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1 Natural History Museum, Cromwell Road, London SW7 5BD, UK
2 Unité des Virus Emergents UMR190 ‘Emergence des Pathologies Virales’, Université de la Méditerranée et Institut de Recherche pour le Développement, Marseille, France
3 Mosquito Research Programme, Uganda Virus Research Institute, PO Box 49, Entebbe, Uganda
4 Centre for Ecology and Hydrology Oxford, Mansfield Road, Oxford OX1 3SR, UK
5 Department of Biology, The Pennsylvania State University, University Park, PA 16802, USA
Correspondence
Shelley Cook
s.cook{at}nhm.ac.uk
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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The GenBank/EMBL/DDBJ accession numbers for the COI gene sequences from mosquito species obtained during this study are GQ165759 [GenBank] –GQ165807 [GenBank] , that for Culex flavivirus Uganda is GQ165808 [GenBank] , that for Nakiwogo virus is GQ165809 [GenBank] and that for cell fusing agent virus strain Rio Piedras is GQ165810.
Three supplementary tables listing primer sequences used, flaviviral abbreviation codes and accession numbers, and mosquito accession numbers and Barcode of Life Datasystem (BOLD) reference numbers are available with the online version of this paper.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; nomenclature for the aedine mosquitoes follows Reinert (2000) and Reinert et al. (2004, 2006, 2008)] and characterized by Cammisa-Parks et al. (1992), previously represented the only known ‘insect-only’ flavivirus, appearing to replicate uniquely in insect cells (Kuno, 2007). In recent years, Cook et al. (2006) reported the first isolation of CFAV, from a natural mosquito population in Puerto Rico. The virus was found in several different species and in mosquitoes of both sexes. Two strains of a second member of the ‘insect-only’ group, designated Kamiti River virus (KRV), were isolated from Neomelaniconion mcintoshi mosquitoes from Kenya {Crabtree et al., 2003; Sang et al., 2003; these authors stated that KRV was isolated from ‘Ae. macintoshi’ from flooded dambos in Central Province, Kenya. However, Aedes (Ochlerotatus) macintoshi (Marks) [Ochlerotatus macintoshi in the classification of Reinert et al. (2008)] is an Australian mosquito, and the species in this case is actually the Rift Valley fever vector Neomelaniconion mcintoshi (Huang) (originally named Aedes mcintoshi), whose larvae are frequently found in dambos}. Taken together with theoretical work that suggested the existence of over 2000 unknown mosquito-borne flaviviruses (Pybus et al., 2002), as well as the discovery of DNA sequences related to flaviviruses in the genomes of St. aegypti and Stegomyia albopicta, which probably resulted from integration events following infection of each mosquito species by a virus (or viruses) related to the CFAV group, it was proposed that further ‘insect-only’ flaviviruses were likely to be discovered in natural mosquito populations (Crochu et al., 2004). Indeed, sequences related to CFAV been discovered have more recently in St. aegypti mosquitoes from Thailand (Kihara et al., 2007). A third member of the ‘insect-only’ group, Culex flavivirus (CxFV), was first isolated in 2007 from Culex tritaeniorhynchus and Culex quinquefasciatus in Japan and Indonesia (Hoshino et al., 2007). Further strains have subsequently been isolated from Cx. quinquefasciatus in Guatemala (Morales-Betoulle et al., 2008) and Mexico (Farfan-Ale et al., 2009), and from Cx. quinquefasciatus and Culex restuans in Texas, USA, and Trinidad (Kim et al., 2009). Finally, flavivirus RNA has also recently been discovered in phlebotomine sandflies from Algeria (Moureau et al., 2009).
We now report the discovery, isolation and characterization of two novel flaviviruses of the ‘insect-only’ group from the Entebbe area of Uganda: a novel lineage, tentatively designated Nakiwogo virus (NAKV), and a new strain of CxFV. The individual mosquitoes from which these strains were isolated, identified retrospectively from a backbone phylogeny generated using the ‘barcode’ region of voucher specimens from the area, were specimens of Mansonia africana nigerrima and Cx. quinquefasciatus, respectively. To date, insect-only flaviviruses have been isolated from Culex, Stegomyia and Ochlerotatus mosquitoes, in common with the majority of the mosquito-borne flaviviruses. This work represents, to our knowledge, the first isolation of a novel insect-only flavivirus from a Mansonia species and the first isolation of a strain of CxFV from Africa. In addition, we include in our analyses, for the first time, the full genome sequence from CFAV Rio Piedras, isolated previously from Puerto Rico (Cook et al., 2006).
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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), using different methods to maximize species diversity. Centers for Disease Control (CDC) fan-augmented light traps were supplemented with dry ice for approximately 8 h trapping periods between dusk and dawn and placed at various heights in urban, rural and forest environments. Modified backpack aspirators and hand-held aspirators were used to sample resting mosquitoes. Specimens were placed on dry ice immediately upon collection at the sampling sites. Upon arrival at the laboratory, samples were sorted on a chill table according to trap, location and sex and placed in individual wells in microtitre plates before storage at –80 °C.
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). Samples of 65 µl from five individual mosquitoes were pooled for RNA extraction. Total nucleic acid (NA) extraction was conducted via the MagNApure LC System using a Culture Cells kit and standard manufacturer's protocol (Roche Diagnostics), or using a Biorobot EZ1, with Virus Mini kit v2.0 (Qiagen).
A 5 µl aliquot of each pooled RNA extraction was used in a one-step real-time QuantiTect SYBR-Green RT-PCR assay, according to Moureau et al. (2007), with primers PF1S and PF2R-bis (see Supplementary Table S1, available in JGV Online). Second-round PCR on 2.5 µl cDNA, using primers PF3S and PF2R-bis, used the following conditions: 94 °C for 2 min; 40 cycles of 94 °C for 30 s, 50 °C for 45 s and 72 °C for 1 min; followed by a final extension of 72 °C for 7 min and cooling to 20 °C for 2 min. To avoid any potential contamination, positive cDNA samples from first-round RT-PCR were manipulated separately from all other samples for second-round PCR. Negative controls comprising both NA extractions from non-infected mosquitoes and reactions in which water replaced RNA were performed through all stages. Positive controls comprised Rio Bravo, Japanese encephalitis and Montana myotis leukoencephalitis viruses (RBV, JEV and MMLV, respectively).
Second-round PCR products were analysed visually by electrophoresis through ethidium bromide-stained 2 % agarose gels under UV light. Products were purified by using a QIAquick Spin PCR Purification kit (Qiagen). Amplicons were sequenced on both strands with an ABI 377 automated sequencer (Applied Biosystems).
For those samples that gave positive results using the primer set PF1S–2Rbis–3S on RNA extractions from pooled mosquitoes, NA were re-extracted from the original individual mosquito homogenates by using a Biorobot EZ1, with Virus Mini kit v2.0 according to the manufacturer's instructions (Qiagen). One-step SYBR green first-round RT-PCR was followed by second-round PCR using the PF1–2bis–3 system as described for the pooled samples. Second-round PCR products were analysed visually, purified and sequenced as described above.
Further investigation of individual samples and virus isolation.
Individual mosquito homogenates were investigated further based on (i) congruent positive sequences in both pooled sample and individual mosquitos and (ii) the resulting 157 nt NS5 sequence clustering with other known ‘insect-only’ flaviviruses.
For the individual mosquito homogenates chosen for virus isolation work, 100 µl homogenate was centrifuged at 13 000 r.p.m. (16 100 g) for 20 min. Subsequently, the supernatant was diluted in a 1 ml final volume of L15 medium without fetal bovine serum (FBS), but enriched with antibiotics (ml–1: 100 IU penicillin G, 100 mg streptomycin, 100 mg kanamycin, 2.5 µg amphotericin B) and inoculated onto C6/36 cells in single 12.5 cm2 flasks. After incubation at room temperature for 1 h, 4 ml fresh 3 % FBS L15 medium was added. The flask was incubated at 28 °C. A negative-control flask was handled under identical conditions. Flasks were examined daily for the presence of cytopathic effect [i.e. cell fusion and/or syncytium formation similar to that seen in the prototype CFAV, as described by Stollar & Thomas (1975)] and 400 µl of each supernatant medium was extracted and tested by real-time RT-PCR. A series of four passages on C6/36 cells was carried out for each positive isolate.
The strategy for sequencing of the entire open reading frame (ORF) followed Grard et al. (2007). Initial RT-PCR amplifications targeted regions encoding the E, NS3 and NS5 proteins using degenerate primers (Crochu et al., 2004; Gaunt & Gould, 2005; Gaunt et al., 2001; Moureau et al., 2007). Specific primers were then designed to complete gaps via long-range RT-PCR (cMaster RTplusPCR system; Eppendorf) and amplicons were sequenced using the long PCR product sequencing (LoPPS) method, a shotgun-based approach applied to long PCR amplification products (Emonet et al., 2006, 2007). Primer sequences are available from the corresponding author upon request.
Transmission electron microscopy.
Tissue-culture samples showing cytopathic effect were prepared for electron-microscopic examination. Negative-stained electron-microscopic specimens were prepared by drying culture supernatant medium, mixed 1 : 1 with 2.5 % paraformaldehyde, onto Formvar/carbon-coated grids and staining with 2 % methylamine tungstate.
Investigation for the presence of virus-specific DNA.
It has been previously shown that DNA forms of CFAV are produced during infection of a number of different mosquito cell types (Cook et al., 2006). Therefore, for flaviviral isolates, total NA were extracted from C6/36 cultures of each virus at passage 5, day 6 post-infection by using a Biorobot EZ-1. Both supernatant medium and sedimented cells were tested using the primer pairs detailed in Supplementary Table S1. First, sedimented cells and supernatant medium were tested by using standard PCR with no RT step. Second, the same primer pairs were used for a one-step RT-PCR (Promega). Third, NA from sedimented cells were treated with recombinant DNase I according to the manufacturer's instructions (Roche). These DNase-treated samples were then tested in a classic PCR with no RT step.
Phylogenetic analyses of viral sequences.
Sequencher v4.8 (Gene Codes) was used to combine reverse and forward viral sequences. Sequences were then compared with those of all other members of the genus Flavivirus available to date. Supplementary Table S2 (available in JGV Online) lists the flaviviral sequences that were used in analyses. Datasets were prepared by using Se-Al (available at http://tree.bio.ed.ac.uk/software/seal/) for (i) nucleotide data for the region encoding the NS5 protein, (ii) amino acid alignment of the complete ORF and (iii) nucleotide and amino acid sequences for the region encoding the E protein for the insect-only flaviviruses, as this currently comprises the greatest number of species available for this group. All nucleotide analyses were repeated both including and excluding third-codon positions (as these were likely to experience site saturation).
For the NS5 region, the dataset was primarily limited to those flaviviruses for which ORF data were also available, to allow comparison between phylogenies. Nucleotides were aligned by using MUSCLE (Edgar, 2004) with manual adjustment to maintain correct reading frame. The model of nucleotide substitution and parameter values were selected via MODELTEST (Posada & Crandall, 1998) and used to estimate maximum-likelihood (ML) phylogenetic trees in PAUP (Swofford, 2000). Values for the substitution matrix, base composition, gamma distribution of among-site rate variation (
) and the proportion of invariant sites (I) are available from the authors on request. A bootstrap-resampling analysis was conducted using 1000 replicate neighbour-joining (NJ) trees based on the ML substitution matrix. Phylogenetic analyses were also performed using the Bayesian method available in MrBayes v3.1.2 (Huelsenbeck & Ronquist, 2001) with a minimum of 10 million generations and a burn-in of 10 %. Stationarity was assessed at effective sample size >400 using Tracer v1.4.1 (part of the BEAST package; Drummond & Rambaut, 2007). For the full ORF dataset, amino acids were aligned by using MUSCLE and phylogenetic analyses were performed in MrBayes, with a minimum of 10 million generations and a burn-in of 10 %. For the E region, nucleotides were aligned based on alignment of amino acid sequences in MUSCLE. ML phylogenetic analyses conducted by using MODELTEST and PAUP were identical to those carried out for the NS5 region. In addition, Bayesian analyses were conducted on the amino acid dataset in MrBayes as described above, after removing 17 sites of ambiguous alignment using GBlocks (Talavera & Castresana, 2007). Final datasets comprised 60 sequences (2514 sites) for the NS5 region (corresponding to nucleotide positions 7790–10270 of YFV; GenBank accession no. X03700
[GenBank]
), 58 sequences (3801sites) for the ORF and 27 sequences (417 sites) for the E region (corresponding to amino acid positions 1184–1531 of YFV; GenBank accession no. U17067
[GenBank]
).
Extraction, amplification and sequencing of voucher mosquito DNA.
Voucher mosquito specimens had previously been identified using morphology (Edwards, 1941; Service, 1990). A 130 µl volume of Chelex 100 Resin 15 % (w/v) (Bio-Rad) was added to 20 µl mosquito homogenate. This was incubated at 95 °C for 20 min and centrifuged at 13 000 r.p.m. (16 100 g) for 15 min. Further analysis was conducted using 50–60 µl supernatant and, of this extraction, 1.0–2.5 µl was used in PCRs. DNA was also extracted, amplified and sequenced from those individual mosquito homogenates found to be positive for novel flavivirus strains.
The COI gene was amplified by using primers UEA3 (5'-TATRGCWTTYCCWCGAATAAATAA-3') (Lunt et al., 1996) and Fly10 (5'-ASTGCACTAATCTGCCATATTAG-3') (Sallum et al., 2002) according to Cook et al. (2006) (hereafter referred to as the ‘Fly’ region). An additional overlapping region, the ‘barcode’ section of the COI gene, was amplified using the primers LCO1490 (5'-GGTCAACAAATCATAAAGATATTGG-3') and HCO2198 (5'-TAAACTTCAGGGTGACCAAAAAATCA-3') [both from Folmer et al. (1994)] with a primer concentration of 10 µM and reaction conditions of 5 min at 95 °C; 40 cycles of 30 s at 95 °C, 30 s at 48 °C and 45 s at 72 °C; followed by a final extension time of 5 min at 72 °C. COI sequences obtained during this work have been deposited in GenBank with accession numbers GQ165759
[GenBank]
–GQ165807.
Phylogenetic analysis of mosquito sequences.
Sequencher v4.8 was used to combine reverse and forward sequences from each mosquito and final datasets were compiled using Se-Al. These sequences were added to those currently available from GenBank and the Barcode of Life Datasystem (BOLD) (Ratnasingham & Hebert, 2007). For all voucher specimens, COI sequences from a minimum of five field-collected individuals per species were included in analyses where possible. In the case of sequences from public databases, five sequences per species (preferably from different geographical locations) were again included where available. Sequences from flavivirus-positive mosquito samples were also included. Datasets for each region comprised 1152 bp from 112 individuals and 636 bp from 338 individuals for the ‘Fly’ and ‘barcode’ regions, respectively. Analyses were conducted separately for each region, due to the fact that taxonomic coverage of mosquito species in public databases varies for each. Following confirmation that all species groups of five were monophyletic, analyses were then repeated using single representatives per species for ease of presentation, except in the case of clades including flavivirus-positive mosquitoes, in which case sequences from multiple vouchers or public data for that species were included if possible. These ‘single representative per species’ datasets comprised 138 and 46 individuals, respectively, for the ‘barcode’ and ‘Fly’ regions.
In each case, nucleotide sequences were aligned by using MUSCLE (Edgar, 2004) with manual adjustment to maintain correct reading frame, the model of nucleotide substitution and parameter values were selected via MODELTEST (Posada & Crandall, 1998) and used to estimate ML phylogenetic trees in PAUP (Swofford, 2000). Bootstrap-resampling analyses were conducted by using 1000 replicate NJ trees based on the ML substitution matrix. For sample H3E6 in the barcode region, the ML phylogeny of Culex and Culiseta sequences only was also estimated for ease of presentation, and is shown in Fig. 6 (see Supplementary Table S3, available in JGV Online). Trees were rooted by using Chagasia (Fly region) or Culiseta (barcode subset).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Upon further testing of the 20 individual samples that contributed to the four positive pools, three individual mosquito homogenates gave congruent positive sequences. No congruent sequences were amplified from individual mosquitoes contributing to the fourth positive pool and virus isolation from both the pool and the relevant homogenates was unsuccessful. Of the remaining three potential flavivirus strains, two individual sequences were identical and originated from mosquitoes in the same trap. Isolation was attempted using only one of these two homogenates. Therefore, in total, two flavivirus strains were taken forward for further characterization and isolation work. Nevertheless, retrospective identification was conducted for all three individual mosquitoes for which flavivirus-positive results were obtained via RT-PCR.
Virus isolation and characterization
The first novel strain (H4A1) was isolated from a female mosquito, UG134-26, collected in a CDC light trap supplemented with dry ice on 24 February 2008 near Entebbe (latitude 0.07555867, longitude 32.4574212; Fig. 1
). The second strain (H3E6) was isolated from a female mosquito, UG134-14, from the same trap. Notably, the third virus-positive sample, H4D1, which was identical at the nucleotide level to H4A1 for the PF3S/PF2R-bis amplicon, was isolated from another female mosquito, UG134-26, also captured in the same trap.
The ORFs of isolate H3E6, designated CxFV Uganda, and isolate H4A1, tentatively designated Nakiwogo virus (NAKV, named after the location of the trap), are 10 092 and 10 122 nt long, respectively (GenBank accession numbers GQ165808
[GenBank]
and GQ165809
[GenBank]
). The topology of the insect-only group, as well as its position relative to the other members of the genus, was similar in both the NS5 and ORF phylogenies (Figs 2 and 3, respectively). CxFV Uganda appears to be related most closely to CxFV from Mexico, with an uncorrected p-distance of 9.7 % in NS5. NAKV appears to form a sister group to the CxFV group, with an uncorrected p-distance between NAKV and CxFV Uganda of 40.5 %. In contrast, in the E region phylogeny shown in Fig. 4, CFAV is related more closely to CxFV than is NAKV.
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, flavivirus-like particles were visible in infected C6/36 culture for both CxFV Uganda and NAKV. In common with other members of the genus, the spherical virions are enveloped with clear projections and approximately 40 nm in diameter. Cytopathic effect in C6/36 cells infected with NAKV was moderate and similar to that seen in the prototype CFAV (Stollar & Thomas, 1975), with the formation of large syncytia. In contrast, CxFV Uganda was associated with a reduction in cell density and modification of cell shape from circular to triangular.
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. For both H4A1 and H3E6, results were consistent with RNA forms of each flavivirus being present in both culture supernatant and pelleted cells. DNA forms were also present, but in sedimented cells only.
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). Further analyses, using single representatives from each species except in the case of species related to the flavivirus-positive mosquitoes (in which case, multiple representatives are included where possible), are shown in Fig. 6. Sample H3E6, from which CxFV Uganda was isolated, grouped with sequences from field-collected Cx. quinquefasciatus voucher specimens in both phylogenies. Mosquitoes comprising samples H4A1 and H4D1 grouped with a field-collected voucher specimen of Ma. africana nigerrima for the ‘Fly’ region. Primers LCO1490 and HCO2198 did not amplify the barcode region for the mosquitoes that comprised samples H4A1 and H4D1 and, for ease of presentation, we show the Culex subset phylogeny, including sample H3E6, for this region of the COI gene.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Both the NS5 and ORF ML analyses indicate that CxFV Uganda is related most closely to CxFV from Mexico, with strong support for this clade. The larger E region dataset suggests that CxFV isolates from the Caribbean and Guatemala also fall into this group. A second CxFV lineage contains isolates from Japan and North America, which fall as sister groups. Previous studies had also identified these two main clades, namely Trinidad/Guatemala and Asia/Texas, as potential subtypes or genotypes of CxFV (Kim et al., 2009). The addition of CxFV Uganda from Africa, which falls into the first of these groups, supports the hypothesis that CxFV may have been introduced multiple times into the Americas, (i) from Africa to the Caribbean, potentially via routes similar to the introduction of other mosquito-borne flaviviruses such as YFV (Gould et al., 2003) and (ii) from Asia to North America (Lounibos, 2002). As such, the distribution of CxFV may reflect that of the Culex pipiens complex, which is cosmopolitan and ubiquitous, with multiple introductions of this mosquito species into the New World. Notably, although members of the Cx. pipiens complex differ in physiology, ecology and behaviour, sibling species are very difficult to distinguish from one another via morphological methods. Until molecular protocols for the accurate identification of Culex species across the range of the complex are fully developed, there is the potential for misidentification of the vectors of CxFV and, as such, it is difficult to draw conclusions regarding the relative prevalence and distribution of CxFV in Cx. pipiens and Cx. quinquefasciatus. As shown in Fig. 6, although the Cx. quinquefasciatus clade is monophyletic and the molecular identification of the mosquito species from which CxFV Uganda was isolated is robust, resolution of the Cx. pipiens plus Cx. quinquefasciatus clade is not high and the development of a molecular backbone phylogeny for these species in Uganda using an additional gene, for example ITS2 (Severini et al., 1996), is recommended for future work.
NAKV tentatively forms a novel species of flavivirus that appears to be related most closely to the CxFV group in the NS5 and ORF phylogenies. CFAV and KRV then form a sister group to the NAKV/CxFV lineage. In contrast, in the E region phylogeny, CFAV is related more closely to CxFV than NAKV. Although this is compatible with potential interspecies recombination, interpretation of the E region tree is not unambiguous, in particular the position of the root, because the high genetic distances involved mean that other members of the genus Flavivirus were not incorporated into this analysis. It is therefore clear that additional data, particularly comprising ORFs for all available insect-only flaviviruses, would be required in future to test this more fully via formal tests.
Notably, both CxFV Uganda and the two samples of NAKV (i.e. homogenates H4A1 and H4D1, which were both positive via RT-PCR, with identical sequences) were isolated from the same trap, but from different mosquito species, namely Cx. quinquefasciatus and Ma. africana nigerrima, respectively. Both of these mosquito species were trapped in other locations and are present across Uganda; hence, although sampling was not designed to investigate virus distribution, this suggests that the presence of insect-only flaviviruses is patchy and potentially linked to a common source of infection. This would be in agreement with the hypothesis that insect-only flaviviruses can be transmitted vertically (Cook et al., 2006). Indeed, evidence exists that West Nile virus can be transmitted vertically by Culex and Stegomyia mosquitoes (as Aedes albopictus and Ae. aegypti) (Baqar et al., 1993). Although CxFV Uganda and NAKV were both isolated from female mosquitoes, these were collected by using CDC light traps supplemented with dry ice, therefore biasing sampling towards host-seeking females. Hence, the possibility that the viruses may also have been present in male mosquitoes, as described for CFAV in Puerto Rico (Cook et al., 2006), cannot be discounted.
The development of a molecular ‘backbone’ phylogeny for the mosquitoes present in a given region of study, as conducted here, is essential for the screening of potential viral vectors, because many aedine and Culex species can only be identified by dissection of male genitalia. Without the development of a molecular system, particularly in species-rich tropical sampling areas, identification of entire adults via hand lens or microscope over a chill block in the field prior to viral screening may result in misidentification. This study has shown that primers LCO1490 and HCO2198 did not amplify the barcode region for Ma. africana nigerrima. Barcode primers have, to date, primarily been used for potential vectors of malaria, i.e. species of Anopheles. Future work will aim to tailor the system to all taxa of the family Culicidae.
Overall, considering that (i) many undiscovered insect-only flaviviruses are likely to exist in nature, (ii) flavivirus RNA has recently been discovered in phlebotomine sandflies (Moureau et al., 2009) and (iii) the transmission dynamics of the insect-only flaviviruses remain unclear, but may involve vertical transmission, it is clear that this group requires further investigation. Whilst there is no evidence that they cause disease in humans, the high prevalence of the flaviviruses in general raises a number of issues for further consideration. First, future work should aim to investigate the effect of insect-only flaviviruses on different mosquito cell lines and on all life stages of vector species to clarify potential mechanisms of infection and vertical transmission. Second, the possibility of competitive exclusion amongst co-circulating flaviviruses within the vector population, including the effect of infection by insect-only flaviviruses on vector competence for other flaviviruses, also requires investigation via experimental-infection studies. Finally, given that insect-only flaviviruses appear to infect both mosquitoes (Culicidae) and phlebotomine sandflies (Psychodidae), it is advisable to screen potential vector species of both families from any given locality via an integrated approach.
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ACKNOWLEDGEMENTS |
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Received 8 June 2009;
accepted 30 July 2009.
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