Department of Zoology, University of Oxford, South Parks Road, Oxford OX1 3PS, UK
Correspondence
Susanna Twiddy
susanna.twiddy{at}zoo.ox.ac.uk
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ABSTRACT |
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Introduction |
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Although RNA recombination was shown to be possible two decades ago, until recently it had only been demonstrated in a small number of RNA viruses (King et al., 1982; Lai, 1992
). Poliovirus was the first RNA virus in which homologous recombination was demonstrated, and since then this process has been detected in a variety of other RNA viruses, including Western equine encephalitis virus (Hahn et al., 1988
), human immunodeficiency virus (Robertson et al., 1995
) and foot-and-mouth disease virus (King et al., 1985
). Until 1999, however, there was no evidence for recombination in flaviviruses, although the possibility was considered (Blok et al., 1992
; Kuno et al., 1997
; Monath, 1994
). Accordingly, the vast majority of work on flaviviruses, including vaccine studies and phylogenetic analyses in which genotypes were identified and sometimes correlated with disease severity (Chen et al., 1990
; Leitmeyer et al., 1999
; Rico-Hesse, 1990
), has rested on the implicit assumption that evolution in the family Flaviviridae is clonal, with diversity generated largely by the accumulation of mutational changes. Recent studies have shown this assumption to be invalid, as homologous recombination has now been demonstrated in pestiviruses (bovine viral diarrhoea virus) (Becher et al., 2001
), flaviviruses (all four serotypes of DEN virus) (Holmes et al., 1999
; Tolou et al., 2001
; Uzcategui et al., 2001
; Worobey & Holmes, 1999
), hepaciviruses (GB virus C/hepatitis G virus) (Worobey & Holmes, 2001
) and most recently in hepatitis C virus (Kalinina et al., 2002
). Given the implications of recombination for virus evolution (Worobey et al., 1999
) and the development of vaccines or virus control programmes, it is clearly important to determine the extent to which recombination plays a role in flavivirus evolution.
For the purposes of this study, we carried out a survey of recombination in the viruses comprising the genus Flavivirus. We chose to use sequence data from the envelope (E) gene because it encodes the most important antigen with regards to virus biology and humoral immunity. Therefore, large-scale genetic changes in this region, as might be brought about by recombination, could have significant impact on virus phenotype. Secondly, the vast majority of published gene sequence information for flaviviruses in GenBank consists of E gene sequences. The methods used were the graphical detection of conflicting phylogenetic signals (sliding-window analysis), followed by maximum-likelihood (ML) phylogenetic analysis to define recombination breakpoints and to test their significance. In most cases, the results obtained were consistent with what is known of the biology of the different viruses within the genus Flavivirus.
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Methods |
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ML trees were constructed for each data set using PAUP* (Swofford, 2002), assuming the HKY85 model of nucleotide substitution and incorporating a gamma distribution of among-site rate variation with eight rate categories (parameter values available from the authors on request). Figs 1
3 show the resultant phylogenetic trees for JE, WN and SLE viruses (as trees for the other viruses have been published recently they are not included here but are available from the authors on request).
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Breakpoint analysis.
Once the closest parental sequences were identified, we used an ML method to estimate recombination breakpoints and assess their significance (program LARD) (Holmes et al., 1999). This method finds the optimal recombination breakpoints, given a sequence alignment of a putative recombinant and its two parents, by breaking the alignment into two regions at each possible breakpoint and reconstructing a separate ML tree for each region. The two likelihoods obtained from each region are then combined to give a recombination model likelihood score for that particular partitioning of the alignment. The highest combined likelihood score is expected when the alignment is broken at the actual recombination position, since the two trees reconstructed either side of the breakpoint, in this case, accurately reflect the true phylogenetic history of the separate recombinant regions. Breakpoints estimated in this manner can be tested for their statistical significance by comparing the recombination model to the likelihood obtained from the unbroken alignment (i.e. the no-recombination model) using a likelihood ratio (LR) test and a Monte Carlo approach using sequences simulated without recombination under the appropriate model of nucleotide substitution.
Phylogenetic trees and bootstrap support.
After breakpoints were identified, separate ML trees were constructed for each putative recombinant region and the phylogenetic position of the recombinant was compared between regions. Phylogenetic conflicts were assessed using the percentage of bootstrap replicates supporting the conflicting phylogenetic positions. Bootstrapping (1000 replicates) was conducted using NJ trees with distances estimated using the ML model of nucleotide substitution defined previously.
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Results |
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Three putative recombinants were identified in the analysis of the JE virus E gene. There have as yet been few rigorous studies of the phylogenetic relationships among JE virus strains or attempts to classify them into genotypes' (Chen et al., 1990; Ritchie et al., 1997
; Tsarev et al., 2000
). In the absence of an accepted convention for classification of JE virus strains, we reconstructed an ML tree from our data set and identified four major clusters of strains, which we have denoted IIV and which are represented in the trees of Fig. 5(ce)
. Interestingly, there appears to be some correlation between the phylogenetic groupings in this tree and the regions of either epidemic or endemic activity reported, as suggested previously by Chen et al. (1990)
. Group I consisted mainly of strains from China, India, Japan and Taiwan, areas where JE virus transmission follows a pattern of seasonal epidemics, whilst groups II, III and IV were largely made up of strains from Thailand, Indonesia, Malaysia and Australia, more Southern regions where JE virus transmission is endemic (Burke & Leake, 1988
). All of the putative recombinant sequences described in this paper are mosaics of genotypes I and II. Breakpoint analysis for all of the putative JE virus recombinants confirmed the recombination crossover points from the diversity plots; P values were <0·005 in each case.
Two of the putative JE virus recombinants, Korea 82 (K82P01) and Korea 91 (K91P55) appear to have parental strains originating in Japan and Korea. In the case of K82P01, the first half and the final 400 bp of the E gene sequence are related to genotype I strains isolated from the 1980s in Japan and Korea, with the central section bearing greater similarity to Korean and Japanese isolates from the 1990s (genotype II). For K91P55, approximately the first 400 nt resemble the genotype I Korean and Japanese strains, with the rest of the sequence being more closely related to the 1990s genotype II isolates. The third putative JE virus recombinant was isolated from Thailand (Thailand 82, KPP034-35CT). At the 5' end of the E gene this strain is closely related to another genotype I Thai 93 strain (ThCMAr6793); however, after approximately 300 bp it becomes more similar to a genotype II strain originating in China (SA-14). The phylogenetic relationships for the different regions of the E genes in JE virus are shown in Fig. 5(ce) and the topological shifts for each of the recombinants have strong bootstrap support.
Finally, a putative recombinant was identified among the SLE virus strains analysed. Specifically, Guatemala 94 (GMO94), a Central American strain, is identical to an Argentinean isolate (CorAn9124) at the 5' end of the E gene; however, after approximately 350 bp it diverges from this South American strain and, after a putative recombination crossover point between nt 427 and 428, it bears a strong similarity to an isolate from the southern United States (TNM4-711K). The hypothesis that recombination occurred in this case is strongly supported using LARD (P<0·005). Again, no generally accepted classification of subtypes of SLE virus exists (Kramer & Chandler, 2001), although it is clear that there are genetic differences between strains isolated in the United States, Central America and South America (Fig. 3
). In the ML trees showing phylogenetic shifts, we attempted to include strains that represented the full diversity observed in natural isolates of SLE virus (Fig. 5f
). These trees show clearly the different topological position of Guatemala 94 (GMO94) in the different regions of the gene, with the topological shift supported in each case by very high bootstrap values.
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Discussion |
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Phylogenetic and biological consequences of recombination have been discussed in depth in previous studies (Worobey et al., 1999; Worobey & Holmes, 1999
) and therefore will not be addressed here. In the current study, the most notable result was that recombination was detected in most, but not all, of the mosquito-borne flaviviruses and in none of the tick-borne flaviviruses. Although these differences could be caused by ascertainment bias, as recombination was detected most frequently in those viruses for which most data were available, it is also possible that they reflect biological and ecological factors related to virus transmission.
Conditions for recombination
For recombination to occur, several conditions must be met. Principally, either vector or host must be co-infected with greater than one strain of the virus in question. This requires either that hosts are fed upon by more than one infected vector and are productively infected by at least two strains of virus at the same time, or that vectors engage in multiple feeding on viraemic hosts, allowing the possibility of vector co-infection with greater than one strain of virus (although the nature of TBE virus transmission is such that the criteria for vector co-infection with TBE virus are somewhat different; see below). The limitations of the methods used to detect recombination impose a further set of conditions; it is extremely difficult to identify recombinant sequences in cases where the sequences analysed are very similar to one another. In practice, this means that only recombinant sequences whose parents are from different genotypes are readily identifiable. Therefore, for detectable recombination to occur, co-circulation of different genotypes must take place, usually requiring mobility of host and/or vector species. In addition, sampling effects can play a major role. In general, the longer the sequence used in the analysis and the wider (and denser) the geographical and temporal sampling, the more likely it is that a recombinant sequence will be identified.
Mosquito-borne flaviviruses in which recombination was detected
Of the mosquito-borne flaviviruses studied, DEN viruses have the highest frequency of recombination. As expected, they fulfil most or all of the criteria outlined above. The principal vector of DEN virus, the A. aegypti mosquito, has been shown to engage in multiple feeding, allowing co-infection of more than one strain in the vector (Gould et al., 1970). In addition, as in all mosquito-borne flaviviruses, virus replication takes place in both the vector and the vertebrate hosts (principally humans in the case of DEN virus) to a sufficient level to allow mosquito-human-mosquito transmission to occur. In all four serotypes, distinct genotypes' are observable. Furthermore, the contemporaneous circulation of different genotypes in a particular geographical region has been documented in DEN-2 (Twiddy et al., 2002
) and must presumably have occurred in the other three serotypes, as recombination has been demonstrated in each (Holmes et al., 1999
; Tolou et al., 2001
; Uzcategui et al., 2001
; Worobey et al., 1999
). Given the global distribution of DEN virus and the exceptional mobility of the human host species, the relatively high frequency of recombination in DEN virus is not surprising.
Although JE virus is also the agent of an important human disease, its natural transmission cycle is between Culex mosquitoes and wild and domestic birds, with pigs as an amplifying host and humans a dead-end host (Burke & Leake, 1988). As with the Aedes vector of DEN virus, the genus Culex has been demonstrated to engage in multiple feeding (Wekesa et al., 1997
), so there is a potential for co-infection of hosts and/or vectors. Furthermore, the JE virus phylogeny presented here shows co-circulation of genotypes III and IV in Southeast Asia in the early 1980s and of genotypes I and II in Korea in the late 1980s/early 1990s, the period in which two of the three JE virus recombinants identified in this study were isolated in Korea. The presence of strains isolated in Japan and Korea in genotype II also suggests that strains may have been introduced into the northern epidemic region from the southern endemic region, probably by birds, the likely agents of the continuing westward spread of JE virus (Solomon et al., 2000
). Finally, the available sample of JE virus sequences is biased towards genotype I, which comprises sequences from the northern range of JE virus transmission and a study conducted using a more representative sample from both transmission regions might be expected to uncover more recombinant JE virus strains.
Recombination was also detected in SLE virus, which, like JE virus, is transmitted by Culex mosquitoes, with birds being the principal vertebrate host. Although all of the sequences are very similar (<10 % nucleotide difference between any two strains), there are discernible differences between strains circulating in the United States and those isolated from Central and South America (Fig. 3). Isolates that cluster in the United States have been recovered from Brazil and Panama, indicating that long-distance transfer of strains is possible, with migrating birds being the most likely agent of geographical dispersal (Monath & Tsai, 1987
). Indeed, the recombinant virus identified here GMO94 does itself appear to be the product of a strain from Central America and a strain from the southern region of the United States. As yet, there are no isolates from the United States that clearly have a South American origin, lending some support to the hypothesis that maintenance of the virus in North America is predominantly local, with virus overwintering in either chronically infected birds or diapausal mosquitoes (Kramer & Chandler, 2001
).
Mosquito-borne flaviviruses in which recombination was not detected
WN virus, another member of the JE virus complex, has a transmission cycle similar to that of both SLE and JE viruses (i.e. avian-Culex-avian) and has been the subject of considerable interest since the New York outbreak of 1999 (Ebel et al., 2001; Lanciotti et al., 1999
). Our analysis failed to find any evidence of recombination in WN virus, although detectable recombination could, in theory, occur, as the requirements for vector co-infection are fulfilled and the close relationship of the New York 1999 strains of WN virus to those isolated in Israel in 1998 and 2000 bears witness to the ability of the virus to be transmitted over long distances (Petersen & Roehrig, 2001
). In addition, distinct genotypes identifiable on the tree of the WN virus E gene (Fig. 2
) appear to have been co-circulating in Israel in 2000 and in Romania in 1996. However, the data set of WN virus sequences is heavily biased towards strains from Israel and New York from 1998 to 2000, among which there is little diversity. Inclusion of sequences from more of the regions where WN virus occurs [including Kunjin virus sequences, which have been suggested to be a subtype of WN virus rather than a separate virus species (Savage et al., 1999
), and for which no E gene sequence data of >300 bp is currently available] may lead to the discovery that recombination does occur in this virus.
Last among the mosquito-borne flaviviruses, we analysed the available E gene sequences from YF virus. Again, there is no reason to suppose that co-infection in host or, more likely, vector could not occur and there is sufficient genetic diversity within the YF virus population thus far sampled to identify at least three major genotypes, previously dubbed I, IIa and IIb (Chang et al., 1995). These three groups are geographically distinct, with two (I and IIa) found in sylvatic cycles in East/Central and West Africa, respectively, and the third (IIb) occurring in the forests of Central and South America, with many Caribbean islands also at risk (Vainio & Cutts, 1998
). More recently, Mutebi et al. (2001)
suggested that each of the African clades could be divided further, with two West African genotypes and two in East/Central Africa, and a single strain from Angola, representing a third African genotype. That mixing of these genotypes via long-distance transfer of virus strains can occur was amply demonstrated in the 17th and 18th centuries, when the mass transport of slaves from West Africa to the Americas was thought to have also introduced YF virus to the New World. More recently, an African strain of the virus was isolated in Trinidad in 1979. However, movements of YF virus hosts on the scale of those that took place during the slave-trading years have not been seen since and the current transmission cycle of the virus is predominantly sylvatic, with non-human primates the major vertebrate host. Most human cases of YF virus infection now occur either among rural workers whose occupation brings them into contact with sylvatic vectors in either Africa or the Americas, or in the form of small outbreaks in villages in the YF virus zone of emergence in Africa (Vainio & Cutts, 1998
). Neither of these groups of people is likely to engage in extensive travel between the areas in which the African and New World strains of YF virus circulate, so recombination between these genotypes is probably infrequent. Recombination is more likely to have taken place between the West and East/Central African genotypes, whose geographical ranges certainly adjoin and probably overlap (Mutebi et al., 2001
; Vainio & Cutts, 1998
), although no evidence for recombination has been found as yet. However, the data set is small and further sampling, particularly of sylvatic isolates from regions where these two genotypes are likely to co-circulate, may reveal inter-genotypic recombinants.
Tick-borne flaviviruses
The final group of flaviviruses analysed were the TBE viruses. No evidence of recombination was detected in this group. However, the biology of these viruses is such that the likelihood of recombination identifiable by the methodologies used in this survey is low. The principal mechanism of transmission for the TBE virus group is tick-to-tick transmission via co-feeding on a host with low or undetectable viraemia (Jones et al., 1997; Labuda et al., 1993
; Randolph et al., 1996
). Therefore, the frequency of co-infection in ticks is dependent upon the likelihood of a previously infected tick co-feeding with at least one other tick that is infected with a different TBE virus strain. As Ixodes ticks feed only infrequently (in general, once as larva, nymph and adult) (Kettle, 1995
) and as TBE virus infection prevalence in I. ricinus ticks in Central Europe has been reported to be in the region of 0·2 % (Randolph et al., 1999
), the probability that any tick is co-infected with more than one virus strain must be low. Similarly, co-infection in the vertebrate host would require transmission to an already viraemic host by a tick infected with a different strain. This is a relatively unlikely prospect as in those vertebrate hosts that develop a systemic viraemia, its duration is short and mortality is high (Gilbert et al., 2000
; Randolph et al., 1996
).
Second, although co-infection may occur, it is highly unlikely that any resulting recombinant strains would be detectable using the methods employed in this survey. Zanotto et al. (1995) observe that there is very little variation in strains of TBE virus in any single geographical area. Ticks themselves are essentially immobile in terms of geographical dispersal of virus strains and although they may be carried some distance by larger vertebrate hosts such as deer (or humans), these tend to be dead-end hosts. The natural hosts of TBE virus, at least in Central Europe, are small rodents, with correspondingly small geographical ranges, so that co-circulation of genetically diverse strains of TBE virus is likely to be extremely rare. When these factors are considered, it is not surprising that, even with a relatively large sample of virus strains, no putative recombinants were identified.
Overall, our study demonstrates that recombination, at least in the mosquito-borne flaviviruses, is a relatively frequent occurrence. In addition, we propose that aspects of the biology of the flaviviruses, such as life-history of vector or vertebrate host, might explain the patterns of recombination frequency that we have observed. Given the drastic effect that recombination can have on phylogenetic studies, it is highly desirable that sequence analysis should be carried out on whole genomes wherever possible, so that putative recombinants anywhere in the genome can be identified and the possibility of misclassification of strains reduced. At present, there is a clear shortage of such whole genome data; this needs to be addressed in the immediate future.
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ACKNOWLEDGEMENTS |
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Received 14 June 2002;
accepted 16 September 2002.