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Review Genetic diversity and evolution of hepatitis C virus 15 years on Peter Simmonds Correspondence Peter Simmonds PeterSimmondsedacuk Centre for Infectious Diseases University of Edinburgh Summerhall Edinburgh EH9 1QH UK In the 15 years since the discovery of hepatitis C virus HCV much has been learned about its role as a major causative agent of human liver disease and its ability to persist in the face of hostcell defences and the immune system This review describes what is known about the diversity of HCV the current classification of HCV genotypes within the family Flaviviridae and how this genetic diversity contributes to its pathogenesis On one hand diversification of HCV has been constrained by its intimate adaptation to its host Despite the 30 nucleotide sequence divergence between genotypes HCV variants nevertheless remain remarkably similar in their transmission dynamics persistence and disease development Nowhere is this more evident than in the evolutionary conservation of numerous evasion methods to counteract the cells innate antiviral defence pathways this series of highly complex virushost interactions may represent key components in establishing its ecological niche in the human liver On the other hand the mutability and large population size of HCV enables it to respond very rapidly to new selection pressures manifested by immunedriven changes in T and Bcell epitopes that are encountered on transmission between individuals with different antigenrecognition repertoires If human immunodeficiency virus type 1 is a precedent future therapies that target virus protease or polymerase enzymes may also select very rapidly for antiviralresistant mutants These contrasting aspects of conservatism and adaptability provide a fascinating paradigm in which to explore the complex selection pressures that underlie the evolution of HCV and other persistent viruses Introduction Since its discovery 15 years ago Choo et al 1989 Kuo et al 1989 hepatitis C virus HCV has been the subject of intense research and clinical investigations as its major role in human disease has emerged Globally HCV is estimated to infect 170 million people 3 of the worlds popula tion and creates a huge disease burden from chronic progressive liver disease HCV has become a major cause of liver cancer and one of the commonest indications for liver transplantation reviewed by Hoofnagle 2002 Seeff 2002 Pawlotsky 2003b HCV infection can be treated but this is costly and requires longterm medical support and followup current therapies are impractical for the majority of HCV carriers worldwide The development of a protective vaccine remains at best a distant prospect HCV is an enveloped virus with an RNA genome of approximately 9400 bp in length Most of the genome forms a single ORF that encodes three structural core E1 E2 and seven nonstructural p7 NS2NS5B proteins Fig 1 Short untranslated regions UTRs at each end of the genome are required for replication of the genome a process that has recently been found to additionally require a cisacting replication element in the coding sequence of NS5B You et al 2004 Translation of viral proteins is dependent on an internal ribosomal entry site in the 59 UTR which comprises a complex RNA structure element that interacts directly with the 40S ribosomal subunit during translation initiation Pestova et al 1998 HCV is classified in the family Flaviviridae although it is differs in many details of its genome organization from the original vectorborne members of the family HCV is additionally distinct and somewhat unusual for an RNA virus in being able to establish persistent infections in the majority of exposed individuals This phenomenon has attracted the greatest interest in HCV research not least because longterm chronic infections underlie its disease manifestations and effective therapy must break this ongoing cycle of replication in the liver Understanding the mechanism of persistence is also of fundamental immunological interest and as discussed represents an important new paradigm in which to explore the genetic basis for this highly adapted interaction with its host The evolution of HCV is shaped by distinct selection pres sures that are associated with on one hand the historical events underlying the adaptation of HCV to its human host Published online ahead of print on 19 August 2004 as DOI 101099 vir0804010 00080401 G 2004 SGM Printed in Great Britain 3173 Journal of General Virology 2004 85 31733188 DOI 101099vir0804010 that have ensured its successful ongoing transmission On the other hand HCV is capable of very rapid adaptive changes that are associated with de novo infection of each individual in response to immunological selection pres sures to antiviral therapy HCV also accumulates sequence changes as a result of neutral sequence drift over time and this process rather than the adaptive changes accounts for much of the sequence diversity that is observed between its different genotypes This review will describe the different mechanisms of evolutionary change their relationship with selection see the next section and the importance of neutral and adaptive evolution in the diversification of HCV and its persistence and treatment resistance Virus sequence change The evolution of viruses resembles that of all organisms it is a process that is ultimately dependent on mutations in their genetic material In many ways however viruses differ from commonly studied organisms such as the geneticists mouse or fruit fly particularly in their speed of sequence change large population size and the nature of the selection pressures that they encounter In popular use the word evolution describes the process of adaptive change whereby organisms change in their phenotype such as body shape or behaviour in response to external sometimes changing selection pressures and by competition with other organisms for limited resources in a shared environment Random mutations from copying errors or chromosomal damage occasionally and entirely by chance might improve organism fitness allowing the mutated gene to spread and eventually to predominate in the population where the advantage it confers in terms of reproductive success is significant In this model the evolution of distinct species of animals plants and bac teria results from large numbers of incremental changes in phenotype that are associated with adaptation to the wide range of separate contemporary and previous environments Surprisingly this Darwinian type of evolution makes very little contribution to the genetic diversity of organisms when measured at the level of DNA or RNA sequences Although Fig 1 Diagrammatic representation of the HCV genome top panel showing positions of structural and nonstructural proteins The lower panel plots sequence diversity between different HCV genotypes in windows of 150 bp across the genome This analysis reveals highly conserved regions in the 59 UTR and core regions and high viral diversity in envelope genes and NS5A 3174 Journal of General Virology 85 P Simmonds highly controversial when proposed Kimura 1968 King Jukes 1969 neutral theory demonstrates that the majority of sequence change in and between species has no significant effect on phenotype ie it is neutral Kimura 1983 Nucleotide changes in coding and non coding sequences that have little or no effect on organism fitness become fixed in the population by chance Thus geographically isolated members of species can become very different genetically whilst often remaining unchanged morphologically and behaviourally The frequency of fixa tion of neutral changes can be predicted to be relatively constant over time and underlies the remarkably close correlation between sequence divergence of certain genes such as haemoglobin with the established chronology of splitting of different mammalian species and orders over the past 150 million years Evidence for both Darwinian and neutral evolution can be found in the sequences of HCV One possible example of adaptive change in HCV is the rapid evolution of the hypervariable region of the E2 envelope glycoproteins to prevent recognition by antibodies that are induced by infection see the section entitled Sequence variability within genotypes In contrast neutral sequence drift undoubtedly accounts for much of the genetic diversity that is observed between geographically or epidemiolo gically separated populations of HCV This process of divergence resulting from the fixation of neutral sequence changes does not alter the phenotype of the viruses greatly Despite the 30 sequence difference that is observed between genotypes of HCV see the section entitled HCV genotypes each retains a similar replication cycle in human hosts Indeed their shared abilities to establish persistent infections in humans with high infectivity titres in blood and to cause only slowly progressive and largely asymptomatic infection are key factors in their ongoing transmission This lack of phenotypic innovation over an extremely long period of divergent evolution demonstrates perhaps how the evolution of HCV is shaped and con strained entirely by its close adaptation to the particular ecological niche it inhabits the human liver HCV genetic diversity and genotypes Genetic variability of HCV exists at several different levels Most obvious is the substantial genetic divergence of the main genotypes of HCV which frequently show specific geographical ranges in the human population and asso ciations with particular risk groups for infection Below this and arising from sequence drift over a much shorter period is the variability that is observed between individual variants or strains Much of the sequence diversity that is observed between such strains such as the 58 divergence observed between variants in epidemiologically unlinked infections by HCV genotypes 1a 1b and 3a reflects processes of neutral sequence drift over time after the introduction of HCV into new risk groups in the 20th century Some of the sequence divergence may represent phenotypically selected changes that are associated with adaptation for replication in individuals with different immune responses to infection see the section entitled Sequence variability within genotypes Finally HCV diversifies measurably within an infected individual over time forming what has been described as a quasispecies This preexisting genetic variability combined with an extremely large replicating population size of HCV in a chronically infected individual provides a large pool of genetic variants that can adapt to new selection pressures such as immunological recognition and antiviral treatment HCV genotypes Comparison of nucleotide sequences of variants recovered from infected individuals in different risk groups for infection and from different geographical regions has revealed the existence of at least six major genetic groups On average over the complete genome these differ in 3035 of nucleotide sites with more variability concentrated in regions such as the E1 and E2 glycoproteins whereas sequences of the core gene and some of the nonstructural protein genes such as NS3 are more conserved Fig 1 The lowest sequence variabi lity between genotypes is found in the 59 UTR where specific sequences and RNA secondary structures are required for replication and translation functions Despite the sequence diversity of HCV all genotypes share an identical complement of collinear genes of similar or identical size However contrasting with this general observation is the marked variation in their capability to express a further protein that is generated by a transla tional frameshift at codon 11 of the core gene Walewski et al 2001 Xu et al 2001 Varaklioti et al 2002 both the frameshift site and potential size of this novel coding sequence are very poorly conserved between and within genotypes This contrast with the evolutionarily conserved nature of so many other aspects of HCV replication supports the idea that this gene is more likely to be a computational artefact that has arisen from RNA structure imposed constraints on thirdcodon position variability in the core gene Tuplin et al 2004 Each of the six major genetic groups of HCV contains a series of more closely related subtypes that typically differ from each other by 2025 in nucleotide sequences compared with the 30 divergence between genotypes Fig 1 Simmonds et al 1993 Some such as genotypes 1a 1b and 3a have become distributed very widely as a result of transmission through blood transfusion and needle sharing between infecting drug users IDUs over the past 3070 years and now represent the vast majority of infec tions in Western countries Fig 2 These are the genotypes that are encountered most commonly in the clinical setting and for which most information has been collected on response to interferon IFN and other antiviral treatments see the section entitled Biological differences A different pattern of sequence diversity is observed in parts of Africa and SouthEast Asia Here there are close httpvirsgmjournalsorg 3175 HCV variability and evolution associations between genotypes and specific geographical regions Fig 3 For example infections in western Africa are caused predominantly by HCV genotype 2 Mellor et al 1995 Ruggieri et al 1996 Jeannel et al 1998 Wansbrough Jones et al 1998 Candotti et al 2003 whereas those in central Africa such as the Democratic Republic of Congo and Gabon are caused by genotypes 1 and 4 Bukh et al 1993 Stuyver et al 1993 Xu et al 1994 Fretz et al 1995 Mellor et al 1995 Menendez et al 1999 Ndjomou et al 2003 In both regions there is a remarkable diversity of subtypes for example 20 of 23 HCVseropositive blood donors in Ghana western Africa were infected by genotype 2 but each corresponded to a different and previously undescribed subtype Candotti et al 2003 This diversity is reproduced in Guinea Benin and Burkina Faso central western Africa where 18 different subtypes of genotypes 1 and 2 were found in samples from 41 HCVinfected individuals Jeannel et al 1998 These field observations reflect both the huge genetic diversity of genotypes 1 2 and 4 and also its probable longterm presence in human Fig 2 Evolutionary tree of the principal genotypes of HCV that are found in indus trialized countries and their main epidemiolo gical associations with specific risk groups These genotypes of HCV are believed to have become prevalent over the course of the 20th century The tree was constructed by using the neighbourjoining method as implemented in the MEGA package using JukesCantorcorrected distances between complete genome sequences Fig 3 Evolutionary tree of all known sub types and genotypes of HCV including those found in highdiversity areas of geno types 1 2 and 4 subSaharan Africa and 3 and 6 SouthEast Asia HCV variants still fall into six distinct clades but with far greater numbers of genetic variants corres ponding to subtypes in industrialized coun tries The tree was constructed by using the neighbourjoining method as implemented in the MEGA package using JukesCantor corrected distances between partial NS5B sequences 320 bp 3176 Journal of General Virology 85 P Simmonds populations in these parts of Africa Taking this geo graphical mapping further genotypes 3 and 6 show similar genetic diversity in southern and eastern Asia Tokita et al 1994a b 1995 Mellor et al 1995 The model that is suggested by these genotype distributions is that HCV has been endemic in subSaharan Africa and SouthEast Asia for a considerable time and that the occurrence of infection in Western and other nontropical countries represents a relatively recent emergence of infec tion in new risk groups for infection Simmonds 2001 Ndjomou et al 2003 In the 20th century parenteral exposure to bloodborne viruses became frequent through the widespread adoption of blood transfusion since the 1940s the medical use of often unsterilized needles for injections and vaccinations a practice that continues in many developing countries and most specifically to industrialized countries injecting drug use and the sharing of injection equipment These new routes for transmission plausibly account for the epidemiological and genetic evidence for recent epidemic spread of HCV over the past 50 years in Europe Egypt and elsewhere Pybus et al 2001 2003 Cochrane et al 2002 Ndjomou et al 2003 However one of the puzzles about the origins of HCV is the absence of obvious transmission routes in those areas where the greatest genetic diversity is observed Trans mission by either sexual contact or from mother to child is inefficient Wasley Alter 2000 Pradat Trepo 2000 Thomas 2000 and there is little historical evidence for the type of widespread parenteral exposure that fuelled the epidemic in Western countries However recent field investigations in southern Burkino Faso centralwestern Africa where genotypes 1 and 2 are prevalent and highly diverse in sequence have shown associations between HCV infection with previous sexually transmitted diseases STDs circumcision excision and scarification practices D Jeannel personal communication Although the asso ciation with STDs has not been documented in studies carried out in Western countries it is possible that lack of mucosal integrity during STD episodes may facilitate the entry of HCV into the genital tract Determining how long HCV has been in these endemically infected populations would clearly be of value in understanding its epidemio logy in these regions see the section entitled Genotype origins Even less is known about the earlier divergence of the six major genotypes of HCV the origins of infection in humans and the underlying basis of the current geographical distribution of genotypes Areas where HCV is endemic and highly diverse correspond closely to those where hepatitis B virus HBV is also prevalent reviewed by Simmonds 2001 and also represent regions where human and ape population ranges overlap However in contrast to HBV there is currently no evidence that HCV or HCVlike variants infect Old World ape or monkey species Makuwa et al 2003 Therefore despite tempting analogies with the introduction and spread of human immunodeficiency virus type 1 HIV1 and HIV2 infec tions in humans through crossspecies transmission of simian viruses from chimpanzees and mangabeys Gao et al 1992 1999 it would be highly speculative and currently unjustified to imagine that HCV originated in these human populations as a result of similar crossspecies transmission On the other hand it has been discovered that a very distantly related HCVlike virus GB virus B Simons et al 1995 infects tamarins andor other New World primates The existence of this homologue in such a distantly related primate species certainly allows for the possibility that HCV or HCVlike viruses may indeed be distributed more widely in primates than was thought previously Largerscale serological and PCRbased surveys of a much larger range of primates in Africa Asia and South America are required to resolve this issue Sequence variability within genotypes Several studies have described the rapid sequence drift of HCV over time a process of diversification that leads ultimately to the existence of identifiably separate strains or isolates within human populations By comparing HCV sequences from sequential samples from chronically infected indivi duals or from those infected by a common source rates of sequence change were measured to be 14461023 nucleotide changes per site per year over the whole genome or 41 and 7161024 changes per site per year in the NS5 and E1 regions respectively Okamoto et al 1992 Smith et al 1997 In coding regions of the genome changes occur predominantly at synonymous sites sites that do not alter the encoded amino acid and are therefore likely to represent the accumulation of phenotypically neutral changes The expectation from neutral theory that such diversification should occur at a constant rate over time is implicit in attempts to use this rate to estimate times of spread of HCV in specific trans mission networks such as IDUs Pybus et al 2001 2003 Cochrane et al 2002 and indeed its extrapolation to calculating times of introduction of specific genotypes such as 1a 1b 3a and 4a into new risk groups for infec tion in Western countries Smith et al 1997 For exam ple the current sequence diversity and phylogenetic tree structure of genotype 4a in Egypt is compatible with the introduction of HCV into that population through parenteral treatment for schistosomiasis Bilharzia tragi cally with nondisposable and poorly sterilized needles in the 1950s and 1960s Frank et al 2000 Ray et al 2000 Pybus et al 2003 The increasing sequence diver sity within genotypes 3a 1a 1b 2a and 2b respectively suggests times of introduction of these viruses at increas ingly earlier times in the 20th century this may have been associated with other parenteral risk factors for infection such as injecting drug use blood transfusion largescale immunizations and syphilis treatment Mortimer 1995 Whilst regions of the genome such as NS5B have been used frequently for epidemiological reconstruction other parts of the genome such as the hypervariable regions httpvirsgmjournalsorg 3177 HCV variability and evolution HVRs of E2 and NS5A show much greater variability and much more rapid amino acid sequence change over time This variability may arise through specific selection Darwinian mechanisms operating on the virus that are associated with immune escape for example the HVR in E2 may be a target for neutralizing antibody and persistence may therefore require continuous virus sequence change to evade Bcell responses Weiner et al 1992 Kumar et al 1993 Taniguchi et al 1993 Farci et al 2000 Kantzanou et al 2003 For HIV1 it is known that initial infection is accompanied by a number of amino acid changes in class I binding motifs in potential Tcell epitopes such as the ArgRLys or Gly change in two different immunodominant epitopes in p24gag that are recognized by the B27 allele Kelleher et al 2001 and by B58 and B5801 Leslie et al 2004 In the latter case it has been demonstrated that the immune selection mediated by B8 and B5801 occurred only with likely significant fitness cost to the virus it invariably reverted to the original wildtype sequence on transmission to other individuals with different human leukocyte antigen HLA types By analogy it is therefore possible that many of the amino acid polymorphisms that are observed in HCV are also driven sequentially by selection from different major histo compatibility complex class I or II alleles or by antibody recognition that is encountered during a viruss passage through human populations Indeed direct evidence for the occurrence of immuneselected changes in cytotoxic Tlymphocyte CTL epitopes has been obtained on experimental infection of chimpanzees Erickson et al 2001 Also supporting this hypothesis is the observation that sequence change was slower in individuals with defects in T or Bcell immunity Booth et al 1998 interpreted as indicating reduced immune selection on CTL or Bcell epitopes Indeed recovery from infection is associated with strong and sustained CTL responses around the time of primary infection Cooper et al 1999 Lechner et al 2000 Thimme et al 2001 a time where there is evidence for specific changes in CTL epitopes and accelerated sequence change in the coding sequence in those who become chronic carriers Chang et al 1997 Cantaloube et al 2003 Sheridan et al 2004 Immune selection may also underlie the high degree of sequence variability between and within genotypes in the NS5A region Fig 1 and lead to differences in the ability of different variants and genotypes to evade intracellular defences see the section entitled Interaction with the host cell Quasispecies The processes of neutral and adaptive evolution of HCV operate during the course of chronic infection within an individual leading to both continued fixation of nucleotide changes over time and the develop ment of variable degrees of sequence diversity within the replicating population at a given time point Sequence diversity is generated continually during virus replication as RNA copying by the virally encoded RNA polymerase NS5B is errorprone and the replicating population is so large For example ongoing error rates of between 1 in 10 000 and 1 in 100 000 bp copied which are typically found for RNA polymerases reviewed by Domingo et al 1996 Drake et al 1998 combined with a rate of virus production of up to 1012 virions per day Neumann et al 1998 would produce a highly genetically diverse popula tion of variants containing mutants that differed at every nucleotide position and every combination of paired differences from the population mean or consensus Even though the consensus sequence may be close to the fitness peak at any one time the existence of a large and diverse population would allow rapid adaptive Darwinian changes in response to changes in the replication environ ment This might take the form of evolving immune responses that select against viruses with specific T or Bcell epitopes it might also confer resistance to anti viral agents The rapid and reproducible independent appearance of specific amino acid changes that are asso ciated with the acquisition of HIV1 resistance to reverse transcriptase and protease inhibitors is a dramatic demon stration of Darwinian evolution of the quasispecies In the future this may be reproduced in HCV infections that are treated with the new generation of protease inhibitors such as BILN 2061 and RNA polymerase inhibitors Lamarre et al 2003 Pause et al 2003 Trozzi et al 2003 Lu et al 2004 Sarisky 2004 Recombination Recombination occurs in many families of RNA viruses its occurrence requiring both epidemiolo gical opportunity and biological compatibility In positive stranded RNA viruses recombination generally occurs through a process of templateswitching during RNA genomic replication To detect such occurrences a single cell must be infected with two or more genetically identi fiable variants of the virus In vivo this requires both coinfection of the same individual with more than one such variant and substantial overlap in their geographical distributions in order to enable recombinant forms to be detected The genotype epidemiology and natural history of infec tion with HCV clearly fulfils both of these criteria A wide range of genotypes circulates in the main risk groups for HCV in Western countries including 1a and 3a in IDUs and 1b 2a2c and 4a throughout the Mediterranean area In these areas infection is often characterized by multiple exposures around the time of primary infection such as frequently repeated needlesharing with several infected individuals over short timeintervals in the case of IDUs and the contamination of blood products such as factor VIII clotting factor concentrates with multiple HCV positive plasma units Indeed even ongoing chronic HCV infection does not protect from reinfection in experimentally challenged chimpanzees Farci et al 1992 or in HCVcontaminated blood or bloodproduct reci pients such as thalassaemics and haemophiliacs Kao et al 1993 Jarvis et al 1994 Lai et al 1994 3178 Journal of General Virology 85 P Simmonds The viability and pathogenicity of inter and intragenotype recombinants are more difficult to assess and are likely to vary considerably between virus families Amongst the monopartite positivestranded RNA viruses recombina tion is bestdocumented in the family Picornaviridae Recombination between different enteroviruses in species B is known to generate a huge number of naturally occur ring recombinant viruses with novel combinations of capsid serotypedetermining and nonstructuralproteins Santti et al 1999 which can show subtle differences in patho genicity in mouse models Harvala et al 2002 Whilst the coding regions of these evidently compatible enterovirus B sequences differ by approximately 25 nucleotide or 9 amino acid recombination between enterovirus species which differ by 4042 in nucleotide sequence and by 4045 in amino acid sequence are not observed possibly because these sequence differ too much to be biologically compatible There is little experimental information on the potential viability of inter or intragenotype recombinants of HCV although it has recently been observed that most com binations of genotype 1a and 1b sequences in the non structural region of the genome fail to generate a viable replicon Gates et al 2004 implying the existence of incompatibilities between variants that show approxi mately 20 sequence divergence Despite these in vitro observations recombinant forms of HCV have been observed in nature including a variant formed from structural genes of genotype 2 and nonstructural genes from genotype 1b that was found in infected IDUs in St Petersburg Russia Kalinina et al 2002 2004 and a possible 1a1b recombinant in Peru Colina et al 2004 Despite the number of studies that have been carried out to investigate this issue the true frequency of recombination of HCV may have been considerably underestimated For example recombination would not be detected easily between variants of the same subtype such as between two infecting genotype 1a strains in an IDU Similarly it would be difficult to document intersubtype recombinants where HCV is highly diverse such as within genotype 2 in western Africa because in such regions we lack a full catalogue of sequence variants within which to observe recombination events The existence of widespread recombination would place a considerable limitation on the use of genotyping assays that are based on single genome regions such as the 59 UTR or core gene Should more recombinant viruses emerge in the coming years as a result of increasing geographical overlap in genotype distributions this would cause major problems in the interpretation of genotyping assay results For recombinant viruses only those assays that genotyped samples in regions of the genome that determined IFN susceptibility would be able to predict treatment response which is one of the main applications of genotyping assays see the section entitled Interaction with the host cell Classification The Seventh Report of the International Committee on Taxonomy of Viruses ICTV currently classifies HCV and GB virus B as members of the genus Hepacivirus within the family Flaviviridae Heinz et al 2000 The six main genotypes of HCV have been desig nated clades reflecting their phylogenetic group Fig 3 As proposed previously based on this type of phylo genetic analysis Mellor et al 1995 Mizokami et al 1996 de Lamballerie et al 1997 new genotypes such as 7a 8a 9a and 11a have been assigned as members of clade 6 and genotype 10a has been assigned to clade 3 The ICTV report acknowledges the existence of the extremely large number of subtypes within the main HCV genetic groups and the difficulty in establishing criteria for their classification and nomenclature Fig 3 Indeed in the future it might be more appropriate to regard the subtype tier in previously published HCV classification proposals as being of significance only in Western countries and other regions where HCV has spread recently Here discrete subtypes are identifiable as they are descendants of founder viruses that have been seeded into these new transmission networks as exemplified by those analysed in Fig 2 The existence of identifiable subtypes within the main clades of HCV is therefore an epidemiological phenom enon that is associated with recent spread Thus there seems to be little justification for the continued efforts to catalogue and name individual subtypes up to the letter r in the case of genotype 4 httphcvlanlgovcontent hcvdbcombinedsearchsearch in highdiversity areas such as central Africa In my opinion future assignment of subtypes is only worth pursuing where it is epidemiologi cally relevant and should therefore be restricted to those that are distributed widely and show specific geographical risk group or other epidemiological associations Another remaining classification issue is the procedure for identifying and naming inter or intragenotype recom binant viruses For HIV1 designation of intersubtype recombinant viruses or circulating recombinant forms CRFs requires the detection and complete genome sequences of the recombinant in three or more individuals with no epidemiological connection and the demonstra tion of recombination breakpoints in identical positions in each sequence For nomenclature each CRF is numbered sequentially in order of discovery followed by subtype identification letters to indicate its approximate com position This procedure might be adapted easily for HCV in which case the recombinant virus circulating in St Petersburg Russia Kalinina et al 2002 2004 would officially be designated CRF012k1b or RF12k1b as proposed by the authors Genotype origins It is difficult to estimate the length of time that HCV has been present in human populations As described above httpvirsgmjournalsorg 3179 HCV variability and evolution the diversity of variants within genotypes 1 2 and 4 in subSaharan Africa and of genotypes 3 and 6 in South East Asia suggests that HCV may have been endemic in these populations for considerably longer than in Western countries As the evolutionary process of sequence diver gence that led to the diversity of subtypes in these regions is likely to have been predominantly neutral in mechan ism it may therefore be possible to calculate the times of splitting of subtypes and possibly also the times of divergence of the six main clades of HCV through use of published rates of sequence change over time Okamoto et al 1992 Smith et al 1997 Extrapolation of these rates to time the 20 and 30 sequence divergence that is observed between subtypes and genotypes respectively produces relatively recent times of origin that in many ways are difficult to reconcile with the epidemiology of HCV and its global distribution For example the diversity of variants observed in west African genotype 2 sequences predicts a time of origin for this endemic pattern of infection of approximately 200250 years ago whilst different genotypes would have diverged from each other about 100 years earlier Even by using complex methods for correction for multiple substitutions and allowing rate variation between sites the current diversity of genotypes predicts an origin no earlier than 1000 years ago Smith et al 1997 This seems to be too recent for such a widely distributed virus that infects often relatively isolated human populations in equatorial Africa and SouthEast Asia We have argued that there may be far greater constraints on sequence change in HCV genomes than are found in eukaryotic and prokaryotic gene sequences on which the neutral theory was first developed and where a molecular clock has been shown to operate over long periods of evolution We have discovered that the genome of HCV is highly ordered forming complex RNA secondary struc tures throughout the coding sequence of the genome Simmonds Smith 1999 Tuplin et al 2002 Simmonds et al 2004 This characteristic termed genomescale ordered RNA structure GORS is a conserved feature of several genera andor families of positivestranded RNA viruses that infect animals and plants Although the func tion of GORS is unknown its correlation with host persistence raises the intriguing possibility of its role in the modulation of recognition or inhibition of innate celldefence recognition or effector mechanisms that depend on the detection of doublestranded dsRNA Simmonds et al 2004 The requirement for basepairing in such structured viruses severely limits the number of neutral sites in the genome as most sequence changes even at synonymous sites would disrupt RNA folding Given the complexity and large scale of these HCV RNA secondary structures truly neutral sites where sequence changes have no significant effect on virus phenotype may be rare indeed These limitations on sequence change can be illustrated dramatically through simulation of constrained neutral drift on HCV sequences and measurement of its effect on retention of RNA structure Fig 4 Simmonds et al 2004 The coding sequence of a genotype 1b variant was mutated by using an algorithm that introduced random changes into the sequence but preserved specific character istics of naturally occurring virus diversity within HCV Despite the close simulation of expected neutral evolu tionary drift mutated sequences showed marked and pro gressive reductions in RNA structure which was apparent even in sequences that differed by only 2 from the original sequence Fig 4 As GORS is conserved in all genotypes of HCV this loss of RNA structure clearly does Fig 4 Effect of simulated neutral sequence drift on the for mation of RNA secondary structure in the HCV genome modi fied from Simmonds et al 2004 A genotype 1b sequence was mutated through the introduction of nucleotide substitutions that reproduced naturally occurring variability in HCV such as transitiontransversion ratio synonymousnonsynonymous sub stitution ratio and base composition to create variants with 235 divergence from the original sequence x axis The formation of RNA structure in the mutant sequences was com pared in extent to that of the native sequence by using MFOLD y axis expressed as a percentage Mutants differing by as little as 2 from the original sequence showed evidence for disruption of RNA structure formation boxed symbols intro duced sequence drift of 10 produced mutants that were no more capable of RNA structure formation than sequence orderrandomized controls In contrast the diversification of naturally occurring variants of HCV differing by up to 33 from the 1b sequence round symbols preserved RNA struc ture with equivalent levels found in other 1b variants other genotype 1 subtypes and other genotypes Thus RNA structure in HCV is highly evolutionarily conserved and its evident requirement for internal basepairing must constrain even its very shortterm evolution 3180 Journal of General Virology 85 P Simmonds not occur during the natural evolution of HCV Pathways followed during virus diversification over time that retain GORS must therefore be extremely constrained and lead to substantial homoplasy and sequence convergence in the limited number of sites where substitutions can occur without damaging RNA structure The measurable loss of GORS in sequences that have been drifted artificially by 2 indicates that even the very recent evolution of HCV such as within the Irish antiD cohort of women that was used for measuring the rate of HCV sequence change see the section entitled Genotype origins is subject to the same severe constraints Applying a molecular clock to extrapolate times of origin of more divergent HCV variants such as subtypes and genotypes is clearly pointless as the number of neutral sites or the limitations on sequence change at variable sites is not known so there is no denominator with which to calculate and correct for multiple substitutions The con striction of sequence space of viruses such as HCV with GORS implies that many of the branches that are evident on phylogenetic analysis of contemporary sequences that define virus species genotypes or genera occurred at remote times in the past In making the molecular clock based estimates above of 3501000 years for the time of divergence of genotypes we are therefore in danger of telescoping a much longer period of virus evolution into an unrealistically short timeframe A much longer time perspective on HCV evolution pro vided by our understanding of GORS constraints fits much better with the globally distributed nature of HCV and the concentration of specific genotypes with histori cally relatively isolated populations in subSaharan Africa and south Asia As a potential comparison GORS in the widely distributed human virus hepatitis G virusGB virus C appears to have restricted sequence drift to 1113 nucleotide sequence divergence over the course of evolution of modern humans over the last 100 000150 000 years GonzalezPerez et al 1997 Pavesi 2001 Simmonds 2001 The greater sequence diversity between HCV geno types implies times of origin that occurred even further back in the evolution of humans Biological differences The major features of HCV structure replication transmis sion and ability to establish persistent infection are shared between all known variants Indeed viewed purely as a survival machine the widespread distribution of geno types 16 in human populations indicates that that each is equally successful in maintaining infections in human populations Despite this obvious evidence for phenotypic similarity there is growing evidence for genotypespecific differences in persistence and interactions with innate cell defences and the immune system that have important repercussions for current and probable future therapy Treatment response Beginning with observational data the clearest difference between genotypes is in their susceptibility to treatment with IFN monotherapy or IFNribavirin RBV combination therapy Typically only 1020 and 4050 of individuals infected chronically with genotype 1 HCV on monotherapy and combination therapy respectively exhibit complete and permanent clearance of virus infection This longterm response rate is much lower than the rates of 50 and 7080 that are observed on treatment of HCV genotype 2 or 3 infections reviewed by Pawlotsky 2003a Zeuzem 2004 This dif ference has proved to be highly significant in patient management and has led to the use of higher doses and longer durations of treatment for type 1 and type 4 infections in order to achieve acceptable efficacy In numerous multivariate analyses genotypespecific differ ences in treatment response have been shown to be inde pendent of host variables such as stage of disease progression age duration of infection sex and HIV and other virus coinfections It is similarly independent of virusspecific factors such as pretreatment viral load although this also correlates independently inversely with response Despite this wealth of observational data we still lack basic understanding of the mechanism of these differ ences mainly because the in vivo mechanism of action of exogenous IFN or RBV remains largely unknown Insights into mechanisms of treatment resistance might be obtained through investigation of the inhibitory effect of IFN or IFNRBV on the in vitro replication of subgeno mic or fulllength genomic replicons of HCV Lohmann et al 1999 Ikeda et al 2002 Pietschmann et al 2002 Blight et al 2003 Replication of the replicon can be inhibited by the addition of exogenous IFN Blight et al 2000 Frese et al 2001 Lanford et al 2003 at least in part through inhibition of translation Wang et al 2003 This model has however provided only very limited information on treatment resistance mainly as a result of poorly understood current limitations of the model system The range of HCV variants that can be cultured is extremely restricted limited to genotypes 1a and 1b which are both equivalently IFNresistant clinically although a fulllength replicon of the more clinically sensitive geno type 2a has recently been described Kato et al 2003 Secondly their in vitro replication requires or is enhanced by adaptive amino acid changes in NS5A and NS3 Bartenschlager et al 2003 even though these play no role in natural infections and actually attenuate replication in experimentally infected chimpanzees Bukh et al 2002 Mutations in NS5A are particularly problematic as they cluster in a region of the protein that is associated clinically with resistance to IFN therapy and that interacts with the dsRNAdependent protein kinase PKR and other host cell defences as part of an evasion strategy It is therefore unclear whether IFN treatment responses can be modelled realistically in this artificial in vitro system In the future the replicon model will be of great value in httpvirsgmjournalsorg 3181 HCV variability and evolution the development and assessment of antiviral activity of newly developed protease and RNA polymerase inhibitors for HCV therapy reviewed by De Francesco et al 2003 and for investigating the development of antiviral resis tance Lu et al 2004 The model is at present again limited by the lack of availability of replicons from other genotypes particularly as there are concerns that antiviral agents modelled specifically on the active sites of genotype 1b protease or RNA polymerase may not be as active against corresponding sites of other subtypes or genotypes HollandStaley et al 2002 Very recently it was indeed found that nongenotype 1infected individuals were nonresponsive or only weakly responsive to shortterm treatment with the BILN 2061 protease inhibitor Reiser 2004 in contrast to its efficacy in genotype 1infected individuals Lamarre et al 2003 This is consistent with biochemical evidence for a nearly 100fold reduction in binding affinity of BILN 2061 to genotype 2 and 3 proteases Thibeault et al 2004 Genotypespecific differences in response to the new generation of antiviral agents will be a major research priority in the future Natural history In contrast to the clearcut differences between genotypes in their response to antiviral therapy it has been much more difficult to obtain data on the differences in natural history and pathogenicity between HCV genotypes Part of the problem with these investiga tions lies in the nature of the patient cohorts that have been studied to date and the exceptionally long time over which complications of HCV infection present clinically With a few exceptions severity of disease has typically been assessed in crosssectional cohorts recruited from patients who were referred because of overt liver disease such as portal hypertension cirrhosis or abnormalities in liverfunction tests eg elevated alanine aminotransferase levels This biased recruitment towards the minority with clinically apparent disease creates study cohorts that lack the community denominator and information on dura tions of infection with which to estimate the timecourse of disease development More importantly for this dis cussion crosssectional recruitment of hepatitis patients cannot resolve whether some genotypes are more likely to present clinically than others Longitudinal studies where the course of HCV disease over time in individuals with known times of infection is monitored prospectively are few in number and frequently limited to patients who are infected with a single genotype For example natural history studies of the Irish and East German antiD cohorts considered individuals who were infected only with genotype 1b Power et al 1994 Takaki et al 2000 Similarly a prospective study in the USA of individuals who were exposed to HCV by blood transfusion in the 1970s was limited to predominantly genotype 1a or 1b infections Seeff et al 2001 However more genotype diversity is found in several European cohorts in which an early diagnosis of infection was possible through specific risk factors such as haemophilia or in communitybased casecontrol studies In these studies genotype 1 appeared invariably to be more likely to establish persistence and in carriers to be associated with more severe liver disease compared with genotypes 2 and 3 Yee et al 2000 Franchini et al 2001 Mazzeo et al 2003 Resti et al 2003 Surprisingly and in contrast to the probable greater long term pathogenicity of genotype 1 infections with geno type 3 are associated with a higher incidence of steatosis RubbiaBrandt et al 2000 Adinolfi et al 2001 which is thought to result from direct cytopathic damage to hepatocytes from a block in lipoprotein secretion Serfaty et al 2001 As with the many other manifestations of biological differences between genotypes including the vexed question of whether genotype 1 is more likely to cause hepatocellular carcinoma Di Bisceglie 1997 the availability of an in vitro system for investigating differ ences in the replication of different genotypes would be of considerable value in dissecting out the differences in virushost cell interactions that underlie these clinical observations Interaction with the host cell HCV is unusual for an RNA virus in being able to establish persistent infections in humans This has attracted considerable efforts to understand the immunological basis for this phenom enon As described in the section entitled Sequence varia bility within genotypes there is substantial evidence that HCV is subjected to a variety of immunemediated pres sures that shape its evolution driving for example sequence variability in the envelope region to evade anti bodymediated neutralization and potentially of Tcell epitopes to evade cytotoxic Tcell responses However it is now increasingly recognized that the most significant and evolutionarily ancient component of host defences against virus infections lies within the cell Intracellular defences are generally triggered through the recognition of dsRNA replication intermediates through interaction with members of a large family of structurally related dsRNA binding proteins DRBPs Girardin et al 2002 Saunders Barber 2003 DRBPs are coupled to a range of anti viral effector pathways that in vertebrates include PKR mediated induction of apoptosis and modulation of the IFNresponse pathways Clemens Elia 1997 and acti vation of oligoadenylate synthetase resulting in RNase L production and consequent cytoplasmic RNA cleavage Player Torrence 1998 Mammalian cells may addi tionally control RNA virus infections through the actions of the mammalian homologues of Dicer and the siRNA armed RNAinduced silencing complex Waterhouse et al 2001 Gitlin Andino 2003 In common with other RNA and DNA viruses HCV has developed a range of celldefence evasion mechanisms that are centred around the activities of NS5A Tan Katze 2001 Reyes 2002 Macdonald Harris 2004 Whilst NS5A is a necessary part of the virus replication complex it shows additional activities in binding to and inactivating 3182 Journal of General Virology 85 P Simmonds PKR Gale et al 1997 blocking apoptotic pathways through sequestration of p53 modulation of intracellular calcium levels and binding to growth factor receptorbound protein 2 Tan et al 1999 Gong et al 2001 Majumder et al 2001 and induction of antiinflammatory interleukin 8 secretion Polyak et al 2001 Fig 5 It has also recently been shown that the HCV NS34A protease blocks the phosphorylation and signalling function of the antiviral IFN regulatory factor 3 Foy et al 2003 The E2 protein when expressed as a nonglycosylated cytosolic protein Pavio et al 2002 also appears to bind to and inhibit PKR as a result of sequence similarity to the autophos phorylation domains of PKR and to e1F2a Taylor et al 1999 Interestingly the degree of similarity to this homo logy domain was greatest for genotype 1 variants and it was proposed that this contributed to the greater resistance of this genotype to IFN therapy Finally the association of GORS with virus persistence Simmonds et al 2004 suggests that the formation of extensive RNA secondary structure in the genomic strand of HCV plays a role in the evasion of cell defences potentially by facilitating escape from innate responses that are induced by certain structured RNAs Each of these complex cell interactions potentially contributes to host persistence and to the inhibition of secondary Tcell responses to the virus during chronic infection One possible explanation for the differences in the out come of infection between variants and genotypes of HCV is that they interact differently with host cells and achieve varying degrees of effectiveness in counteracting cell defences Most obviously the greater similarity of the E2 protein of genotype 1 to the phosphorylation domains of PKR and e1F2a has been suggested to explain its greater clinical resistance to treatment see above However fur ther studies have generally not confirmed this hypothesis with little correlation between the E2 sequence and res ponse between genotypes or subtypes 1b 2a 2b 2c 3a and 4cd 4a Saito et al 2003 Watanabe et al 2003b Quer et al 2004 More promising evidence for a relationship between virus sequence and persistencetreatment resistance was demon strated in the region of NS5A that interacts with PKR Long before its function was known it was observed that there was a clustering of amino acid changes in NS5A during IFN treatment An association was also found between treatment response and possession of the socalled proto type NS5A sequence in the region where mutations occurred Enomoto et al 1995 Prototype IFNsensitivity determining region ISDR sequences were also associated with higher circulating virus loads in untreated patients Watanabe et al 2003a As the ISDR colocalizes with the part of NS5A that interacts with PKR Fig 5 it was suggested that PKR evasion was a key determinant in the persistence of HCV and potentially other aspects of virus host interaction Since the original study several groups have sought to reproduce the findings of a dependence on ISDR sequence of treatment response in other patient cohorts Despite highly variable results between studies a recent meta analysis of all the available data has demonstrated a clear correlation between the prototype ISDR sequence and treatment resistance and as a corollary a large number of diverse amino acid changes in nonresponders Witherell Beineke 2001 It has also been shown that the same differential response exists in HCV genotype 2a and 2b infections Murakami et al 1999 In trying to unravel the mechanism of this interaction it remains curious that whilst the prototype ISDR sequence of NS5A is found specifically in individuals who resist IFN therapy there is Fig 5 Diagram of the NS5A gene showing the ISDR and regions of the protein that are known to interact with PKR and other cellular pathways associated with innate cell defences against viruses Sequence changes in the ISDR correlate with sensitivity of the virus to IFN therapy potentially representing a tradeoff between functional activity and immune selection Sequences of other regions under apparent selection during therapy are shown in red httpvirsgmjournalsorg 3183 HCV variability and evolution no evident selection for this sequence in viruses with non prototype sequences that are treatmentsensitive One theory is that the sequence in NS5A is under strong immune selection and shows varying degrees of freedom to mutate towards the most biologically active pro totype sequence for each genotype NS5A is indeed known to contain a high concentration of T and Bcell epitopes Zhang et al 1994 RodrıguezLopez et al 1999 Lee et al 2000 Dou et al 2002 and it is possible that immune selection in many individuals drives the ISDR or neighbouring sequence away from the prototype in individuals with certain HLA types that target epitopes in this region A poorly functioning NS5A protein may make the infecting virus more sensitive to intracellular antiviral responses and thus to a greater likelihood of spontaneous viral clearance as well as increased susceptibility to IFN therapy in those who remain viraemic Similar immune mediated selection may underlie the observation of treatmentinduced amino acid changes in other functional regions of NS5A such as V3 and a second region at posi tions 22822302 marked with an asterisk in Fig 5 Nousbaum et al 2000 Sarrazin et al 2002 The balance in this tradeoff between NS5A function and immunological recognition may differ between genotypes of HCV For example the reason that infections by HCV genotypes 2 and 3 are generally much more responsive to IFN treatment may be because a far greater proportion of individuals recognize the prototype NS5A protein immunologically Subsequent evolution of the infecting virus with a functionally impaired NS5A protein makes it less able to resist the further assault of exogenously administered IFN used for therapy Human population specific differences in the frequencies of HLA types in different study groups may also go some way to explaining why the association of prototype ISDR and potentially sequences in other NS5A regions with treatment resistance varies so much between studies in Japan and Europe Witherell Beineke 2001 Summary In summary the evolution of HCV is a highly dynamic process It occurs both through multiple processes of adap tive selection that drive sequence change such as those resulting from the host immune response and potentially from antiviral treatment and through drift in which phenotypically neutral sequence changes accumulate over time without altering the phenotype or behaviour of the virus However despite its potential to 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