EVOLUTION, EPIDEMIOLOGY, AND DISPERSAL OF FLAVIVIRUSES REVEALED BY MOLECULAR PHYLOGENIES Ernest A. Gould,” Xavier de Lamballerie,* Paolo M. de A. Zanotto,t and Edward C. Holmes? “Insitute of Virology and Environmental Microbiology, Oxford, OX 38R, United Kingdom ‘Unite des Virus Emergents, cule de Médecine, 13005 Marseile cedex 05, France Departamento di Microbiologio, 1374, Braz SDepariment of Zoology, Universy of Oxtord, Oxford, OXT 3P5, United Kingdom 1. Introduetion A. General Properties ofthe Flaviviruses B. Geographie Distribution ofthe Flaviviruses C. Early Evolutionary Conclosions from Sequence Comparisons IL Molecular Phylogenetic Analysis ‘A. Thek-Vectored Viruses 15. Mosquito-Veetored Viruses ©. Nonvectored Viruses IIL, Correlation of Tree Topology with Epidemiology and Ecology IV, What Ie the Origin and Ago of tho Genus? 'V. Flavivirus Dispersal Strategies A, Tiek-Vectored Viruses B, Mosquito-Veetored Viruses ©. Nonvectored Viruses Vi. Conclusions References 1. Iyrropuction Fora long time, it has been assumed that the opportunity for genetic variation among the arboviruses is significantly constrained by their need to replicate in both vertebrate and invertebrate hosts. In this sank, the flavivinises present an interesting case for analysis hecanse the genus Flavivirus contains both arthropod-vectored and nonvec- tored viruses. This review will examine our current knowledge of fla- vivirus phylogeny and try to illustrate the significance of arthropods, habitats, and vertebrates, including humans, in their evolution, epi- demiology, and dispersal. The comments and assumptions we shall make may seem controversial among some traditional flavivirologists but they might spark deeper investigation in the future. n stabs sn ny ee,72 E.A. GOULD ETAL, A. General Properties of the Flaviviruses The genus Flavivirus contains approximately 70 antigenically related Viruses many of which infect both vertebrate and invertebrate species. They are genetically distinct from viruses in the other recog- nized genera (6, 41) within the family Flaviviridae, viz., Pestivirus, Hepacivirus. By combining antigenic and phylogenetic data based on either the viral envelope (E) gene the nonstructural genes NS5 and 'NS3. or the whole genome (3, 24, 35, 56) and also taking into account the vertebrate and invertebrate hosts, as well as the diseases with which each virus is associated, the flaviviruses were recently classified by the International Committes for the Taxonomy of Viruses (ICTV) into 12 distinct groups (20). For the purpose of virus identification in this review, and taking into account more sequence data than was previously available, a similar but not identical scheme of subdivision of the flaviviruses is presented in Table 1. This modified scheme (1) places Kadam (KAD) virus in the ‘TYU group as opposed to the TBE virus complex, to recognize ts phy- logenetic position; (2) provides more detail relating to the tick-borne virus group; (3) identifies several viruses as distinct species rather than subtypes of other viruses; and (4) identifies a Kedougou virus group. Although these taxonomic arrangements or subdivisions may require modification as more sequence data become available in the future, most of the current groups show similar relationships to those originally proposed on the basis of serological tests (6, 41) and as will be shown helow, correspand closely with their knawn epidemiological characteristics. The virions are approximately 50 nm in diameter, spherical in shape, and contain a lipid envelope. The single-stranded positive aenae RNA, which is about 11 kb in length, is enclosed by a single capsid (C) protein. The envelope of mature virions contains two virus-encoded proteins: the membrane (M) protein, which is postiranslationally cleaved from the premembrane protein (immature virions) and the E protein which hemagglutinates red blood cells, mediates receptor binding and acid pH-dependent fusion activity, as well as inducing the neutralizing and/or protective humoral immune response (39). Infected cells contain seven nonstructural proteins; NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5. The precise roles of many of the proteins have not yet been defined but the NS1 protein may be involved in virion assembly or release (28) and has also been impli- cated in virus replication (16-18) and signal transduetion and mem- brane anchoring (22). The NS3 protein appears to have severalFLAVIVIRUSES 73 ‘TABLE I VISION OP THE FLAVIVIRUSES BaSKO PRIMARILY on Sequence Dara su 1 Tick-borne viruses ‘Mammalian tick-borne virus group Louping il virus i Trish subtype (Ise) British subtype (BSE) Spanish subtype (Sse) Turkish subtype (TSE) Tiok- borne encephalitis virux (TRE) European subtype wre} Siberian subtype (STBE) Far Bactorn eubtype \PETBE) Omsk hemorrhagic fever (OHF) Langat virus (Len) yasanur Forest disease virus RED) Karshi virus «Ks Royal Farm virus RF) Powassan virus «ows Gadgets Gully virus ccay) Seabird tick-borne virus group yuleniy virus eryvy Meaban virus (MBA) Saumarner Reef nirus (SRR) Kadam virus? (KAD) 2a, Mosquito-borne viruses (Aedes spp. associated) Yellow fever virus group Bans virus aan) Uganda § virus ‘us agra vie save) Potshum virus ror) Saboyaviras® sab) Boubou virus (B00) age Hil virus Bm Yellow fover virus a) Wesselsbron snus vst) Sepik virus ‘ser? Kedougou virus? ° (KED) (continues)4 B.A GOULD BT AL. TABLE I (continued) 2a. Moxquito-borne viruses (Aedes app. associated) ‘Dengue virus group Dengue virus 1 END, Dengue virus 2 (DEN-2) Dengue virus 3 (DEN) Dengue virus 4 (DEN) ‘Spondweni virus group ‘Spondweni virus (spo) Zika virus (aK) 2h Macouitachame rirueee (Onley app a ‘roa virus group ‘Aroa virus (AROA) Iauape virus acu) Naranjal virus aL) Bussuquara virus Bus) \Neaya virus group Bagaza virus aa) Israel turkey meningoencephalitis virus an Neaya virus «vray ‘Tembueu virus amu Mheus virus Le) Rocio virus (Roc) ‘St Louis encephalitis virus (SLE) Japanese encephalitis virus group Alfuy virus (aur) Murray Valley encephalitis virus (uve) Japanose encephalitis virus aR) Due virus wsv) Koutango virus (Kou) Kunjin virus aus) West Nide virus (wn) Yaounde virus (xAo) Cacipacore virus cro) ‘Kokobera virus group Kokobera virus (KOK) Derayora virus (rn (continues)FLAVIVIRUSES cc VABLII (continued) 3, Nosknoun vector viruses Batu Cave virus wwe) Phnom Penh bat virus PPB) Carey Isiand virus cr Bukalasa bat virus (BB) Dakar bat virus DB) Rio Bravo virus (RB) Montana myotis eukoencephalits virus iL) ‘Modoe virus group Cowbone Ridge virus «cry Modoc virus imop) Sal Vieja virus sv) Jutiapa virus uur) San Perlta virus (sp) poi virus (apo YYokose virus group Bude but virus BB) Yohose virus (YOK: Sokoluk virus rod ‘Tentative species in the genus Tamana bat virus (TAB Cal fusing agent (CRAY | No direct evidenee of transmission in mosquitoes Tentative assignment. functions including serine proteinase activity when the N-terminal one-third of this protein is complexed with NS2B, resulting in process- ing of the polyprotein. The C-terminal portion has an RNA helicase domain and RNA triphosphatase activity that is probably involved in the formation of the 5'-terminal cap structure of the viral RNA (44), ‘The NSB protein is the RNA polymerase (493) Many flaviviruses can replicate in both vertebrate and invertebrate (mosquito or tick) cells. The positive-sense RNA serves as the messen- gor for nogative strands that are then used as templates for additional genome-length positive-sense RNA molecules. The detection of replica- tive intermediates containing both double-stranded and single-76 E.A. GOULD ETAL, stranded molecules as well as replicative forms, i.e., duplex RNA mol- ecules, implies a semiconservative mechanism for RNA synthesis, ‘Translation of the RNA leads to the production of a polyprotein that is processed by cellular and viral proteases to produce the structural and nonstructural viral proteins. Virus assembly probably takes place in the rough endoplasmic reticulum. Immature virions are then trans- ported through the membrane systems of the host cell to the surface where exocytosis occurs. Immediately prior to virion release, the PrM protein is cleaved by a furin-like protease to gonerate mature virions. B. Geographic Distribution of the Flaviviruses Viruses in the genus Flavivirus are found on every continent but individually they have distinct geographic distributions, determined by a combination of factors such as the ecological habitat in which they reproduce, and the vertebrate and invertebrate hosts that they infect: Precise information describing the geographic location where each virus was isolated, their antigenic relationships with one another, and the vertebrate and invertebrate hosts with which they are associated has been available for many years (23, 50). The tick-borne flaviviruses can be divided into two main groups: the tick-borne encephalitis (TBE) complex viruses, most of which infect radents, other forest animals, humans, and Trodes spp., and a second group, which is associated with seabirds and their ticks. With the exception of Powassan (POW) virus and a closely related strain designated Threo Arch virus, all recognized TBE complex viruocs are found only in the Old World. In the natural environment, Powassan virus is known to have life cycles in both ticks and mosquitoes, and Uhis might account for Une wider geographic distribution of this virus compared with other TBE complex viruses. As a general rule, the TBE complex viruses do not share overlapping habitats with other flaviviruses, L<., they exhibit niche-like characteristics in the forests of Malaysia, India, Asia, and Europe. There are reports (11), that ‘TBE complex viruses were isolated from the brains of ducks that have migratory flight paths between Russia and Slovakia, Unfortu- nately, the sequences of these bird-associated TBE complex viruses have not been determined. If this becomes possible in the future, it will be interesting to see whether or not the bird-associated viruses resemble, most closely, Slovakian or Russian strains of TBE complex viruses. A subgroup of the TBE complex viruses causes encephalomyelitis in sheep. These viruses are found on the sheep- rearing hillsides in southern Europe (34), Turkey (12), Greece (34),FLAVIVIRUSES 7 and on the moorlands of the British Isles (38, 43) and Norway (13), where sheep are farmed. The British virus, louping ill, is the most comprehensively described of these sheep-associated viruses. ‘A second and smaller group of tick-borne viruses mainly infects seabirds and their associated ticks, particularly Ornithodorus spp. ‘These seahird.associated viruses (see Table 1) which are found only in the Old World, are geographically widely separated, in northwestern France (Meaban virus), far eastern Russia (Tyuleniy virus) and the Great Barrier Reef of Australia (Saumarez Reef virus). Kadam virus, which was isolated from Rhipicephalus pravus, feeding on cows in Uganda and Hyalomma dromedarri, feeding on camels in Saudi Ara- bia, is also included in Uhis group of viruses aud may represent an early land-based, mammalian-associated, predecessor of the seabird- associated viruses. Heologically, the mosquito-borne flaviviruses can be divided into two ‘major categories: those usually but not exclusively, associated with Aedes spp., which have been assigned to four groups in Table 1 and those associated primarily with Culex spp., which have also been assigned to four groups. The geographic distribution of mosquito-borne Mlaviviruses is determined by the natnral habitats of the vertebrates and the mosquitoes that they infect. All Aedes spp. associated viruses occur naturally in the Old World. Among these, only yellow fever (YF) and dongue (DEN) virus havo also boon found in the New World. Yel low fever shows a relatively restricted geographic distribution, being mainly confined to the central tropical regions of Africa in the Old World and to the Caribbean islands and central and south America in the New World. Perhaps the most baffling characteristic of YF is its failure to become established in tropical Asia where there are suscepti- ble mosquitoes and nonhuman primates. Many theories have been put forward to explain the geographic localization of this virus. These include (1) the presence in Asia of related viruses which either out compete the virus or provide a residual background immunity pre- venting its establishment, (2) low vector competence of Asian strains of Aedes aegypti, (3) the relatively low frequency and concentration of virus introduced into Asia, compared with Latin America, (4) YF virus in the eastern regions of Africa, i.e., the virus most likely to be dis- persed eastward, conld he significantly different. from the strains that ‘occur in central and west Africa, (5) genetic immunity to YF virus in Asians and/or Asian primates, (6) YF virus has retained its sylvatic nature and therefore does not adapt readily to new environments. Although the first possibility is often considered the most likely, none of these theories adequately explains the failure of YF to become estab-8 £.A. GOULD ETAL, lished in tropical Asia, although a combination of some or all may be near to the truth. Dengue virus. on the other hand. is distributed worldwide throngh- out the tropics wherever Aedes aegypti breed. Unlike the TBE com- plex viruses, the four DEN virus serotypes show overlapping geographic distributions and frequently produce dual infections in humans (26, 32), which in some cases result in intratypic genetic recombination (55). The increasingly wide dispersal of dengue virus throughout the tropics reflects the growing impact that human activ- ity, in the form of urbanization, transportation, commercialization, and tourism, is having on flavivirus dispersal. Other Aedes spp. asso- ciated viruses, such as Spondwent, Zika, Wesselsbron, Banzi, Uganda §, ete. tend to have more restricted geographic distributions and are found in Africa or Asia in forest habitats, or in environments containing wild or farmed animals. Until 1999, individual Culex spp. associated viruses were found either solely in the New World (e.g., St Louis encephalitis virus, Caci- pacore virus) or only in the Old World (e.g., West Nile virus, Japanese encephalitis virus). West Nile virus was previously found in Africa, southern Rurope, Asia, and Australasia but. recently this situation changed when West Nile (WN) virus was discovered to have arrived and become established in North America (5, 27). The inadvertent introduction of WN virus into the New World possibly roprocents another example of the impact of modern transportation systems on the emergence of virus diseases. As with some of the Aedes spp., many Culex spp. associated viruses also frequently sliow geographic overlay in Asia, Australasia, or the Americas. Moreover, several of the Culex spp. associated flaviviruses have also been isolated from bats and rodents in the natural environment, demonstrating the wide range of species that can support these viruses. It is interesting that some Culex spp. associated flaviviruses such as Murray Valley encephalitis, Alfuy, Kokobera, and Stratford are found only in Australia and nearby regions of southeast Asia, Since these viruses represent recent evolu tionary lineages within the Culex clades. and since several other related viruses, e.g., dengue, JE, and possibly WN/KUN, have been introduced into Australia, it seems reasonable to assume that they represent surviving introduced lineages that have adapted to the local ecologies. Viruses such as West Nile clearly have greater adaptabil- ity/survivability and can therefore be tracked along their presumed disporsal routes back to Africa. The flaviviruses that have no known vectors (NKV), are associated either with rodents or bats. The individual bat-associated NKV virusesFLAVIVIRUSES 79 are found either in the New World or in the Old World but none so far has been found in both regions. On the other hand, and with the excep- tion of APOT virns, rodent-associated NKV viruses have only been iso- lated in the New World where they have retained very restricted dispersal patterns. It is known that neither bats nor birds use migra- tory pathways across the major oceans, ie., the most common long dis- tance migratory flights are generally in a northerly and southerly direction. This probably accounts for the apparent lack of mixing between Old and New World NKV viruses even though there have clearly been introductions at some time in the past in one direction or the other, perhaps by rodents on ships. Nevertheless, bats undoubt edly contribute significantly to localized spread of flaviviruses, over major land masses. C. Early Evolutionary Conclusions from Sequence Comparisons ‘The first phylogenetic comparisons of tick- and mosauito-borne fla- viviruses were reported in 1989 (33). These authors demonstrated the coincidence between flavivirus relationships based on serological and sequence data Subsequently, Shin et a! (47) compared flaviviras sequences based on C, M, and E genes and predicted the distinet evolu- tionary divergence of tick- and mosquito-borne viruses from a common flavivirus predecessor. Aa will be shown later, this has subsequently been supported in more detailed phylogenetic investigations. It was also noted that the degree of relative similarity among nearest neighbors of individual TBE complex viruses is higher than that with the mosquito- borne viruses, Based on this observation, the authors proposed that the tick- and mosquito-borne flaviviruses had been subjected to different selective constraints during their evolution. At about this time, phyloge- netic analysis of sequences based on the E-NS1 junetion of a large num- ber of DEN-2 viruses led to the conclusion that sylvatic West African dengue viruses could be distinguished from epidemic strains and also that some dengue epidemics in the New World had arisen as the result. of the introduction of DEN-2 viruses from Asia. Subsequently. nhyloge- netic trees constructed from each individual gene of 11 different fla- viviruses revealed similar topologies (4). These data were interpreted as, implying that the flaviviruses have evolved exclusively by divergent ‘mutational change and it was concluded that genetic recombination had not played a significant role in this evolution. As will be discussed later, thie is now known to be incorrect. ‘The authors (4) also compared the transitions and transversions of & portion of the genomes of 40 DEN-2 virus isolates and interpreted the80 E.A.GOULD ETAL, data as implying that sylvatic dengue viruses (.e., DEN-2 strains from West Africa) have evolved under stronger selective constraints (fewer nucleotide and amino acid differences but equal numbers of transi- tions and transversions) compared with dengue virus strains that cir- culate freely in other parts of the world among human populations (predominantly silent transitions). In addition to ite significance for dengue virus epidemiology, this observation could also have important implications for yellow fever virus, which has distinct sylvatic and urban cycles. A phylogenetic Lree subsequently cousiructed from he complete E gene sequence data of 8 tick-borne and 10 mosquito-borne flaviviruses (52) extended the phylogenetic analyses of flaviviruses, confirming the monophyletic origin of the genus and the distinct diver- gence of tick- and mosquito-borne viruses. Subsequently, on the basis of the close antigenic relationships and comparative similarities between strains of LI and TBE complex viruses, it was concluded that LI virus isolated in Norway was probably introduced from Scotland relatively recently, and it was also proposed that the TBE complex viruses had evolved, as a “genetic continuum” (13), implying correla- tion with temporal and spatial parameters. Phylogonotie analycee of dengue (45, 46, 53), Wost Nile (2), and yol- low fever virus (30), showed the extent of genetic variation within the envelope gene of individual virus species and enabled conclusions regarding Uke origin, persistence, and geographic diepercal of these viruses. For example, phylogenetic analysis of DEN-2 virus in 1991 (45) showed that these viruses formed distinct clusters according to their geographic origin. Moreover, it was proposed that the strain of DEN-2 virus responsible for dengue hemorrhagic fever in the Ameri- cas originated in Asia and was introduced presumably by transporta- tion across the Atlantic Ocean. The first detailed phylogenetic analysis of the genus Flavivirus showed that of the mosquito-borne viruses analyzed at that time, YF virus diverged first, followed sequentially by DEN-4, DEN-2, DEN-3, and DEN-1 virus in the Aedes spp. group (Fig. 1). Interestingly, the Culex spp. group of viruses appear to have origi- nated after the first divergence of the Aedes spp. group and can there- fore be considered descendants of the Aedes spp. viruses. Among the Culex spp. viruses, SLE had the most divergent lineage, followed more recently by WN/KUN and MVEVJE virus (85). Extensive genetic varia tion within geographically closely related southeast Asian dengue strains contrasted with the very close genetic relationships of some dengue viruses shown to be present both in South America and eouth- east Asia (29). These results show directly that DEN-2 virus has been dispersed throughout the tropics in very recent times, reflecting com-FLAVIVIRUSES 81 mercial activities and the movement of large numbers of military per- sonnel across the major oceans. (On the basis of sequence comparisons. YF virus has been erouned var iously into east African, central/west African, west African, and south American subtypes (9, 30, 54). The degree of genetic similarity between Latin American and African YF viruses also confirmed that YF virns was introduced to the New World relatively recently. Within these YF groups, the extent of genetic variation is relatively limited in comparison with the more variable dengue viruses, implying that YF virus has retained its sylvatic nature and has been evolving less rapidly than the dengue viruses. However, this possibility does not take into account the limited number of samples that have been studied. As the speed and frequency of nucleotide sequencing increased, more detailed phylogenetic analyses became possible and these are dealt with below. II. Moecunar Prrvtocenetic ANaLxsis A. Tick-Veetored Viruses Viruses in the tick-borne encephalitis complex have always been con- sidered difficult to distinguish by conventional serological methods and serology provides only a fow clues aa to the precice nature of their evolu tion. Prior to 1995, relatively little of the characteristies of tick-borne flavivirus evolution was known from detailed phylogenetic analyses of represeutative viruses in the gemus favivirus. Analysis of the complete envelope (E) gene sequences of 22 flaviviruses (35) confirmed the com- mon origin and the early divergence into mosquito- and tick-borne viruses, The phylogenetic tree Ug. 1) showed that the tick-borne viruses diverged into two major clades, one consisting of TYU and SRE virus, a monophyletic sister group of another clade comprising the tick- borne encephalitis (TBE) complex viruses. The TYU group, together with Meaban virus, which was included in later trees, are found in seabirds and their associated ornithodorus spp. ticks. Figure 1 shows that these viruses diverged early in the evolution of the genus. ‘The tree also revealed striking differences in the evolutionary char- acteristios hetween the TRF complex and the mosquito-borne viruses and emphasized the enormous influence of the tick vector (Ixodes spp. on the evolution and dispersal of the viruses in the TBE virus complex. Data based on E gene sequence commencing with POW virus at the deepest node, confirmed that the TBE complex viruses have evolved continuously with time until the emergence of louping ill (LI) virus,oe i ee wove | L_apemee, - i ereen les) Tick Fo. 1, Neighborjoining phylogenetic tre illustrating the evolutionary relationships of viruses in the genus Flavivirus. The tree was constructed using the Ist and 2nd codon posi tone for 41 E gone, Al horisontal branch longths are drawn to scale (0. proportional to ‘the number of accumulated nucleotide substitutions). Percentage bootstrap support values fre shown above the branches. The root fr the genus separating tick- and mosquito-borne {groups was determined based on the use of CRA virus as an outgroup. 82FLAVIVIRUSES 83 2. A cladogram (56) for the TBE virus complex E genes shown geographically from west to east and presented with respect to LI virus. the most recent lineage in the TBE virus complex. The gradually increasing genetic distance between theoe with inereasing geographic distance of the viruses from each other, in the northern hemisphere, measured from a point in Scotland where LI Viruses are most frequently found. The TBE complex viruses have therefore evolved as a cline across the forests and sheep-rearing hill- sides of the northern hemisphere and this was illustrated (Fig. 2) by Zanotto et al. (56). Independent evidence to support the concept of a clinal distribution was reported following analysis of the envelope gene of TBE complex viruses from western and central Europe (10) and far east Asia (19). Three lineages have been identified, those cor- responding to European, Siberian, and far east Asian subtypes and these have been incorporated into Table 1. Moreover, by assuming that all flaviviruses have evolved during a continuous period in the past and by knowing the dates of isolation of these viruses, it was possible to compare the relative rates of variation of tick-horne and mosquita- borne viruses. The results showed that the tick-borne viruses have evolved significantly more slowly than the mosquito-borne viruses (66). This will be discussed in more detail below. reconstructed maximum likelihood tree with an increased number of viruses, based on partial NS5 gene sequence with the third codon 008 correlates diroetly84 E.A.GOULD ETAL. position and the hypervariable loop excluded (Fig. 3), places KAD virus in the seabird-tick associated virus clade, with POW virus as the earliest lineage in the clade for TRE complex viruses. Gadgets Gully (GGY), Royal Farm (RF), and Karshi (KSI) virus (24) were shown to be lineages within the TBE complex. Caleulatione based on cotimated substitution rates and dates of virus isolation (38) suggested that LI virus in the British mainland was probably introduced from Ireland about 400 to 500 years ago and then continued lo evolve on the northern hillsides of Britain during the past 200 to 300 years. This is further supported by veterinary reports of sheep encephalomyelitis on the Scottish hillsides and on the Devonshire moors following their introduction during the 19th and 20th centuries, respectively. The phylogenetic tree (Fig. 4) based on envelope gene sequence data, includes all recognized sheep cencephalomyelitis viruses, i.e., those from Turkey, Greece, Spain, Ire- land, Wales, England, Scotland, and Norway, and illustrates their divergence from the other TBE complex viruses that are associated with rodents in forests. Estimations of the times of emergence for the entire TBE complex viruses (57) predict that the earliest lineage of the TBE complex viruses, i.o., POW virus, soparatod from the TYU group between 4000 to 6000 years ago. Although it is recognized that such estimates must allow a substantial margin for error, the most recent glaviativu lasted until abvut 10,000 Ww 12,000 years ayy eucomipascinng extensive areas of the northern hemisphere. One can therefore imag- ine post-ice age viruses from the warmer biogeographic regions of Africa and Southeast Asia dispersing northward into far east Asia and ‘westward into Europe creating the cline that has been described. The cline therefore illustrates the characteristic way in which these viruses establish niches and then remain genetically homogeneous. Strains of LI virus in the United Kingdom are remarkably similar, showing >97% amino acid identity in the B gene among strains that were isolated more than 50 years apart (38). The Spanish equivalent of LI virus, SSE virus, is distinct (95% identity with LI virus, 95% iden- tity with TBE viruses, and 95% identity with TSE virus) and occupies its own niche on the sheep-rearing hillsides in the Basque region of Spain. A unique tripeptide sequence in the E gene can distinguish each ofthe antigenically very closely related LI-like viruses found in Turkey (12), Greece and Spain (34), the United Kingdom and Norway (13), and Ireland (38). The exquisite specificity of molecular analysis for distin- guishing virusca, io also clegantly illustrated by the discovery that ‘Negishi (NEG) virus, which was apparently isolated from two children during an epidemic of Japanese encephalitis in 1948 in an urban areaPLAVIVIRUSES 85 Sw Comex Fi. 3. Maximum likelihood tree of most flaviviruses, based on partial NSS sequence ata with the third codon position and the hypervariable loop excluded. Sequences were assumed to evolve according to the JKYSS substitution model with the rate of transi tions and transversions and the extent of amongesite variation in substitution rate ji. Gamma distribution) astimated Fram the date86 E.A GOULD ETAL. 10% Divers Fic. 4. Maximum likelihood phylogenetic tree (38) of the E gene from 24 tick-borne ‘avivirases. Branch lengths are drawn to scale and all nodes supported by more than 75% bootstrap support are indicated. The tre is rooted with the sequence from FETBE virus, Sofin strain. The three main populations of vieus in the British Ialeo (Ireland, Wales, and Great Britain) are indicated, along with those viruses secondarily introduced into Ireland and Norway, and the viruses found in the southwest of England, of Tokyo (1, 40), is unequivocally LI virus (24, 51). LI virus has been isolated many times in the British [oles and was fully characterized nearly 20 years before the first reports relating to Negishi virus. More- over, Negishi virus was never re-isolated in Japan and there is no definitive serological evidence of its presence there. Therefore, the con- cept that LI virus (strain Negishi) exists in Japan is inconsistent with the recognized characteristic of Ll virus as the etiological agent of tick- borne sheep encephalomyelitis on the sheep-grazing uplands of the British Isles (43). It remains to be explained how a strain of LI virus apparently travelled thousands of miles from Britain to cause two fatal infections in children in a region of Japan where there were no sheep and suitable ticks to transmit the virus. B. Mosquito-Vectored Viruses Figure 1, which uses data based on flavivirns F. gene sequence, illus: trates the marked contrast in phylogenetic topology and therefore epi- demiological characteristics, between the mosquito-borne viruses and tho TBE complox virusoe. Tho TBE complex viruses have diverged con- tinuously through time, generating new serotypes continually and pro- ducing an asymmetric topology with a characteristic stepwise branchingFLAVIVIRUSES 87 process, In contrast, the mosquito-borne viruses show a two-phase pat- tern of evolution in which a slow growth phase is followed by one of rapid growth and only small numbers of distinct virus serotypes have survived from the ancestral nodes in each of the major mosquito-borne clades. For the mosquito-borne viruses there are no observable lineages from each of the main serotypes until relatively recently when there is a major increase in eladogenesis. This is also supported by the serological data that show close antigenic relationships between the TBE complex virnses, representing continuons lineage generation throughout. the recent past. On the other hand, the mosquito-borne viruses show greater antigenic and genetie diversity implying higher rates of evolu- tion and phylogenetic gapa in which lincages have dicd out. The higher rates of evolution of the mosquito-borne viruses are consistent with esti- mates of the relative rates of evolution based on knowledge of times of virus isolation and calculated rates of nucleotide substitution. Tt was estimated that the tick-borne viruses are evolving about 0.56 times as fast as the mosquito-borne viruses (56). This is probably accounted for by the different vector biology of the tick- and mosquito-borne viruses, which will be deseribed below. Estimations of the times of divergence of the mosquito-borne viruses show that many of the viruses currently causing epidemic outbreaks represent very recent lineages. In the DEN and JE serotypes the period of intense cladogenesis occurred during the past two centuries (21, 57), reflecting the recent availability of new and susceptible hosts, i.e., humans, coupled with inereased worldwide movement and mixing of vectors, viruses, and hosts. C. Nonvectored Viruses ‘The evolution of NKV viruses has not been extensively studied but the deepest split on the tree is between the vectored and nonvectored viruses, All the other viruses in the family Flaviviridae are nonvec- tored, suggesting that this is also the ancestral state for the genus In other words, flavivirus vector-transmission evolved from nonvec- tored transmission. The split between vectored and nonvectored fla- viviruses appears to have occurred before the separation of the tick- and mosquito-borne viruses, probably more than 5000 years ago. The fact that some NKV viruses (EB, YOK, SOK) diverged with the mos- quito-borne viruses and then separated to form a distinct NKV group. implies that there has been secondary loss of vector-borne transmission in these viruses and reflects the great adaptability of the flaviviruses. This is also evident from the fact that several mos-88 E.A. GOULD ETAL. quito-borne and even some tick-borne flaviviruses have been isolated from bats in the natural environment. ‘The phylogenetic topology of the NKV viruses resembles the mos- quito-borne viruses with a discontinuous pattern of evolution but the most recent NKV lineages show less diversity, probably reflecting their specific adaptation to the sylvatic environment, although it may merely reflect the lack of adequate sampling. IIL. CorRELATION oF TREE TOPOLOGY wit EPIDEMIOLOGY AND EcoLoGy The flaviviruses are an excellent example of the influence of arthro- pods and their vertebrate hosts on virus evolution. They display a very wide range of epidemiological characteristics and the phylogenies reflect this as an arrangement of viruses that correlates very closely with their epidemiology and ecology. ‘The ‘YU and the ‘I'BE complex viruses are dependent tor their life cycles on different tick vectors, i.e., ornithodorus or ixodes spp. and appropriate vertebrate hosts, i.e., seabirds and rodents, respectively. These viruses diverged relatively early in the evolution of the fla- viviruses and have subsequently evolved to show strikingly different epidemiological and ecological characteristics. Interpretation of their evolutionary characteristics may change as more related viruses are identified. Nevertheless, Tyuleniy (TYU), Saumarez Reef (SRE), and ‘Meaban (MEA) virus, none of which are thought to be human pathogens, may have their roots in Africa as represented by Kadam (KAD) virus. They can be isolated from ornithodorus spp. that infest the nesting grounds of seabirds. They have radiated to very different regions of the world, i.e., far eastern Russia, the Great Barrier Reef, and northwestern France. Serological evidence shows that the seabirds may be infected by these viruses when they are bitten by infected ticks but no clinical signs of infection have been noted, indi- cating a long-term association between the birds and the viruses. The TBE complex viruses are more closely associated with Lxodes spp. These viruses are human pathogens and are usually found in infected ticks surviving in vegetation that provides a micro-climate with relative humidity close to saturation throughout the year. The forest undergrowth, in many parts of Asia, Europe, and North Amer- ica, as well as the upland sheep-grazing pastures of the UK and parts of southern Europe, provide such conditions. The postulated continu- ous evolution of the TBE complex viruses, north-eastward and thenFLAVIVIRUSES. 89 westward across Asia and Europe (56), is now further supported by the ‘more recent sequence data (24) for Karshi (KSI), Royal Farm (RF), and Gadgets Gully (GGY) virus. These viruses represent an early part of the cline, which is a feature of the TBE complex viruses. Gadgets Gully virus was isolated from Macquarie Island in the Southern Ocean, Since this virns is also associated with migratory seahirds, and their related ticks, it was suggested (48) that it could represent a natural link between subaretie and subantarctie TBE complex viruses and pos- sibly also, a more gonetieally distant link between the scabird-ascoci ated TYU group and the rodent-associated TBE complex viruses, In the natural environment, the mosquito-borne viruses are known Ww be primarily, but not exclusively, assuciated with either Aedes ur Culex spp. This division of the viruses on the basis of their associated ‘mosquito species correlates precisely with the tree topology. Thus, there are clades corresponding either to the Aedes spp. viruses or to the Culex spp. viruses, The Aedes spp. viruses tend to bite primates in forest or savannah environments, and/or humans and herded animals in urban or rural environments. Some of these viruses (yellow fever and dengue virus) are also associated with the development of hemor- thagie disease in humans. The Culex spp. viruses. on the other hand preferentially bite birds and rodents in forest environments and are also attracted to pigs, horses, ducks, ete., which form an essential part of the human food chain in rural Asia. In contrast with the Aedes spp. viruses, many of the Culex spp. viruses (SLE, JE, MVE, WN) are more characteristieally associated with encephalitic disease in humans. Whether or not such divisions in disease characteristics have occurred through selection of specific genetic determinants remains to be confirmed by detailed bioinfor- matic analysis. Another significant feature revealed in the phylogenetic tree (Fig. 3: is the fact that the Aedes spp. viruses were all originally isolated in the Old World, and only YF and DEN virus can be found in the New World. ‘They are believed to have been introduced during the past three or four centuries following commercial trading on slave ships, etc., across the Atlantic Ocean. Thus, the Aedes spp. viruses almost certainly have their evolutionary origins in the Old World. Moreover, although the tree is not at all certain in this region, the branching pattern of the ‘mosquito-borne viruses shows that the first node of the mosquito- borne virus phylogeny led to Aedes spp. viruses and at least three NKV viruses, EB, YOK and SOK. The branching pattern at the next more recent node leads to another Aedes spp. clade and the Culex spp. clade. ‘The same conclusions were derived from the phylogenetic trees con-90 E.A. GOULD ETAL. structed using sequences representing either the entire flavivirus genome or individual genes (3). Thus the branching patterns of all these trees suggest that the Culer spp. virnses are descendants af the Aedes spp. viruses. If this proves to be correct, it could have important, implications for the temporal and spatial origins of the mosquito- transmitted flaviviruses. Onc the Culex epp. viruses diverged from the Aedes spp. viruses, they then became established in both the Old World and the New World but with the exception of WN virus (see later) none of these viruses became established in buth the Old World. and the New World. Clearly, this demonstrates that although birds may be responsible for dispersal of these viruses across major land ‘masses, they do not appear to have contnibuted significantly to disper- sal of the viruses between the Old World and the New World. The alternative possibility, that viruses have been moved regularly across ‘oceans by birds but the viruses have failed to become established when introduced to new environments, seems unlikely in view of the fact that the flaviviruses do exhibit extensive adaptability. Three NKV viruses that infect bats, EB, YOK, and SOK virus, diverged away from the other NKV viruses and separated with the mosquito-borne viruses, before evolving as a distinct clade, following secondary loss of vector transmission. The larger group of NKV viruses, which did not separate with the mosquito-borne viruses, sub- sequently diverged into rodent- and bat-associated viruses. However, once again it is clear that regardless of the type of vertebrate host with which they became associated, they were dispersed either into the Old or into the New World, not into both regions. IV, Waar IS THE ORIGIN AND AGE OF THE GENU Currently, there is insufficient phylogenetic information for a pre- cise answer to the question of the origin of the flaviviruses. Neverthe- less, most of the tick-borne viruses, all of the Aedes spp. associated viruses. and many of the NKV viruses, have sylvatic life eycles in the Old World, implying their presence over a long period of time. Cell fus- ing agent (CFA), which is a very divergent lineage, has been used to root Mavivirus trees on the basis of its similar genome strategy. This, insect virus was isolated from African Aedes aegypti and appears to be nonpathogenic for this mosquito species (7), again implying long-term relationahipo between the virus and the hoat. Therefore, although it io recognized that only limited numbers of virus samples are available for analysis, at this stage, the Old World appears more likely than the New World to be the origin of the genus Flavivirus. This is also sup-FLAVIVIRUSES, 91 ported by the fact that several flaviviruses are known to have been dis- persed to the New World from the Old World but there is currently no evidence of the opposite direction of dispersal. For how long has the genus Flavivirus, as we recognize it, been evolving? In a discussion of virus evolution rates (14), it was stated that “The fast mutation of some viruses with RNA genomes has led some virologists to conclude that most (RNA genome) viruses we know today probably arose since the last Ice Age!” Clearly these authors thought that this concept was nonsense. Is it nonsense, or had the authors misinterpreted the original conclusion? The viruses that “we know today” are still circulating in the environment. Most of the viruses from which they arose, probably do not circulate today. The genus Flavivirus comprises viruses that are recognizable today and it is the lineages that these viruses represent that we can attempt to date. Many other related flaviviruses will have emerged but subse- quently become extinct. Therefore, the concept that viruses at the deepest nodes of the tree, emerged since the last Ice Age is not at all unrealistic. In fact, because of the high rate of variation in positive stranded RNA viruses, which do not integrate with cellular DNA or co- evolve with the host, if viruses such as yellow fever or Tyuleniy had emerged before the last Ice Age, they would almost certainly be genet- ically unrecognizable today. ‘Using either logical argument based on historical facts or by caleu lating nucleotide substitution rates and then estimating dates of diver- «gence from known times of virus isolation, one can attempt to estimate the divergence tines for the recognizable viruses in the genus Fe vivirus. Taking the approach based on facts and logic first. The sug- gestion that Ll virus has probably been present in the UK for no more than three or four centuries seemed ridiculous less than 10 years ago, but the evidence for its recent introduction to the UK is now com- pelling. The term Jouping ill is an ancient word and was used in the 18th century to describe a disease of sheep, occurring in the Border counties of England and Scotland where sheep were farmed inten- sively on the hillsides (37). Sheep were then introduced north of the Forth-Clyde valley onto the Seottish moorlands and the disease fol- lowed quite rapidly. Phylogenetic trees show clearly that Scottish LI viruses have mare recent evolutionary lineages than Irish and Welsh LI viruses (38). In each of these geographic regions, grouse are highly susceptible to infection by LI virus even though the grouse have clearly been associated with moorland environments for many een turies, as shown by the fact that they have adapted a specialized diges- tive tract for the digestion of heather (31). The very high virulence of LI virus for grouse implies its revent intruduetiou to the mourlands (8),92 B.A. GOULD ETAL. Further support comes from the evidence that sheep could not have been successfully farmed either on moorlands or even on any uplands during most of the 16th century hecanse Britain and Burope experi- enced a “Little Ice Age.” If LI or an ancestral TBE complex virus was present in the British Isles before this time, its epidemiological char- acteristics would have been very different from those seen from the 18th century onward. Moreover, throughout Europe and Asia, Ixodes ricinus is essentially a forest and woodland tick and only prospers on moorlands if there are sufficient suitable mammalian hosts, such as sheep or deer. Despite significant investigation, there is no evidence that either LI virus or an antigenically closely related TBE complex ‘virus is, or ever was, present in the British forests or woodlands. These facts therefore strongly support the phylogenetic explanation for the appearance of LI virus in the British mainland (.e., an early sheep encephalomyelitic virus was introduced from Europe, probably from Spain, through Ireland during the past few centuries). Since the west- ern European TBE complex viruses are clearly very closely related ancestors of LI virus and the sheep encephalomyelitic viruses in Spain, Turkey, and Greece, it follows that the TBE complex viruses have alco omorged relatively recently (i.c., contusion), rathor than many thousands of years ago. This line of argument can be followed throughout the entire tick-borne encephalitic group of viruses. They lhaye ewerged coutinuvusly aud as far ae vant be ances tained fevus Hie phylogenetic topology, at about the same rate. Thus, the viruses in the entire clade must have emerged in the past few thousand years rather ‘than over a period of hundreds of thousands of years during which northern Europe and Asia were frozen over. If these estimates for dates of evolution are significantly in error, then it could be argued that the TBE complex viruses emerged nearer the equator when infected animals, birds, and accompanying ticks migrated southward to avoid the cold conditions imposed by the Ice Age in north- em Europe and Asia. After this cold period of more than one hundred thousand years, one would then have to propose that the viruses returned ta northern Europe and Asia many thousands of years later with the returning animals, birds, and ticks. This possibility seems extremely unlikely in view of the very clearly defined cline that has been doseribed for the TBE complex viruses in the northern hemisphere. It is also unlikely because hundreds of thousands of years ago, there were no sheep grazing on the uplands of the Old World equatorial regions. ‘What about the mosquito-borne viruses? All phylogenetic trees show that the mosquito- and the tick-borne viruses evolved from an ancestral virus (i.e., at the same time in the past). Because the dynamics of the life cycles differ (see later), he mosquilo-burne viruses are believed Wo beFLAVIVIRUSES 93 evolving at least twice as rapidly as the tick-borne viruses (57). Indeed, because of their higher rate of evolution, the mosquito-borne viruses at the most recent nodes of the phylogenetic trees. are likely to have even ‘more recent lineages than the tick-borne viruses and this is now sup- ported in studies of dengue phylogenies (21). Finally, the gonetic distance between the most widely divergent fla- viviruses, is near to 70% in the most conserved genes. This approaches the maximum degree of genetic distance between viruses that will pro- duce reliable alignments and robust phylogenies. The available mos- quito-borne, tick-borne, and nonvectored viruses produce a balanced topology within these limitations, implying approximately similar times for evolution of these three major virus categories. On the basis of these arguments it is therefore not unreasonable to conclude that. the lineages represented by the viruses in the genus Flavivirus are likely to represent viruses that have evolved during the past 5000 to 10,000 years (i.e., since the last major Ice Age across the northern hemisphere). Estimates were made for several tick- and mosquito- borne viruses (38,56) and they are in general agreement with the con- clusions drawn from the arguments presented above. Although it is reengnired that the precision with which ench estimates ean he made is likely to be low for viruses in the deeper nodes of the tree, it is unlikely that they are incorrect by several orders of magnitude. V. FLavivinus DisPeRsAL STRATEGIES ‘There are probably far more flaviviruses than are currently recog- nized and this must be borne in mind when interpreting the data. Nev- ertheless, the dispersal strategies employed by the major groups of viruses within the genus Flavivirus are remarkably characteristic for each group and reflect very closely the ecological associations of the viruses. It was argued above that the flaviviruses have a common ori- gin and possibly originated in the Old World. Since many of these viruses have sylvatic eycles in Africa. this will be their assumed point of origin to illustrate their most likely dispersal strategies. However, it is recognized that the evidence at this time is weak and some schools of opinion might quite justifiably place the origins of these viruses in Asia or perhaps less likely, in Australia! A. Tick-Vectored Viruses ‘The seabird-tick associated viruses (TYU, SRE, and MEA) diverged as a munophyletie sister group Ww the TBE complex viruses. From Une94 E.A. GOULD ETAL. estimated divergence times, they represent the most ancient lineage to date of the tick-borne flaviviruses. The genetic distance between each of these individual species is greater (approximately 60% amino acid identity) than that between most of the TBE complex viruses (approx- imately 70% amino acid identity), presumably reflecting greater bio- geographic separation due to the migratory nature of tho seabirds. ‘These viruses can be isolated from ticks that are found in the nests of the migratory seabirds. Since one of the earliest nodes of the tick- borne clades includes an African virus (KAD), one could predict that during the past 5000 years, infected ticks have been dispersed by seabirds, from the presumed African (or Asian) origin, to northwest France (MEA), to the eastern coast of Russia (TYU), and to the Great Barrier Reef (SRE). It is unlikely that each of these radial dispersions took place in a single migratory flight. Indeed, it is much more likely that the viruses were, and still are being, carried along the migratory pathways in stages. If this is the case then there should be more TYU serogroup viruses to discover on the appropriate migratory routes. ‘At the deeper nodes of the tree, the dispersion charaeteristies of the ‘TBE complex viruses resemble those of the TYU serogroup viruses (i.e., the carliost virusos are fund at great geographic distances fram each other), It is quite possible that these early viruses were dispersed by mechanisms similar to those for the TYU serogroup viruses. For exam- ple, Macquaric Island, the source of GGY virus, ia oituated several hun dred miles off southern Australia, and is visited by very few humans. This virus can be found under the rocks and debris that are used by the penguins and other seabirds. Because of their antigenic relatedness wilh ‘TBE complex viruses in the northern hemisphere, it was suggested that GGY virus could have been introduced to the island by Storm Petrels (Oceanites oceanicus) or the Arctic 'lorn (Sterna Paradisaea), which have reciprocal breeding patterns between the Arctic and Antaretie (48). Once the TBE complex viruses reached the forests and woodlands of Asia (POW, KSI, RF, KFD, LGT, etc.) the opportunity arose for continuous or progressive dispersal along the defined corridors provided by the forests. All phylogenetic trees show an asymmetric topology for the more recent ‘TBE complex flaviviruses, indicating that their evolution has been con- tinuous, rather than interrupted, In other words, the fareast Asian TBE complex viruses are the ancestors of the central Furopean virases, which in tum are the ancestors of the western European and ultimately the most westerly, virus (louping ill in the British Isles). ‘One of the most important factors in this clinal evolution is the tick. Ixodes spp. have a complete life cycle that may last from 2 to 7 years. During this time, the tick will feed 3 times, for about 5 days each time,FLAVIVIRUSES: 95 and will therefore spend less than 1% of its life on a vertebrate host. If the larvae become infected, they may remain infected for life and will feed twice more. as nymphs and as adults, providing 2 opportunities to transmit the virus to a vertebrate host. Rodents and deer are common vertebrate hosts for ticks that transmit TBE complex viruses. These animals show no clinical signs of disease and develop only 2 low-level viremia, and it was therefore originally believed that they may not provide a reservoir for transmission of virus to other ticks. However, ‘we now know that virus transmission between ticks, as they eofe nonviremie animals, is an efficient method of ensuring the virus life cycle is perpetuated even if the vertebrate hosts are immune to TBE virus (25). For the virus to persist within a system dominated by non- viremic transmission it is necessary for the larval ticks to be feeding at the same time and in the same feeding groups as the infected nymphs, a process known as coincidental feeding (42). Current work suggests that the degree of coincidental feeding could be determined by local cli- matic factors that influence the relative timing of tick emergence and as such predict the focal distribution of TBE. The tick is relatively immobile, spending nearly all its time under stones and rotting vegetation in the forest. ndergrowth. Tt emerges and moves almost exclusively in the vertical plain, as it ascends the vegeta- tion to quest for a bloodmeal by attaching to a foraging animal. These animale aleo have very localized territories and therefore the virua ia spread only slowly across the forest floor. Therefore the tick, the verte- brate host, and the climate each play central roles in the gradual east to west dispersion and eontinuuus evolution of the TBE complex viruses across the forests of Asia, and central and western Europe. The predicted pattern for the epidemiology, evolution, and dispersal of the sheep encephalomyelitis viruses represents an example of humans’ impact on wildlife species in the natural environment. The following hypothesis offers a rational explanation for the first appear- ance of sheep encephalomyelitis viruses. Many of the regions near the southern borders of forests in Asia and Europe provided ideal upland grazing for sheep and goats. These animals provided an amplification host for Ixodes spp. which were dispersed between the nearby forests and the grazing areas, by the sheep/goats and wildlife species (rodents and deer, ete). This localized distribntian of the ticks on the grazing Jand brought the ticks into contact with TBE complex viruses in the forests. As the western European TBE complex virus from the forest rodents was introduced to the grazing sheep and goate, a genetic vari ant was selected that was highly neurotropic for the introduced sheep/goat species. This selection appears to have occurred on the don96 E.A.GOULD ETAL. upland grazing slopes of Greece and Turkey. Presumably the sheep/goat encephalomyelitis virus was subsequently introduced onto the sheep-erazing regions of northern Spain by the exchange of ani- mals and then to the moorlands of Ireland and Britain by the intro- duction of infected sheep. The phylogenetic trees imply that the evolutionary process took place from east to west over a poriod of a few hundred years and this is supported by the arguments proposed ear- lier for the dates of emergence of LI virus in the UK. B, Mosquito-Vectored Viruses In contrast, mosquito-transmitted viruses have entirely different dispersal characteristics and this is largely due to the different behav- ioral patterns of mosquitoes and their vertebrate hosts, compared with ticks and their hosts. Whereas the tick bloodmeal usually takes sev- eral days to complete, mosquitoes feed on vertebrate hosts within a few seconds. If the mosquito becomes infected, the virus then repli- cates and within 8 to 10 days reaches a high level of infectivity in the brain, body, and salivary glands of the mosquito. Within a few weeks, the mosquito may feed again, transferring the virus to a vertebrate host, which if susceptible will reproduce the virus to high titers in the target organs, develop a viremia, and transmit this virus to uninfected ‘mooquitocs that feed during the viremie otage. The mosquito borne viruses are therefore replicated through many cycles in a relatively short period of time, Mosquitoes are more mobile than ticks and may disperse (he virus over significant distances within a few hours. More- over, the hosts that are infected by the mosquitoes may also be more mobile than the corresponding forest animals that serve as hosts for ticks. Overall, the life eyele and the factors that determine dispersion are much more dynamic in mosquito-transmitted viruses than tick- borne viruses. This is reflected by (1) the wider geographic dispersal of many individual mosquito-transmitted virus species, (2) the fact that mosquito-borne viruses show overlapping distributions, and (3) the ‘mosauito-borne viruses show ereater levels of genetic variation and higher estimated evolution rates particularly those that cause human epidemics, and the structure of the phylogenetic trees suggests periods of rapid population growth. Despite these generalizations, many of the Old World Aedes spp. associated viruses have remained essentially syl- vatic and show restricted geographic dispersal presumably because of their adaptation to local vector-vertebrate ecology. ‘Yellow fever virus has a geographically limited distribution. Epidemic outbreaks occur in central and west Africa, the Caribbean, and centralFLAVIVIRUSES 97 and South America. Cases of YF anywhere else are introduced by indi- viduals infected in a YF region, and then travelling to other parts of the world, Yellow fever introduced in this way, does not lead to subsequent infections. In the natural environment, the virus can be isolated from Aedes spp. that bite and infect monkeys living in the tree canopy of the ‘tropical rainforests and in the savannah that borders the equatorial forests. In Africa, many species of monkey show no clinical symptoms although they replicate and serve as reservoir hosts for the virus that is then transmitted to noninfected mosquitoes feeding on the infected monkeys. A similar virus life eycle, between mosquito and monkey, also occurs in the New World rain forests but the monkeys frequently become sick and die as the result of becoming infected, which is taken to indicate that YF virus was introduced into the Americas relatively recently. The considered wisdom is that the virus was transported from Africa on the slave boats that traveled frequently to the Americas dur- ing the past three or four hundred years. The fact that cases of YF were also seen in many seaports in Europe and even in northern ports of the United States, where slave boats were frequent visitors, supports this argument. Dispersal of YF out of Africa is therefore very restricted and is almost entirely attributable to human commercial activities, partic larly the slave trade, during the past few centuries. Human YF infections occur throughout the year in the humid equa- torial foreste. Humans are bitten by infected aylvatie Aedes app. vee tors that usually feed on monkeys. The sylvatic cycle of virus, being transmitted between monkeys and mosquitoes, is referred to as jungle fever when it involves humans. Yellow fever epidemies in Africa occur toward the end of the rainy season when Aedes aegypti densities are at their highest. Humans infected in the savannah or rain forests take the disease to urban dwelling Aedes aegypti. These mosquitoes then spread the disease through the human population. This form of YF (human-mosquito-human), is referred to as urban fever (49). In tropi- cal America, the urban form of YF has not been reported for many years although in some of the heavily populated parts of tropical ‘America it seems to be only a matter of time. The dispersal characteristics of dengue virus serotypes contrast significantly with YF virus. Whereas YF virus appears to have retained its aylvatic nature, the dengue viruses are better suited to the urban environment and epidemic dengue no longer seems to require a aylvatic reservoir host. All 4 dengue serotype viruses circu- late and cause human cpidemics throughout most tropical regions of the world wherever high densities of Ae aegypti are present. On the basis of their phylogenies, the dengue viruses represent more recent98 EA. GOULD ET AL. evolutionary lineages than YF virus. It is therefore tempting to pos- tulate that an ancestral dengue lineage arose in Africa, where Aedes spp.competent for dengne virns transmission were present, and the 4 recognized serotypes subsequently emerged and diverged as they dis- persed across Asia and then into the New World, exploiting modern transportation to aid their diepersal. In common with YF, other viruses in the Aedes spp. clades (for example, SEP, EH, UGS) show more restricted geographic dispersal presumably because, like YF vicus, they are preferentially adapted to the prevailing vectors, ver~ tebrates, and local ecology. Not surprisingly, the dispersal characteristics of the Culex spp. asso- ciated viruses contrast strongly with those of the Aedes spp. viruses mainly due to the fact that most Culex spp. viruses have life cycles that involve birds, Strains of many of these viruses (for example, SLE, JE, WN) can be isolated over wide geographic areas, because they are carried by migrating birds. Nevertheless, it is possible to predict defined dispersal patterns for them. Using the arguments presented above, we can presume an Old World origin, probably Africa or Asia, for these viruses. One can speculate that the ancestral virus(es) was dispersed by a combination of birds and spreading human popnlations with their associated farming practices, across Africa and Asia during the past two or three thousand years. As the dispersion occurred, vari- ants continued to evolve and become established in various regions of the Old World. In some cases, for example, WN or JE virus, dispersion was very efficient and these viruses can now be isolated over very large geographic regions. In other cases, for example, KOK and STR, the viruses were more suitably adapted to local species in northeast Australia and neighboring regions of southeast Asia. How did the Culex spp. associated viruses disperse to the New World? It seems most likely that during the past few hundred years, either a single virus or several related viruses were transported from the Old to the New World, presumably aboard commercial ships. ‘These introduced viruses then became established in the Americas and continued their evolution independently of the Old World Culex spp. associated viruses. The phylogenetic trees imply that SLE and CPC represent early New World descendants of the Old World ances- tral viruses. However, as pointed ont earlier, even thongh there are Culex spp. viruses in both the Old and the New World, WN virus is the only one that has become established simultaneously, on both sides of the Atlantic Occan and this occurred only very recently. This supports the belief that birds do not routinely disperse Culex spp. associated flaviviruses across the major oceans.FLAVIVIRUSES 99 It is thought that WN virus was inadvertently introduced into the New York area of the United States after being carried by or in (1) an infected human. (2) an infected mosquito, (3) the luggage of an air- plane passenger, (4) an infected bird or animal, imported illegally into North America, (5) an infected bird blown off course from the Old World. Although any of these alternatives is thearetically possible, the fact that WN virus appears to have been successfully introduced to the ‘New World on only one oceasion argues against its introduction by an infected bird flying in from the Old World. C. Nonuectored Viruses The NKV viruses fall into two distinet groups, those that diverged with the Aedes spp. associated viruses, which are found exclusively in the Old World. At the deeper nodes of the tree, these viruses separated from the mosquito-borne viruses to form a distinct NKV clade of bat- associated viruses. Despite the potential for the bats to disperse the viruses over wide geographic areas, each virus has apparently remained localized in the region where it was first identified, implying adaptation ta the local ecalogy. The ather NKV viruses diverged inde- pendently of the arthropod-transmitted viruses (Fig. 3). They then diverged again, into rodent-associated viruses, which are exclusively New World, and bat-associated viruses, which are found either in the ‘New World or the Old World, Since the divergence between the rodent and bat NKV viruses seems to have occurred early in the evolutionary history of the genus Plavivirus (at least a few Unvusand years ayo), we are faced with the problem of deciding how they subsequently became dispersed. Two possibilities immediately spring to mind. Firstly, an carly ancestral flavivirus with a natural hfe cycle involving bats and/or rodents emerged in the Old World and was introduced to the New World by bats with long distance flight patterns, before becoming, established in rodents and bats in the New World. A second possibility is that most of the evolution within the NKV clades has occurred in the Old World and the descendant viruses were only introduced into the New World during the past few centuries as the commerical ships plied their trade across the oceans. Although this idea may not sit com- fortably with some doubters, itis not inconsistent with the quite con- vincing evidence that many of the mosquito-borne viruses have been introduced into the New World by similar mechanisms. Other possibil- itios will almost certainly be propoved but until more viruses have been identified and their sequences determined, speculation is the only definite option!100 B.A. GOULD ETAL. VI. Conctustons Taken together, the data presented above suggest that an early NKYV-type flavivirus was capable of infecting a wide range of verte- brate and invertebrate species. This virus probably originated in the Old World, Early descendants infected rodents and bats both of which were capable of either transmit- ting the virus directly from vertebrate host to vertebrate host or indi- rectly via an arthropod vector. The evolutionary pathways that then developed and which have been defined above were determined by the specific interactions between virus, vector, vertebrate host, and associ- ated ecology. Despite the clear evidence that many of the flaviviruses are transmitted between vertebrate hosts by arthropods, it is known that they can also be transmitted orally (15) and transplacentally (36). The methods by which the NKV viruses are transmitted have never been adequately defined but in addition to those proposed above, other possi- ble routes include, urine, feces, aerosols, and blood. 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