key: cord-0035569-y3d9ghel authors: Hewson, Roger title: Molecular Epidemiology, Genomics, and Phylogeny of Crimean-Congo Hemorrhagic Fever Virus date: 2007 journal: Crimean-Congo Hemorrhagic Fever DOI: 10.1007/978-1-4020-6106-6_5 sha: c325cc9dc2fa2ced1845ea31004be3cf95e39cca doc_id: 35569 cord_uid: y3d9ghel Crimean-Congo hemorrhagic fever virus (CCHFV) constitutes a group of viruses of the genus Nairovirus (family Bunyaviridae). Like all members of the Bunyaviridae, the genome of CCHFV is composed of tripartite single-stranded RNA. These segments, designated small (S), medium (M), and large (L), minimally encode the nucleocapsid (N), envelope glycoproteins (Gn and Gc), and RNA-dependent RNA polymerase (RdRp), respectively [38]. Asia 2, Europe 1, etc., which has been employed as a simple description of genotype ( Fig. 5-1) . Furthermore, these studies also show that similar genotypes are found in distant geographical locations ( Fig. 5-2) , supporting the idea that virus or infected ticks may be carried over long distances during bird migration [10] . Anthropogenic factors, such as the trade in livestock, may have also played a role in the dispersal of CCHFV. Thus, molecular epidemiological observations support a global and dynamic reservoir of CCHF virus. Sequence information on L segments has lagged behind those of both S and M segments primarily due to the technical difficulties in working with these very long molecules. Nevertheless, several data from strains is available and while the number of alternative strains is on a different scale to those of S segments, there is evidence that the S and L segments from the same strains have similar evolutionary history (Fig. 5-3) . For M segments however, the situation is different and it enables an insight into the ways CCHFV have evolved. The driving force for evolution is provided by genetic change and variation in genomes. These lead to phenotypes which are molded by selective forces, thus genomes gradually change with their changing environments. RNA viruses, with their large population sizes, swift, and mutation-prone replication rates are generally considered capable of rapid evolution [16] . Additional evolutionary processes of (i) recombination, and for viruses with segmented genomes (ii) reassortment, also offer potentially important routes of generating genetic diversity. The genomes of arthropod-borne RNA viruses however, need to function and maintain high fitness in both arthropod and vertebrate host cells. This maintenance on two fronts is frequently thought to constrain the evolutionary processes acting on arbovirus genomes [44] . Thus, low levels of genetic diversity are frequently observed for arboviruses. The genome of CCHFV is interesting since, as well as showing features of high genetic stability [13] , it also shows features of high flexibility [8] . CCHFV is often described as an emerging virus [22, 47] . Studies of its genetic fine structure aimed at developing a better understanding of the ways it can change and evolve are helping to illuminate its nature as an emerging pathogen. Complete genome entries of several CCHFV are now available in GenBank, and analysis of these sequences are enabling evolutionary hypothesis to be inferred and tested. Genetic homologous recombination -the formation of chimeric RNA molecules from sequences previously separated on different molecules -is an important means of variation open to RNA genomes. Indeed, it is clear that homologous recombination has been an important process that has shaped the evolution of RNA viruses per se [46] . However, the contribution of its effects 46 and the rate at which it occurs vary for different virus families. For example, it is known to be frequent in retroviruses [19] , less common but periodic for positive-strand RNA viruses [24] , but relatively infrequent in negative-strand RNA viruses [4, 32] . Yet, cases of recombination in the latter group do occur and evidence of it in the Bunyaviridae [39] and Arenaviridae [1] is well documented. Such reports have encouraged the search for recombination in CCHF viruses. Noteworthy evidence, including the demonstration of phylogenetic incongruence, often regarded as the best support for recombination [34] , has been illustrated for the CCHF S segment [26] . Similar evidence for recombination in either of the M or L segments was not detected. A very recent study [8] also supported this latter observation in the majority of M and L segments. In addition, however, an analysis employing similarity plots, bootscanning and the informative sites tests, highlighted the possibility of recombination events within L segments of the Asian groups [8] . Interestingly, the cases of recombination are phylogenetically ancient and there is evidence that the sequences in question have diverged considerably after recombination. This suggests that tion of isolation. From these data, it appears likely that the L segments also conform to the same grouping pattern as observed for S segments, although there are fewer L segment sequences. Additional sequence data provided by very recent work has enabled more comprehensive analysis and shows some exceptions to this idea. Nevertheless, while the more recent tree topologies of L and S segments are not analogous, they remain very similar. recombination in CCHFV is a rare event and while it is difficult to estimate precise recombination rates, it is apparent that such rates are lower than those of point mutation. Nevertheless, an important consideration borne out of such work is that inferences about recombination events should only be entertained when molecular analysis have been constructed from complete segment sequence data. Additional consideration should also be given to the quality of published sequence data. A noteworthy example is provided by strains; (i) STV/HU29223 from European Russia (Stavropol) and (ii) Uzbek/TI10145 from Uzbekistan, which present some the of best evidence of genetic recombination in CCHFV as observed by phylogenetic incongruence [12] . However, this conclusion should be treated with caution as there is also evidence that the observed recombination may be an artifact [29] . RNA viruses with segmented genomes have the capacity to reassort their genomic segments into new genetically distinct viruses if the target cells are subject to dual infection. Indeed, this ability is believed to play a key role in the evolution, pathogenesis, and epidemiology of important pathogens such as influenza viruses, rotaviruses, and arthropod-borne orbiviruses [20, 25, 30] . Within the Bunyaviridae family as a whole, reassortment has been demonstrated for members of the genera Orthobunyavirus [2, 33, 42] , Phlebovirus [40] , Hantavirus [15, 23, 37] , and Tospovirus [35] , accordingly it is not surprising that segment reassortment in the Nairovirus genus has also been demonstrated [8, 14] . Here, evidence of reassortment in CCHFV is illustrated by a phylogenetic analysis of each strain or segment (Fig. 5-4) . The phylogenetic groupings of S and L segments are consistent and show a correlation with the geography of parent strain isolation; however, the phylogenetic groupings of M segments are different. Distinct groups that were formed in S and L segments by Asia 1 and Asia 2 genotypes, for example, are not matched in the M segment phylogeny (Fig. 5-3) . Although full-length sequence data is limited it is possible to ascertain that reassortment has taken place in the biogenesis of certain strains of CCHFV. For currently available data, the best evidence of reassortment is provided by the Matin strain isolated from Pakistan. If we consider groups for which there is full-length sequence data available on each segment (so that recombination events can be ruled out), then there appear to be strains with five types of S and L segment (Europe 1, . It is likely that other strains have also arisen by segment reassortment. Indeed, very recent work has provided more complete sequence data from a broader range of strains [8] exposing many more examples of segment reassortment. It is clear that the majority of these events involve M segment reassortment, however, L segment reassortment viruses are also observed, albeit at a lower frequency. The reassortment events involving strains from widely separated geographical locations, illustrates that coreplication enabled by the movement and mixing of viruses is quite common. It follows that there may be a global reservoir of CCHFV, with local subreservoirs supporting high levels of virus circulation and permitting frequent coinfection (in which migratory birds play a significant role in virus dispersion). There is evidence that both recombination and reassortment are able to play roles in the evolution of CCHFV, in addition to general genetic drift. Obviously such genetic exchange requires coreplication of two or more strains within the same cell. The most likely coinfection environment where segment reassortment occurs is within ticks, where lasting virus infections persists for extended periods and superinfection with a second strain, during the strict requirement for blood meals, is very likely [28] . Given the currently available data on the low rate of recombination in CCHFV, and particularly the fact that the rate of recombination seems lower than general genetic drift, it appears that reassortment plays the most contributory role to the variability and flexibility of the CCHFV genome. Indeed, the low rate of recombination in negative-strand RNA viruses generally has led to suggestions that genome segmentation and reassortment have evolved to increase their fitness for survival [7, 31] . Specifically, while the high mutation rates of RNA viruses provide the raw material for evolutionary processes [21] , mutations also introduce fitness compromising deleterious effects [6] . Genetic exchange through recombination or reassortment are recognized as adaptive methods of purging such effects [5, 27] , thus in the practical absence of recombination, reassortment is able to take up the reins. In addition, reassortment enables alternative virus genotypes to be selected from a pool of functional segments. The current evidence of reassortment in CCHFV [3, 8, 14] points principally to the exchange of M segments between viruses in mixed infections. In addition, the majority of data on L and S sequences show that in many cases these segments have evolved together as partners. Thus, in mixed virus infections where reassortment is a possibility, partner L and S segments have a propensity to end up in the same virus particle (due to the ostensibly strong interrelationships between the nuclear protein and RdRp) in order to constitute a viable new virus [3] . Some exceptions to this idea have been exposed by the availability of more sequences [8] , and while it is clear that L and S segments trees are not analogous, they remain highly similar. M segments on the other hand seem to be more autonomous and could result in new virus phenotypes. Thus, as CCHFV are dispersed and introduced into new areas in which they are already endemic, the emergence of new CCHFV would principally be the result of M segment reassortment. Glycoprotein spikes encoded by M segments are well known for their ability to influence host range and cellular tropism [11, 41] , furthermore, they are often associated with altered pathogenicity. These mechanisms, together with the likely contact and infection of new hosts, provide a foundation for the appearance of new CCHF disease and the emergence of new viruses [17] . These genomic studies highlight the importance of molecular surveillance to monitor and track the natural fluxes of virus and CCHF disease. A number of key questions can be asked in this context: For example, are certain viral genotypes more associated with severe disease? If so, are certain combinations of segments (or mutations) involved in the production of virulent strains? If there 52 Hewson is a strain basis to disease, is viral genetic diversity increasing so that new strains with novel biological properties (such increased virulence or transmission potential) might appear? A practical conclusion of the evolutionary opportunities open to this virus is that CCHF diagnostic approaches and potential vaccine research strategies should be tested against isolates from all parts of the world, regardless of the intended location of use. 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