Pathogen Molecular Biology and Biochemistry Unit, Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT, UK1
Institut Pasteur de Dakar, Dakar, Senegal2
Unité de Virus Emergents, Faculté de Médecine, Boulevard Jean Moulin, 13005 Marseille, France3
Wellcome Trust Centre for the Epidemiology of Infectious Disease, Department of Zoology, University of Oxford, South Parks Road, Oxford OX1 3FY, UK4
Centre for Ecology and Hydrology (formerly Institute of Virology and Environmental Microbiology), Mansfield Road, Oxford OX1 3SR, UK5
Institute of Poliomyelitis and Viral Encephalitis, Moscow, Russia6
Author for correspondence: Michael Gaunt (at the London School of Hygiene and Tropical Medicine). Fax +44 20 7636 5739. e-mail michael.gaunt{at}lshtm.ac.uk
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Abstract |
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Introduction |
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Flaviviruses are a useful model for studying the evolution of vector-borne virus diseases, since they comprise mosquito-borne, tick-borne and no-known-vector (NKV) viruses (Porterfield, 1980 ). The genus contains about 70 recognized flaviviruses that are antigenically related and have a widespread geographical dispersion. They are positive-stranded RNA viruses with a genome of approximately 10·5 kb. Virions contain three structural proteins, capsid (C), membrane (M) and envelope (E), and infected cells have been shown to contain seven non-structural (NS) proteins, NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5 (Rice et al., 1985
; Rice, 1996
).
The evolution, dispersal patterns and epidemiological characteristics of flaviviruses are believed to have been determined through a combination of constraints imposed by the arthropod vector, the vertebrate hosts, the associated ecology and the influence of human commercial activity. For example, the clinal evolution of the tick-borne encephalitis (TBE) complex viruses across the Euro-Asian land mass reflects the life-cycle and feeding habits of the ixodid tick (Zanotto et al., 1995 ) combined with the appropriate rodent host species and climatic conditions (Randolph et al., 2000
). Similarly, the introduction of goats and sheep onto the hillsides of Turkey, Greece, Spain, Ireland, Norway and the British Isles was followed by the appearance of louping ill (LI) virus (Reid, 1984
; Gao et al., 1993
; Gaunt et al., 1997
; McGuire et al., 1998
). The emergence and expansion of dengue haemorrhagic fever in the tropics has followed an increase in human and mosquito population densities brought about by urbanization and industrialization (Zanotto et al., 1996
). Finally, the trans-Atlantic dispersal of yellow fever (YF) virus, and possibly many other flaviviruses, was thought to have coincided with the transportation of people and mosquitoes from Africa to the Americas on slave ships during the past few centuries (Strode, 1951
; Gould et al., 1997
).
Using detailed molecular phylogenetic analyses, we have attempted to bring all these factors together in order to understand the nature of flavivirus evolution, epidemiology and dispersal. In this study, maximum likelihood (ML) phylogenetic analyses of virtually all of the recognized flaviviruses were performed using partial NS5 gene sequences and the tree was compared with one based on the E gene sequences. The epidemiological and aetiological characteristics of each flavivirus have been mapped onto the phylogeny to reveal a striking pattern of coincidence between the topological arrangement of the viruses and their associated epidemiological characteristics.
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Methods |
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The robustness of the data sets was examined for deviations in nucleotide or amino acid base composition between taxa, as nucleotide or amino acid base homogeneity is a prerequisite for the mutation models used in this study. The NS5 nucleotide alignment required the removal of the third codon position to obtain nucleotide base homogeneity (2-test, P<0·05 for all three codon positions and P>0·99 for codon positions one and two only) (PAUP*, version 4.0; Swofford, 1999
). The E gene amino acid alignment required the removal of cell fusing agent virus, which had sometimes been used in previous analyses, to obtain amino acid homogeneity under the same test (P>0·05 for all other species) (Puzzle; Strimmer & von Haeseler, 1996
). Nucleotide variation was examined using a sliding window analysis (SWAN; Proutski & Holmes, 1998
). The NS5 gene was further investigated for possible saturation using a 10 bp sliding window analysis, which estimated as an entropy function of the nucleotide variation (Var). The sliding window analysis of the mosquito-borne flaviviruses, and each mosquito-borne flavivirus clade described later, identifies a region between nucleotides 342 and 392 of the 693 bp NS5 gene alignment showing the highest level of variation (Var=0·721·21). This region coincided a large alignment gap and proved difficult to align using amino acids; therefore, these nucleotides were subsequently removed. The ML model for NS5 gene nucleotide substitution was determined by testing 40 models of nucleotide substitutions (MODELTEST; Possado & Krandall, 1998
), which described an eight parameter model consisting of the general time reversible (GTR) model of nucleotide substitution (six parameters), an invariant rate parameter (PINVAR) and the alpha parameter of a four category discrete gamma distribution (
; one parameter). The ML model parameters were estimated by an automated reiterative ML heuristic search (PAUP*) and from JukesCantor distances (MODELTEST). Parameter estimates were incorporated into a full heuristic ML search for ten replications. The level of phylogenetic support was determined by bootstrap re-sampling using ML distances incorporating each parameter estimate and a full heuristic search for 1000 replications (PAUP*). In addition, 100 Monte Carlo DNA sequence simulations were performed from the final NS5 gene tree topology and reconstructed using the ML parameter estimates, as described previously (Seq-Gen; Rambaut & Grassly, 1997
).
Evolutionary reconstructions of partial E gene amino acid alignments were performed by ML quartet puzzling using the JTT substitution matrix (Jones et al., 1992 ) for 10000 quartet puzzling steps (Puzzle; Strimmer & von Haeseler, 1996
). Likelihoods were analysed for 0, 4, 8 and 16 discrete
categories and the presence or absence of an PINVAR was assessed using a likelihood ratio test.
The phylogenies obtained from the partial NS5 and E gene sequences of each flavivirus were tested for their association with particular arthropod vectors, disease associations (haemorrhagic and encephalitic) and their geographical distribution using the International Catalogue of Arboviruses (Karabatsos, 1995 ).
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Results |
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Figs 1 and 2
present the phylogenies for the NS5 gene and the E gene, respectively. The topologies showed congruence at all levels where bootstrap or quartet puzzling support was greater than 60%. Equivocal incongruence was observed for SLE, AROA, BSQ and NJL virus monophyly and the DEN viruses between the two phylogenies presented in Figs 1
and 2
.
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Aedes clades and the Culex monophyly
Mapping epidemiological and disease characteristics of the individual mosquito-borne viruses onto the phylogenetic trees revealed a correlation between the principal vector genera (Culex and Aedes species), the principal vertebrate hosts (birds and/or mammals) and the virus tropisms in humans and livestock (neurotropic versus non-neurotropic) (Figs 1 and 2
). The mosquito-borne viruses could be divided into two epidemiologically distinct vector groups, those that were primarily isolated from Aedes species and those that were primarily isolated from Culex species. The 17 flaviviruses that were primarily isolated from Aedes species formed two paraphyletic groups, one containing YF virus and the other containing the DEN viruses. In these two paraphyletic groups, 82% of mosquito-borne flaviviruses are known to be associated with Aedes species (14/17), hereafter denoted as the Aedes clades. The other viruses in these clades, i.e. SEP, POT, BAN and SAB, have been primarily associated with Mansonia species, rodents, Culex species and sandflies, respectively. The major exception is the mosquito species Haemagogus, the sylvatic vector of YF virus in South America, although this does not apply to the urban vector. The flaviviruses primarily isolated from Culex species formed a single clade of 23 viruses, which included JE, SLE and WN viruses as notable examples. The mosquito vector was identified for at least 16 of these viruses (Karabatsos, 1995
). Of these 16, 89% of mosquito-borne flaviviruses in the Culex species monophyly are known to be isolated from Culex species (14/16), hereafter denoted as the Culex clade.
Figs 1 and 2
show that there is a clear correlation between the virus, mosquito vector species and associated host. Both Aedes clades contained viruses that were maintained in sylvatic primate cycles, namely YF, DEN and ZIK viruses, or other mammals, while birds were not strongly associated with any of the viruses in the Aedes clades. Although Aedes clade viruses may infect birds, they are believed to be dead-end hosts. In contrast, none of the Culex clade viruses are maintained in primate cycles. Moreover, a high proportion of flaviviruses in the Culex clade are associated with mosquitobird cycles (at least 12/23 viruses) (Figs 1
and 2
). However, mammals are additionally involved in the persistence of Culex clade viruses, although many are considered dead-end hosts. For example, pigs play a role in the maintenance of JE viruses, while bats could be involved in WN virus persistence (Monath & Heinz, 1990
). Furthermore, AROA, IGU, BSQ and NJL viruses, which form a single clade, could be maintained by rodents.
Mosquito-borne viruses normally associated with neurological disease in humans or livestock, leading to encephalitis in severe cases, were found in the Culex clade and were generally associated with viruses that cycled between mosquitoes and birds. DEN virus from the Aedes clades was the exception, since there are rare cases of DEN encephalitis (Lum et al., 1996 ; Hommel et al., 1998
; Solomon et al., 2000
). In contrast, the mosquito-borne flaviviruses that are normally associated with haemorrhagic disease were exclusive to the Aedes clades and were associated with viruses that cycle between mosquitoes and primate hosts.
Clade robustness
The Culex and Aedes clades showed robust quartet support in the partial E gene phylogenetic tree and robust bootstrap support using the partial NS5 gene sequence for a single Aedes clade containing YF virus. The robustness of the distinction between the Aedes clade that contained the DEN viruses and the Culex clade was separately assessed using a Monte Carlo simulation. The observed phylogeny was subject to 100 Monte Carlo simulations, reconstructed using ML (reconnection limit=1) and manually assessed for alternative topologies. Distinct Culex and Aedes clades were observed in 98% of all simulations, while the SPO and ZIK virus sister group formed a trifurcation with the Aedes clade containing DEN virus and the Culex clade in 9% of these simulations.
There are also significant differences between the relative positions of some flaviviruses, as presented in the NS5-derived tree shown in Fig. 1 and those presented by Kuno et al. (1998)
. For example, ZIK and SPO viruses are positioned together with other Aedes-associated viruses (Fig. 1
); they had previously been placed in different positions among the mosquito-borne viruses by Kuno et al. (1998)
. Secondly, and in contrast with the previous analysis (Kuno et al., 1998
), KED virus showed close phylogenetic relationships with SPO and ZIK viruses (Fig. 1
), confirming the published serological data (Karabatsos, 1995
).
Tick-borne and NKV flaviviruses
Vertebrate host clades were also observed in the tick-borne and NKV flaviviruses for both the NS5 and E gene trees. In the NS5 gene phylogeny, NKV viruses, for which APOI virus was the basal lineage, were subdivided into rodent and bat clades. The rodent clade showing robust bootstrap support contained CR, JUT, MOD, SV and SP viruses, whereas the bat clade showing robust bootstrap support contained BUK, CI, DK, PPB and RB viruses (Figs 1 and 2
). APOI virus is also associated with rodents and, therefore, could be included in the rodent clade. The bat MML virus was also included in the bat clade, despite low bootstrap support. The remaining three NKV (bat) viruses, ENT, SOK and YOK, in the NS5 gene phylogeny form a sister group with the Aedes clade containing YF virus and are maintained by robust bootstrap support. From the point of view of their evolutionary origins, it is important to note that the rodent clade NKV viruses, with the exception of APOI virus, have only been isolated in the New World, whereas bat-associated viruses have been isolated from the Old and the New World, although none has been isolated in both regions of the world.
The TBE complex viruses were primarily associated with ixodid (hard) ticks, mainly Ixodes species and rodent hosts. The second group, consisting of tick-borne seabird-associated viruses (MEA, TYU and SRE), were most frequently isolated from Ornithodorus species or Ixodes uriae. KAD virus, which is associated with Rhipicephalus appendiculatus in Africa and Hyalomma pravus in Saudi Arabia, and GGY virus, which is associated with seabirds and Ixodes uriae, represent early lineages in the two tick-borne clades and possibly indicate a genetic link between the two tick-borne flavivirus groups.
Geographical distribution
The geographical distribution of the mosquito-borne flaviviruses was also examined to see whether or not virus dispersal correlated with either the Aedes clades or the Culex clade. With the exception of YF virus and DEN virus, which are believed to have originated in the Old World but can now also be found in the New World, all other viruses in the Aedes clades are only found in the Old World. On the other hand, the viruses in the Culex clade show geographical clustering, but genetically closely related viruses in the Culex clade have been widely dispersed to the Americas, Africa, Asia and Australasia, i.e. the Old and the New World.
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Discussion |
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It was demonstrated previously (Marin et al., 1995 ; Kuno et al., 1998
) that the Flavivirus genus was monophyletic and three distinct groups of viruses, namely tick-borne, mosquito-borne and NKV viruses, diverge at the deepest nodes. We have now demonstrated that the mosquito-borne viruses are subdivided into the Culex clade and Aedes clades. Moreover, the evolution of the Culex clade appears to have occurred after the separation of the mosquito-borne viruses from the tick-borne and NKV viruses. These observations were supported by the congruence between the NS5 and E gene phylogenies as well as by Monte Carlo simulation and quartet puzzling support.
The dominance of Aedes and Culex species (subfamily Culicinae) in flavivirus transmission is explained by the species prevalence of each of the genera, which contain 975 and 769 species, respectively, and comprise more species than all other mosquito genera combined (1522 species). Aedes and Culex mosquitoes are also among the small number of genera that are globally dispersed. Blood-meal data obtained for Aedes species suggest that mammals are the primary hosts of most species, which could explain the Aedes cladesprimate/mammal association (Mitchell, 1988 ; Christensen et al., 1996
; Clements, 1999
). The feeding patterns of only relatively few species of Culex mosquito are known, although a small number of bird- or mammal-specific species have been identified. Many Culex species feed indiscriminately on both mammals and birds and they include the principal vectors for several flaviviruses in the Culex clade, such as C. annulirostris (MVE virus), C. tritaeniorhynchus (JE virus), C. tarsalis (SLE virus) and C. univittatus (WN virus) (Robertson et al., 1993
; Christensen et al., 1996
; Clements, 1999
). The difference in feeding behaviour between Aedes and Culex mosquitoes provides a clear explanation for the associations between Aedes-borne flaviviruses and mammals or between Culex-borne flaviviruses and birds. Moreover, it explains why the association between the Aedes clades and mammals appears to be unequivocal, while the association between the Culex clade and birds contains a number of notable exceptions.
The second major correlation was between the type of disease produced and the mosquito clade in which each virus appeared. In general, severe infections caused by some Aedes species viruses result in haemorrhagic disease, whereas many Culex species viruses cause encephalitic disease; however, there have been reported cases of DEN (Aedes species-associated) encephalitis, but these seem to be very rare (Lum et al., 1996 ; Hommel et al., 1998
; Solomon et al., 2000
). Until the precise basis of flavivirus pathogenicity has been defined at the molecular level, it is not possible to understand why these different disease associations can be seen in the phylogenetic tree. In contrast with the mosquito-borne flaviviruses, different viruses in the tick-borne virus groups produce encephalitic disease, but OHF and KFD viruses may also produce haemorrhagic disease in humans and this does not appear to correlate with either their phylogenetic or their geographical characteristics.
Phylogenetic divisions between Old and New World flaviviruses were seen throughout the NS5 and E gene phylogenies. In some instances, dispersal of flaviviruses could be readily linked with the vertebrate host, providing evidence of the importance of the host in flavivirus evolution. In the case of viruses that established infections in bats, it is easy to imagine dispersal to remote sites, as the Old World bats from which flaviviruses have been isolated are known to migrate hundreds of kilometres (Shilton et al., 1999 ). On the other hand, individual rodent-associated NKV viruses might be expected to show a more restricted distribution and this is demonstrated by their detection almost exclusively in the New World and by their localized or niche-like distribution.
Virtually all of the tick-borne flaviviruses are exclusively Old World, with the exception of POW virus. The seabird-associated tick-borne viruses were dispersed to geographical areas where they established niches in seabird colonies in both the Northern and the Southern hemispheres (TYU, SRE and MEA viruses) (Chastel et al., 1985 ). At the early period of their evolution, the TBE complex viruses appear to have been dispersed either by seabirds or by rodents and their associated ticks. As they reached the forests of Asia, they became established predominantly in Ixodes species, where they continued their clinal evolution into Europe (Gao et al., 1993
; Zanotto et al., 1995
; Gould et al., 1997
).
The earliest evolutionary lineages in the mosquito-borne virus clades appear to have radiated to geographically distant parts of the Old World and to a wide variety of species, i.e. bats, Aedes species, sandflies and large animals, including simians and humans. Only YF virus and the four DEN virus serotypes, which cause human epidemics, are found in the New World. There is strong evidence to support the notion that YF virus was introduced to the Americas from the Old World during the past few centuries when slaves were transported across the Atlantic Ocean (Strode, 1951 ; Monath & Heinz, 1990
; Gould et al., 1997
).
There are also reasons to believe that DEN viruses have an African ancestry. The other members of the Aedes clade containing the DEN, ZIK, SPO and KED viruses were all isolated from Africa and formed two paraphyletic lineages to the DEN viruses. In addition, the E gene phylogenies of endemic/epidemic and sylvatic DEN viruses show a basal position for Old World sylvatic lineages of DEN1, DEN2 (Africa and Malaysia) and DEN4 (Wang et al., 2000 ). The vector of DEN virus, Aedes aegyti, is also believed to have originated in Africa (Tabachnick, 1991
). There is no reason to believe that DEN virus could not have been shipped to the Americas from the Old World in the same way as YF virus. Therefore, as most of the other Aedes species-associated viruses are found solely in Africa and since the Culex species-associated viruses appear to be descendants of the Aedes species-associated viruses, the mosquito-borne flaviviruses appear to have evolved out of Africa.
In conclusion, the flaviviruses that are recognized today represent a diverse group of viruses that could have emerged and dispersed during the past 10000 years, i.e. since the most recent ice age (Zanotto et al., 1996 ). The characteristic epidemiological groupings of the viruses that are apparent in the phylogenetic trees illustrate the significant influence of the invertebrate vectors, the vertebrate hosts and the particular ecological niches into which these species have evolved.
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Acknowledgments |
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Received 28 February 2001;
accepted 25 April 2001.