1 The Arbovirus Laboratories, Wadsworth Center, New York State Department of Health, 5668 State Farm Road, Slingerlands, NY 12159, USA
2 Department of Biomedical Sciences, School of Public Health, The University at Albany, State University of New York, Albany, NY 12144-3456, USA
Correspondence
Gregory D. Ebel
ebel{at}wadsworth.org
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession numbers for consensus sequences reported in this paper are DQ010338DQ010357.
Supplementary material is available in JGV Online.
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INTRODUCTION |
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The mechanisms that lead to population-level genetic change in WNV and other arthropod-borne viruses (arboviruses) are poorly understood. RNA viruses such as WNV are thought to exist within a host as a genetically heterogeneous mixture of variants that differ from a consensus nucleotide sequence: the term quasispecies' has come to refer to the complex mutant spectrum that surrounds the master viral nucleotide sequence (Eigen & Biebricher, 1988; Eigen, 1993
; Domingo, 1998
). Quasispecies result from the high error rates of most RNA virus-encoded RNA-dependent RNA polymerases (RDRP) (Holland et al., 1982
), as well as their short generation times and large population sizes. A genetically diverse virus population would seem to have adaptive advantages due to the pre-existence of variants within the mutant spectrum that may be more fit in a novel and/or changing environment. This genetic diversity may have important implications for virus populations and hosts. For example, quasispecies structure has been shown to be critical in the failure of hepatitis C treatment (Farci et al., 2000
, 2002
), AIDS disease progression (Essajee et al., 2000
) and the persistence of virus infections at the cellular, organism and population levels (Domingo et al., 1998
). The diversity of the viral mutant spectrum has been shown to be both host- and virus-dependent (Schneider & Roossinck, 2000
, 2001
) and a critical determinant of virus fitness (Martínez et al., 1991
; Ruiz-Jarabo et al., 2002
). Further, quasispecies structure provides a virus population with a molecular memory that exists as minority genotypes within the quasispecies mutant distribution (Ruiz-Jarabo et al., 2000
, 2002
; Domingo et al., 2002
). Quasispecies population structure may therefore be critical to RNA virus perpetuation in nature.
It is unclear, however, whether mosquito-transmitted RNA viruses, which evolve more slowly than other RNA viruses and are constrained by replication in taxonomically diverse hosts (Weaver et al., 1992; Zanotto et al., 1995
), are best described and understood as quasispecies (Smith et al., 1997
; Holmes & Moya, 2002
). In particular, quasispecies theory states that natural selection acts on the whole of the mutant spectrum rather than on the individual genomes composing it (Eigen & Biebricher, 1988
). Intrahost genetic variation is thus necessary for a quasispecies population structure, but not sufficient. If intrahost genetic diversity arises anew following infection by a clonal or highly bottlenecked virus population, the possibility for natural selection to act on the entire mutant spectrum at critical points in its life cycle (and for the transmission of the proposed molecular memory) is negated. This problem is compounded in arboviruses such as WNV that are maintained in nature in an enzootic cycle between arthropods and vertebrates. In complex transmission cycles, the genetic bottlenecks that may accompany transmission between host types may be frequent and the duration of an infection in any given host may be comparatively short. Few studies have examined the relevance of quasispecies to naturally occurring arbovirus populations. The hypothesis that WNV is maintained in nature as a quasispecies was therefore evaluated in this study. In particular, WNV was sampled from naturally infected mosquitoes and birds and intrahost genetic diversity was quantified within each host type. By using these data, we determined whether patterns of intrahost genetic diversity are host-dependent and whether minority (i.e. non-consensus) WNV haplotypes may be preserved throughout the transmission cycle, and also assessed the role of natural selection in shaping intrahost and interhost genetic diversity.
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METHODS |
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WNV detection and quantification.
The infection status of each specimen was determined by quantitative, real-time (TaqMan) RT-PCR as described previously (Shi et al., 2001; Kauffman et al., 2003
). To determine whether low virus titres may bias estimates of genetic diversity via repeated sampling of DNA amplified from a single RNA molecule, WNV RNA and infectious virus were quantified in each specimen by real-time RT-PCR as described previously (Shi et al., 2001
; Kauffman et al., 2003
) and by plaque assay on African Green Monkey kidney (Vero) cells according to standard protocols. Briefly, confluent cell monolayers in six-well culture plates were inoculated with 0·1 ml serial tenfold dilutions of WNV-containing material diluted in BA-1 diluent [M-199H, 1 % bovine serum albumin, 0·05 M Tris (pH 7·6), 0·35 g sodium bicarbonate l1, 100 µg penicillin ml1, 100 µg streptomycin sulfate ml1, 1 µg fungizone ml1]. Plates were incubated for 1 h at 37 °C with 5 % CO2. A primary overlay containing 0·6 % agar in Eagle's minimal essential medium with 10 % fetal bovine serum (FBS) was applied and plates were incubated as described above. After 48 h incubation, a second overlay, as described above except that it contained 2 % FBS and neutral red, was applied to each well. Plates were returned to the incubator and plaques were counted at 72 h post-infection.
Culex species identification.
Females of Culex pipiens and Culex restuans are difficult to differentiate on the basis of morphological characters. To determine the species composition of the WNV-infected mosquito pools that had been identified in the field as either C. pipiens or C. restuans, species present in the pools were identified by using PCR. Genomic DNA was extracted from mosquito-pool homogenate by using a DNeasy Tissue kit (Qiagen) according to the manufacturer's protocol. PCRs containing primers targeting ITS1 and ITS2 sequences specific for C. pipens, C. restuans and Culex salinarius were performed as described by Crabtree et al. (1995). Reactions were amplified in an MJ Research PTC 2000 thermocycler programmed for one cycle at 95 °C for 5 min, 35 cycles at 95 °C for 15 s, 55 °C for 30 s and 72 °C for 1 min, and one cycle at 72 °C for 5 min. Products were visualized on a 1·5 % agarose gel and inspected for band sizes of 698, 506 and 175 bp, representing C. pipiens, C. restuans and C. salinarius, respectively.
High-fidelity RT-PCR, cloning and sequencing.
RNA was extracted from infected specimens by using RNeasy spin columns (Qiagen) and RT-PCR was conducted by using primers designed to amplify the 3' 1159 nt of the WNV envelope (E) coding region and the 5' 779 nt of the WNV non-structural protein 1 (NS1) coding region [forward primer WNV1311 (5'-ATGCGCCAAATTTGCCTGCTCTAC-3'); reverse primer WNV3248 (5'-ATGGGCCCTGGTTTTGTGTCTTGT-3')]. RT of 5 µl RNA was performed with M-MLV-RT (Ambion) and Sensiscript RT (Qiagen) at 45 °C for 40 min. RT reactions were followed by heat inactivation at 95 °C for 5 min. The resulting cDNA was used as a template for PCR amplification. To minimize misincorporations introduced during RT-PCR by Taq polymerase, the resulting WNV cDNA was then amplified with a high-fidelity protocol using PfuUltra (Stratagene), according to the manufacturer's specifications. Amplification was carried out for 40 cycles at 94 °C for 30 s, 50 °C for 30 s and 72 °C for 4 min, and one cycle at 72 °C for 10 min. PCR products were visualized on a 1·5 % agarose gel, amplicons of the appropriate size were excised and DNA was recovered by using a MinElute Gel Extraction kit (Qiagen) as specified by the manufacturer. The recovered DNA was ligated into the cloning vector pCR-Script Amp SK(+) and transformed into XL10-Gold Ultracompetent cells (Stratagene) according to the manufacturer's protocol. The bluewhite colour-screening method was used to select transformed colonies. White colonies were screened by direct PCR using primers specific for the insert of interest. Plasmid DNA was purified by using either a Wizard Plus Miniprep kit (Promega) or a QIAprep Spin Miniprep kit (Qiagen) as specified by the manufacturers. Sequencing was carried out by using five pairs of overlapping primers (sequences available from the authors upon request) and the T3 reverse primer. Sequencing was performed at the Wadsworth Center Molecular Genetics Core (WCMGC) using ABI 3700 and 3100 automated sequencers (Applied Biosystems). Twenty clones per positive bird and per mosquito pool were sequenced. Cloning and plasmid DNA extraction were performed on separate days for each specimen to reduce the likelihood of between-specimen contamination. In addition, each set of clones was sent to the WCMGC separately so that no two sets of clones would be included on the same sequencing run.
Sequence analysis.
Sequences were compiled and edited by using the SeqMan module of the DNAStar software package and a minimum of twofold redundancy throughout each clone was required for sequence data to be considered complete. Twenty clones from each individual bird or mosquito pool were aligned by using MegAlign within DNAStar. The consensus sequence for each sample was determined and the sequence of each clone was compared to the consensus. The percentage of nucleotide mutations (total number of mutations divided by total number of bases sequenced) and the percentage of mutant clones were used as indicators of genetic diversity. Statistics were performed by using the STATA software package and GraphPad Prism version 4.00.
Analysis of divergence and selection.
Analysis of intrahost WNV populations was conducted by using alignments of the nucleotide sequences of 20 clones for each of the 20 WNV specimens described above. Interhost measures of divergence and selection were obtained from an alignment of the 20 consensus sequences obtained from these intrahost populations (designated NY-Suffolk-03 in Table 6) and from an alignment of 67 WNV E coding region sequences obtained during previous studies of WNV in New York described elsewhere (designated NY-99-03 in Table 6
) (Ebel et al., 2001a
, 2004
). To determine the nucleotide divergence present in each WNV population (both intra- and interhost alignments), the mean pairwise nucleotide distance between sequences (
) was computed by using DnaSP (www.ub.es/dnasp) (Rozas & Rozas, 1999
). In addition, the proportion of mutations in each alignment that were non-synonymous [designated pN by Holmes (2003)
] was computed. Finally, the number of non-synonymous (dN) to synonymous (dS) substitutions per site (dN/dS) was compared. The mean number of synonymous and non-synonymous sites per sequence in each alignment was calculated by using the NeiGojobori method implemented in DnaSP and dN/dS ratios were calculated by using Microsoft Excel. dN/dS ratios were set as undefined in alignments without any mutations and as 1·000 in alignments with no synonymous mutations.
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RESULTS |
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To evaluate the possibility that estimates of intrahost genetic diversity may differ when structural and non-structural coding regions were examined, these estimates were compared independently for the portions of the E and NS1 coding regions that were co-amplified during RT-PCR (data not shown). Estimates of nucleotide diversity within the NS1 coding region were slightly higher than those from the E coding region and estimates of predicted amino acid diversity were slightly higher within the predicted E protein sequence. However, these estimates did not differ significantly when considered either independently for mosquitoes and birds or for the dataset as a whole.
Examination of estimates of intrahost genetic diversity derived from mosquito- and bird-derived populations of WNV showed that the percentage of substituted bases and mutant clones was significantly greater in WNV sequences from mosquito pools than from birds, with mosquito populations of WNV approximately twofold more genetically diverse than WNV from birds (Table 3). Although predicted amino acid diversity was also greater in mosquitoes than in birds, the difference was not statistically significant. Comparison of the mutation types observed in mosquitoes and birds failed to identify a host-dependent bias for a particular class of mutation (Table 4
). Transitions and transversions were the most abundant mutations observed. However, insertions, deletions and non-translated mutations were also present in a small proportion of clones.
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DISCUSSION |
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Experimental error was monitored in this study by using a control WNV population that is expected to be highly genetically homogeneous. Experimental errors were introduced at a low but measurable rate that is consistent with the error rate for PfuUltra provided by the manufacturer, 4·3x107 misincorporations per base copied. All three mutations detected in the control studies were C to A transversions, which were detected in our study samples only infrequently (data not shown) and may have been introduced into control sequences during the in vitro transcription process required for the generation of infectious RNA transcripts from the WNV cDNA clone. Estimates for nucleotide diversity in the control WNV population were approximately fourfold lower than in mosquitoes and birds, suggesting that the observed quasispecies diversity was not an experimental artefact. Analysis of predicted amino acids, however, revealed a more significant impact; all of the nucleotide substitutions in the control WNV resulted in amino acid substitutions, leading to artificially high estimates of protein diversity. Monitoring of experimental error through the use of a control WNV population allowed us to conclude that primary nucleotide sequence data are reliable, but failed to confirm the reliability of data on predicted amino acids.
Approximately 0·016 % of bases sequenced, and 19·5 % of clones, differed from the consensus. These estimates of nucleotide diversity are approximately tenfold lower than previous reports of intrahost genetic diversity for Dengue virus (DENV) (Wang et al., 2002; Lin et al., 2004
) and WNV (Beasley et al., 2003
; Davis et al., 2003
). Several factors may explain this lack of concordance. Taq polymerase, which introduces errors at a higher rate than proofreading polymerases such as Pfu (Malet et al., 2003
), was used in the PCR step of several published studies, potentially inflating estimates of nucleotide and amino acid diversity. Importantly, our estimates of intrahost genetic diversity are consistent with studies of RNA viruses conducted by using a similar methodology (Schneider & Roossinck, 2000
, 2001
; Bonneau et al., 2001
). Virological and ecological differences between DENV and WNV may also partially explain the differences between the findings reported here and those for DENV. Although DENV and WNV are both flaviviruses, they belong to different antigenic groups and may have diverged sufficiently to differ in the basic fidelity of their respective RDRPs. Furthermore, DENV and WNV perpetuate in divergent natural transmission cycles: DENV perpetuates between Aedes species mosquitoes and human hosts, whereas WNV perpetuates in nature between birds and Culex species mosquitoes. The implications of divergent transmission cycles on intrahost genetic diversity are not clear at present. However, their impact on the evolution of arbovirus consensus sequences is well described (Weaver et al., 1994
; Zanotto et al., 1996
; Ebel et al., 2001b
).
WNV was more diverse in mosquitoes than in birds. A greater measure of genetic diversity in mosquito compared with bird specimens could be the result of multiple infected mosquitoes within a single pool. To evaluate this possibility, the MIR was determined for C. pipiens/restuans at each collection site. The MIRs reported, and the number of individual mosquitoes in each infected pool, are generally low. Combined, these site- and time-specific MIRs and small pools support the assumption that only one WNV-infected mosquito was analysed in each pool. Greater genetic diversity in mosquito specimens than in bird specimens could also be explained by our use of a single avian tissue as a source of WNV from birds and whole-body homogenates as a source of WNV from mosquitoes. The hypothetical impact of tissue-specific selection and compartmentalization of particular variants within the quasispecies distribution was minimized by limiting our analysis to corvids, which are susceptible to particularly high viraemias (Komar et al., 2003). At the time of death, experimentally inoculated American crows have viraemias up to 107 p.f.u. ml1 (K. A. Bernard, unpublished data). A large proportion of virus detected in the kidney is thus attributable to viraemia and is reflective of circulating virus produced in tissues throughout the bird's body. As WNV infections in mosquitoes appear to be chronic, whereas infections in birds tend to be acute and either (i) resolve upon the initiation of an immune response or (ii) terminate fatally, the increase in genetic diversity in mosquitoes could also be explained by the longer duration of WNV infection in these hosts, even though the underlying mutation rate may be approximately constant. WNV infections in birds may produce extremely high titres compared to WNV infections in mosquitoes. Therefore, although WNV is proportionally more diverse in mosquitoes, a similarly large pool of variants may be present in birds, particularly corvids. The finding of higher quasispecies diversity in mosquitoes than in birds contrasts with a recent study of DENV in mosquitoes and patients by Lin et al. (2004)
, who found that DENV sequences in human sera were more diverse than those obtained from a mosquito pool. As discussed above, the lack of concordance in results may be related to methodological, virological and ecological factors that require further study. These data show, however, that WNV usually exists as a genetically diverse population within hosts and that it is more diverse within mosquitoes than birds. Mosquitoes may therefore provide a source for WNV genetic variation in nature.
In addition to nucleotide substitutions, insertions, deletions and non-translated mutations (substitutions that occurred 3' of an insertion or deletion) were observed in 2·25 % (nine of 400) of the genomes sampled. Insertions and deletions invariably disrupted open reading frames and introduced premature stop codons, resulting in non-viable genomes that are highly likely to be removed rapidly by natural selection and contribute little to the WNV mutant spectrum.
Evidence that WNV may be transmitted between hosts as a genetically diverse population was sought by sampling specimens for analysis from a single transmission focus on Long Island, NY, USA, during late summer 2003. Three clones in two avian samples shared non-consensus mutations. Convergent evolution in the same host type (American crows) might have produced the observed pattern of genetic similarity. The lack of amino acid changes associated with the mutations, however, renders this scenario unlikely. Rather, the several mutations that genetically link the three clones suggest strongly that they are identical by descent and not the product of rapid convergent evolution. The sampling methods used in this study allow for only the most common viral variants within a single infection to be sampled reliably at most, approximately 0·2 % (20 of at least 10 000) of genomes present in each infection was sampled. It is therefore not surprising that a relatively small proportion of the clones in this study had genetic signatures suggesting that minority components of the WNV mutant spectrum might occur in multiple specimens. Conversely, it is surprising that these minority genomic variants were detected at all. Experiments with Bluetongue virus have documented transmission of minor variants between vertebrates and Culicoides sonorensis (Bonneau et al., 2001). To our knowledge, however, this is the first published report providing evidence that an arbovirus may be transmitted as a genetically diverse population in nature.
Data on intrahost genetic diversity in the 20 specimens analysed in this study permitted examination of the selective pressures acting on WNV within and between hosts. The mean genetic distance in each of the intrahost alignments was quite low and was approximately tenfold lower than the mean genetic distance of each interhost WNV population considered. WNV sequences are therefore more diverse between hosts than within an individual host. The proportion of non-synonymous mutations (pN) and dN/dS ratios tended to be low in both intra- and interhost populations, suggesting the action of purifying selection in both groups of sequences. Combined, the low values of and the relative rarity of non-synonymous variation in all WNV alignments examined support the observation of tight constraints on arbovirus sequence variation (Weaver et al., 1992
; Zanotto et al., 1995
; Twiddy et al., 2002
; Holmes, 2003
). The slight elevation in values of
, pN and dN/dS obtained from mosquito infections suggests that constraints on WNV sequence variation may be looser in mosquitoes than in birds. In vivo experimental studies currently in progress will address this issue directly.
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ACKNOWLEDGEMENTS |
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Received 7 March 2005;
accepted 24 April 2005.
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