruses appear to form a well-supported sister group to the mosquito-borne clade, as has been suggested by some previous studies (Billoir et al., 2000). Most striking is the fact that the novel virus is clearly related most closely to CFAV, with the CFAV clade forming a well-supported sister group to KRV. The sequences for mosquitoes A, B and C were identical. The uncorrected pairwise distance between the consensus novel CFAV strain and the GenBank CFAV sequence was 4 % at the complete-genome nucleotide level, suggesting strongly that these constitute strains of the same virus. We have designated the novel CFAV as strain Culebra, named after the offshore island from which the virus was first isolated.
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Cell culture, viral isolation and identification of CFAV-positive mosquitoes
The MCFA_V1S/R primer set used for viral isolation was tested by using nucleic acid extracts from all (non-infected) cell lines used in the current study and constantly generated negative results, demonstrating that they are highly specific to the CFAV sequence and do not allow the amplification of flavivirus-like integrated sequences (Crochu et al., 2004). Viral culture was then attempted on homogenates from all 201 potentially infected mosquitoes, i.e. those individuals that contributed to the CFAV-positive pools (individuals numbered M1–M201). The novel CFAV strain was isolated from the P1 supernatant for 51 individual mosquitoes. Clean sequence was obtained for both the COI and COII genes from 44 of these 51 CFAV-positive mosquitoes and further phylogenetic analyses were limited to these 44 individuals. Sequences of approximately 1295 bp for COI and 792 bp for COII were obtained. This covered most of the COI gene and the entire COII gene for each specimen. No insertions or deletions were identified within the aligned sequences. In addition, the alignment of derived COI and COII sequences was unambiguous, as there was no length variation in the amplified gene region. Figs 3 and 4 show results for the phylogenetic analysis of the COI and COII datasets, respectively, and results are summarized in Table 4. It can be seen that the majority of the mosquitoes fell into one of four clades; these have been designated clades A–D.
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Detection of DNA and RNA forms of the CFAV genome
During preliminary work for the current study using the original reference strain of CFAV, RNA forms of the viral genome were detected in the culture supernatant, but DNA forms were also detected in pelleted C6/36 cells, suggesting that CFAV infection leads spontaneously to DNA forms of the virus genome, as indicated previously (Crochu et al., 2004). This finding was fully confirmed when various mosquito cell cultures were infected with the CFAV reference strain: large numbers of RNA genomes could be detected by RT-PCR for all cell cultures at 72 h p.i., confirming active replication of the virus. DNA forms of the CFAV genome were detected repeatedly from both C6/36 and AA23 A. albopictus cell lines 24 h p.i. After 72 h, cell lysis was complete and prevented further investigation. Infected cells were passaged four times and positive direct PCRs were obtained for all passages. Amplicons were sequenced and found to be identical to the CFAV sequence and therefore clearly distinct from cell silent agent inserts (Crochu et al., 2004). Further analysis of these DNA forms was conducted: CFAV-specific PCR primers designed all along the viral genome were used to perform direct PCR in all structural and non-structural genes and in non-coding regions (see Table 1). After 48 h, all systems provided positive results and the sequences of amplicons were identical to that of CFAV in all regions tested. Infection of A. aegypti (A20) and A. W-albus cells by CFAV led to similar results: DNA forms of the CFAV genome were detected repeatedly after 48 h p.i.
Effect of RT inhibitors on synthesis of DNA forms of CFAV genomes in A. albopictus cells
None of the molecules tested could inhibit the synthesis of DNA forms of the CFAV genome, regardless of the time of addition (1 or 12 h before infection in the case of azydothymidine) or final concentration (50 or 100 µg ml–1) used.
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DISCUSSION |
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), confirming that they are closely related.
It has recently been shown that COI and COII gene sequences can be used to identify mosquitoes reliably at the species level (Cook et al., 2005). In the current study, which includes published sequences from laboratory colonies and wild-caught samples drawn from worldwide locations, Aedes and Culex spp. form well-supported clades. We have shown that CFAV strain Culebra was present in a range of mosquito species, including some Culex species, A. aegypti and A. albopictus. The presence of the virus in the latter of these species is particularly notable, as the original strain of CFAV was found to cause massive syncytium formation in A. albopictus cells, whereas the novel strain of the virus from Puerto Rico was found in mature A. albopictus mosquitoes. The diversity and distribution of mosquitoes in Puerto Rico have not been studied extensively and literature is extremely limited. The most recent and wide-ranging survey comprises a checklist based on morphological characters (Belkin & Heinemann, 1975) and hence more detailed identification of the CFAV-positive mosquito species would require wide-ranging research aimed at (i) an exhaustive survey of the mosquito fauna of the island, (ii) morphological identification by an entomologist and (iii) the determination of molecular markers for the species found. This is clearly a priority for future research. Interestingly, the checklist produced by Belkin & Heinemann (1975) does not include the species A. albopictus, which may therefore represent a relatively recent introduction to the island.
Overall, it is clear that the novel CFAV strain was present in a number of Aedes and Culex species. The use of DENV-inoculated mosquitoes as positive controls for large-scale screening using PF1–PF2–PF3 demonstrates that no other flaviviruses were present in the samples. The CFAV-positive samples were also distributed over a large geographical area, with the virus present in locations across the island of Puerto Rico and on the offshore island Culebra. Importantly, the novel CFAV strain was also found in mosquitoes of both sexes. Together with its wide geographical distribution, this suggests that the novel CFAV strain may be transmitted vertically via oviposition, a mechanism shown to be possible under experimental conditions in the case of WNV (Goddard & Wolstenholme, 1978).
RT-PCR is sensitive, does not require virus amplification and has been recommended for routine arbovirus surveillance (Howe et al., 1992). Indeed, numerous flaviviruses have recently been detected and sequenced by using RT-PCR on RNA extracted from mosquito tissue samples (Jupp et al., 2000). Recently, Kramer et al. (2002) compared diagnostic assays for the detection of St Louis encephalitis virus in mosquito pools for sensitivity, accuracy and specificity. In situ enzyme immunoassay, plaque assay on Vero cells, passage in A. albopictus Skuse C6/36 and C7/10 cells, antigen-capture enzyme immunoassay and RT-PCR were evaluated by using pools of 50 mosquitoes containing one or two experimentally infected individuals. It was determined that RT-PCR was the most sensitive assay, with a detection limit of <0·1 p.f.u. In the current study, we developed a degenerate RT-PCR system that was applicable to bulk samples comprising large numbers of wild-caught mosquitoes. The system was shown to be sensitive to the presence of a sequence with high similarity to known flaviviruses within the sample mosquito population. However, the presence of viral sequence alone does not necessarily mean that infective or replicative virus is present (or at least that live virus is present in levels high enough to be transmitted). Therefore, initial screening was confirmed by cell culture and viral isolation. Hence, in addition to the discovery of a novel viral strain, we have shown that this system represents a viable surveillance tool for all flaviviruses.
DNA sequences related to flaviviruses have recently been found integrated into the genome of Aedes sp. mosquitoes (Crochu et al., 2004). It was suggested that this occurred via infection of mosquitoes by flaviviruses related to CFAV that are yet to be characterized. Our current study supports this hypothesis by demonstrating the presence of a novel CFAV-related virus in a natural mosquito population. In this case, the novel CFAV-related virus is clearly different from those sequences found integrated into genomes of Aedes sp. mosquitoes. Taken together, these observations suggest that a range of such CFAV-related members of the genus may exist in nature. Results from experimental infection using the original CFAV strain are consistent with the existence of a highly efficient RT mechanism in which initiation of copying occurs at the 3' terminus of either the genomic RNA or the intermediate of replication. This provides new insights into the potential mechanism by which integration of a flaviviral sequence from a novel virus into the mosquito genome may have occurred.
The process by which the novel CFAV strain spread to Puerto Rico remains unclear. The time to the most recent common ancestor of CFAV and KRV is estimated to be around 3500 years ago (Crochu et al., 2004) and the majority of flaviviruses infecting Aedes species are found in the Old World (Gould et al., 2001). It therefore seems likely that the novel CFAV strain has spread ‘horizontally’ across the globe, potentially influenced by human migration in a manner similar to the spread of YFV to the New World during the 18th century via the slave trade (Strode, 1951).
As the flaviviruses include members that are categorized as having both vertebrate and invertebrate hosts, those with invertebrate hosts only and those with vertebrate hosts only, the genus Flavivirus represents a useful model for the evolution of vector-borne disease and host specificity. Phylogenetic studies based on the E gene of the flaviviruses indicate that CFAV may represent a basal lineage of the genus that separated from the other flaviviruses before the separation of the mosquito- and tick-borne groups (Cammisa-Parks et al., 1992; Marin et al., 1995). It has further been suggested that arthropod-borne viruses may have evolved from insect-only viruses (Schlesinger, 1971) and, to date, CFAV and KRV represented the only known members of the latter category. The isolation of a novel strain of CFAV provides additional data for genetic comparisons and points to new opportunities for the investigation of flaviviral phylogeny and evolution. Together with recent studies that have suggested the possible existence of over 2000 unknown flaviviruses (Pybus et al., 2002), it is clear that further research aimed at a deeper investigation of the distribution and diversity of flaviviruses related to CFAV is essential.
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ACKNOWLEDGEMENTS |
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Received 31 August 2005;
accepted 9 January 2006.
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