R E V I E W SUMMARY. Reviews in Medical Virology

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1 Reviews in Medical Virology R E V I E W Rev. Med. Virol. 2010; 20: Published online 14 July 2010 in Wiley Online Library (wileyonlinelibrary.com)..660 Recombination among picornaviruses A.N. Lukashev 1,2 * 1 M.P. Chumakov Institute of Poliomyelitis and Viral Encephalitides, Russian Academy of Medical Sciences, Moscow, Russia 2 Institute of Virology, University of Bonn Medical Center, Bonn, Germany SUMMARY Picornaviruses are small non-enveloped positive strand RNA viruses that can cause a wide range of clinical manifestations in humans and animals. Many of these viruses are highly diversified and globally prevalent. Natural recombination has been reported in most picornavirus genera and is a key genetic feature of these infectious agents. In several socially relevant picornavirus genera, such as enteroviruses, aphthoviruses, parechoviruses and cardioviruses, recombination, combined with dynamic global epidemiology, maintains virus species as a worldwide pool of genetic information. It can be suggested that on a short time scale recombination acts to promote virus diversity, and new recombinant forms of picornaviruses emerge frequently as snapshots of this global pool. On a longer time scale, recombination maintains stability of a gene pool of a species by shuffling sequences and thus limiting divergence and speciation. This review covers existing evidence of recombination in most genera of the family Picornaviridae and possible implications for diagnostics, epidemiology and classification. Copyright # 2010 John Wiley & Sons, Ltd. Received: 18 March 2010; Revised: 20 April 2010; Accepted: 11 May 2010 INTRODUCTION Picornaviruses are a diverse and ubiquitous family of non-enveloped positive strand RNA viruses. The growing family Picornaviridae currently comprises 12 genera that infect mammals and birds. The most clinically and economically significant picornavirus genera are Enterovirus, Aphthovirus, Cardiovirus, Parechovirus, Hepatovirus and Kobuvirus. Among picornaviruses are such important pathogens as poliovirus, enteroviruses, HAV, foot and mouth disease virus of cattle and many others. The picornavirus genome encodes a single polyprotein that is cleaved into 3 4 structural proteins and 7 8 Corresponding author: A.N. Lukashev, Institute of Poliomyelitis and Viral Encephalitides, p/o Institut Poliomielita, Moscow Region , Russia. alexander_lukashev@hotmail.com Abbreviations used: CVA, coxsackievirus A; E, echovirus; EV, enterovirus; EMCV, encephalomyocarditis virus; FMDV, foot and mouth disease virus; HEV, human enterovirus; HPeV, human parechovirus; ICTV, International Committee on Virus Taxonomy; IRES, internal ribosome entry site; NPEV, non-polio enteroviruses; NSP, non-structural proteins; NTR, non-translated region; RF, recombinant form; RFLP, restriction fragment length polymorphism; SEV, simian enterovirus; SLCV, Saffoldlike cardioviruses; SVDV, swine vesicular disease virus; TMEV, Theiler s murine encephalomyelitis virus. non-structural proteins (NSP) (Figure 1). Conserved major NSP are 2C, 3A, 3B, 3C and 3D. The 2A, 2B and L proteins are highly diverse in structure and function among different picornavirus genera and do not share a common phylogenetic origin. The open reading frame is preceded by a 5 0 NTR that contains an internal ribosome entry site (IRES) responsible for capindependent initiation of translation. The 5 0 NTR and 3 0 NTR also encompass secondary structure elements responsible for genome replication. Picornaviruses exhibit remarkable plasticity of their genomes. The RNA-dependent RNA polymerase is very error-prone, resulting in roughly one substitution per copied genome [1,2]. Most of these mutations are supposed to be deleterious, and recombination is a process capable of recreating functional genomes from defective ones. Two distinct recombination mechanisms have been suggested. A classic copy-choice model suggests that recombination occurs when the viral polymerase switches template strands in the course of synthesis of the negative RNA strand during replication of two viruses in a co-infected cell [3]. Alternatively, it has been shown that recombination can happen in the absence of replication Copyright # 2010 John Wiley & Sons, Ltd.

2 328 A. N. Lukashev Figure 1. Schematic representation of genomes of the major picornavirus genera between RNA fragments that do not encode a complete polymerase [4]. It was suggested that in this case the recombination mechanism consisted of joining of the broken RNA molecules. Most likely, both mechanisms are valid; however, it is not possible to conclude which of them is predominant in nature. Natural recombination is extremely frequent in picornaviruses. Some time ago current knowledge on recombination among enteroviruses was summarised [5]; however, many new works have been published in the last few years, and several genera of picornaviruses have been recently acknowledged as important human pathogens. This review covers present data on recombination in different Picornaviridae genera and discusses possible implications for understanding the evolution of these viruses. RECOMBINATION IN POLIOVIRUSES Poliovirus, a member of the genus Enterovirus, was the most significant picornavirus for practical healthcare in the 20th century because of pandemic poliomyelitis. Recombination in poliovirus was discovered as early as Cells were infected with two strains of poliovirus that carried mutations providing resistance to either guanidinium or horse serum. Recovery of viruses with double resistance was times higher than could be expected to result only from mutation [6,7]. Recombination in polioviruses could be easily reproduced in vitro and thus became a helpful tool to study the virus genome before the advent of reverse genetics [8]. The most valuable results on poliovirus recombination in vivo were obtained in the course of the polio eradication campaign. Live polio vaccine is a mixture of three attenuated virus serotypes administered simultaneously, which creates ideal conditions for intertypic recombination. As the parental vaccine viruses were known, it was easy to identify recombinant viruses with inexpensive methods, such as PCR-RFLP, and to map recombination points with high precision. Recombinants appeared soon after vaccination and comprised up to 36% of the excreted viruses [9]. It is thus likely that recombination helps to restore fitness of the attenuated vaccine strains [10]. Recombination occurred most frequently in the NSP genome part and less frequently between the 5 0 NTR and the coding genome region [11]. Recombination within the capsid-encoding region seems to be rather an exception than a rule [12]. Circulating wild and vaccine-derived polioviruses readily recombine with other human enterovirus C serotypes [13], which justifies classification of poliovirus within the HEV-C species (see below). RECOMBINATION IN NON-POLIO ENTEROVIRUSES Over 100 serotypes of enteroviruses are currently known and a few more are discovered each year. Human enteroviruses are classified into four species, Human enterovirus A D. Other members of the genus are Human rhinovirus A C, bovine, swine and simian enteroviruses and a number of

3 Recombination among picornaviruses 329 unclassified enteroviruses. Human enteroviruses are ubiquitous and asymptomatic infection occurs frequently in infants [14]. Clinically prominent disease is relatively rare, although enteroviruses are a significant cause of neurological disorders, meningitis, myocarditis, sepsis-like disease of newborns, etc. [15]. It can be also expected that a wider use of modern screening methods would reveal a role of enteroviruses in a range of syndromes not currently associated with virus infection. Non-polio enteroviruses (NPEV) have received much less attention until the last decade when apparent eradication of poliomyelitis in Europe and the Americas liberated the resources of surveillance laboratories. The first systematic study on recombination among non-polio enteroviruses was performed in 1999 [16]. Conflicting phylogenies in genome regions encoding capsid and NSP were subsequently reported in a number of works [17 21]. Common to all studies, all circulating strains of human enteroviruses were recombinant relative to the prototype strains isolated in the 1950s. Phylogenetic grouping of enteroviruses corresponded to the serotype only in the genome region encoding the major capsid proteins VP1, VP2 and VP3. All other genome fragments were highly shuffled by recombination, with the most prominent hotspots mapping to the edges of the P1 genome region encoding proteins VP4 and 2A [22]. Reports of recombination within the P1 genome region were rare and involved viruses of the same serotype [23 25]. Recombination was most frequent within HEV-B species [21]. This conclusion was based on both direct estimation of recombination rate over time and analysis of recombination traces in the completely sequenced genomes. Prevalence of recombination within HEV-A species was significantly lower. Prototype strains of HEV-C species were shown to be recombinant relative to each other [13], and circulating HEV-C strains were shown to readily recombine with poliovirus; however, there has been no systematic study of recombination prevalence in circulating non-polio HEV-C. One of the most prominent non-polio enteroviruses is enterovirus 71 (EV71), a member of the HEV-A species, which is distinguished as a cause of massive epidemics of hand, foot and mouth disease in Asia and the Pacific. Apparent prevalence of recombination among enterovirus 71 strains is lower than in HEV-B, and many groups of strains are monophyletic in distant genome regions [26]. This might reflect true recombination frequency, but could also be affected by biased sampling. Indeed, there are over 100 contemporary EV71 sequences available, but only one complete sequence for each of the remaining HEV-A prototype strains and no complete sequences of the modern HEV-A isolates. In addition, in many studies EV71 strains are compared only to each other, but not to the rest of the species. We noticed that EV71 strains are recombinant relative to other HEV-A species members and carry at least three different variants of the 3CD genome region similar to the prototype strains of CVA5, CVA8 and CVA16 (unpublished observation). Highly mosaic structure of enterovirus genomes relative to each other brought an understanding that classical enterovirus surveillance methods based on the serotype neglect nearly two thirds of the genome. To address this problem, it was suggested to study circulation of recombinant forms (RF), which are combinations of VP1 and 3D without apparent signs of recombination, i.e. viruses that group together on phylogenetic trees for both genome regions. Definition of RFs relies on abundant sequence information, so it is practical only in larger studies. This approach demonstrated that new RFs of echovirus 30 emerge frequently, become dominant over Europe in a few years and then often vanish completely from circulation [27]. It is unclear what allows a RF to spread over large territories and displace other viruses from circulation; it is still more intriguing what makes a RF vanish simultaneously over the whole continent. Human rhinoviruses were only recently added to the genus Enterovirus based on their genetic properties [28]. There are three groups (putative species) of rhinoviruses, Human rhinovirus A C. Unlike enteroviruses, rhinoviruses are acid liable and usually cause a mild respiratory infection. Rhinoviruses are commonly typed by sequencing the VP4/VP2 genome region. There is evidence for frequent recombination between group C and group A rhinoviruses that involves the adjacent 5 0 NTR and VP2/VP4 genome regions [29,30]. Recombination between rhinoviruses of different groups was also observed in the NSP part of the genome [30]; therefore, delineation of rhinovirus species requires further clarification.

4 330 A. N. Lukashev ANIMAL ENTEROVIRUSES Animal enteroviruses are probably as diverse and ubiquitous as their human congeners, but they are much less studied. There is evidence of recombination events among bovine and simian enteroviruses [31,32]; however, lack of sequence data prevents estimating recombination frequencies. A key question for practical healthcare is how frequently enteroviruses change host species and if genetic information can be exchanged between human and animal viruses. There is at least one example of coxsackievirus B5 that crossed the species barrier and became prevalent as swine vesicular disease virus (SVDV) only 50 years ago [33]. Interestingly, the rate of recombination in SVDV has dropped markedly compared to its human progenitor after changing host species. Several simian enteroviruses phylogenetically belong to human enterovirus A species [34], but they are ancestral to human serotypes and there was no evidence of recombination between human and simian enteroviruses [31]. Therefore, the possibility of genetic exchange between human and animal enteroviruses remains to be investigated. Highly prevalent recombination was also reported in teschoviruses, which were formerly classified as porcine enteroviruses [35,36]. As in enteroviruses, breakpoints (locations in the genome where phylogenetic affiliation of a strain changed, indicating recombination) mainly mapped to P1 region boundaries, but they could also be found all over the NSP part of the genome. APHTHOVIRUSES The major member of the genus Aphthovirus is footand-mouth disease virus (FMDV), which causes severe infection in cloven-hoofed animals and is a major veterinary concern. There are seven serotypes of FMDV. Recombination in FMDV was discovered in 1965 using the same experimental approach as for poliovirus [37]. Currently there are over 150 full genome FMDV sequences available, which allows profound recombination analysis. Recombination breakpoints map predominantly to the borders of the P1 genome region, much like in other picornaviruses [35]. Recombination has also been reported within the P1 genome region [38], but it seems to be uncommon as in other picornavirus genera [35]. Interestingly, three FMDV serotypes that are endemic in Africa, SAT1-SAT3, show almost no evidence of recombination with the more common and globally spread serotypes [39]. As several recombination events could still be observed, this is probably an example of spatial separation that has not yet resulted in reproductive incompatibility. It is tempting to hypothesise that evolutionary relations of the endemic SAT and common epidemic FMDV serotypes might have parallels in genetics of common human pathogens, such as enteroviruses, which possibly exchange genetic information with animal viruses. PARECHOVIRUSES Parechoviruses (HPeV) were first isolated in 1950s and were initially classified as echovirus 22 and 23. Analysis of the genome sequence justified their reclassification as a new genus of picornaviruses [40]. Prevalence and diversity of parechoviruses was not truly anticipated until very recently. Parechovirus infection proved to be highly prevalent in humans, with the proportion of seropositives reaching 88% at the age of 2 years [41]. Currently, 14 types of parechoviruses are associated with a wide range of infant diseases [42] including meningitis and sepsis-like disease of newborns [43,44]. In general, recent reports show that epidemiological and clinical features of parechoviruses are similar to human enteroviruses. Recombination is also highly prevalent in parechoviruses [45 48], and recombination breakpoints map to the same genome regions as in enteroviruses. The hotspots are the boundaries of the P1 genome region, recombination in NSP is quite common, and there is no intertypic recombination in most of the P1 genome part. In enteroviruses a recombination hotspot maps to the genome region encoding the minor capsid protein VP4 [22]. In parechoviruses, unlike in most other picornavirus genera, VP0 is not cleaved into VP2 and VP4 proteins. Nevertheless, a recombination hotspot in parechoviruses is found in the 5 0 part of VP0 genome region that corresponds to VP4 in other picornaviruses. As in enteroviruses of the same species, all parechoviruses of different types can recombine. The only exclusion is HPeV type 3 viruses that rarely recombine with any other type [46]. It is intriguing to see if this observation results from a sampling bias or reflects some biological barrier to recombination between HPeV3 and other parechoviruses. In the latter case we might be

5 Recombination among picornaviruses 331 witnessing emergence of a novel parechovirus species. A distinctive feature of recombination in parechoviruses is a clear geographic pattern of the non-structural genome part. A study of partial genome sequences in VP1 and 3D genome regions revealed that the latter genome fragment in strains of different types isolated in Netherlands was distinct from British and Brazilian isolates [49]. A study of full-genome parechovirus sequences suggested that a unique variant of 3ABC genome region was endemic in Brazil for the last century and did not occur elsewhere in the world (Drexler et al., submitted). CARDIOVIRUSES For a long time two rodent cardioviruses, encephalomyocarditis virus (EMCV) and Theiler s murine encephalomyelitis virus (TMEV), were the only known members in this genus. There was no reliable evidence of cardiovirus infection in humans until 2007 when a virus distantly related to TMEV was identified in a faecal sample dating back to 1982 from an infant with fever of unknown origin [50]. The virus was named Saffold virus. In the following 2 years eight types of Saffold-like cardioviruses were found mainly in patients with gastroenteritis [51 54]. Serologic studies indicate that asymptomatic cardiovirus infection is highly prevalent in infants below 2 years old [51]. Evidence of widespread recombination in Saffold-like cardioviruses (SLCV) appeared almost immediately upon their discovery. Most reports indicated recombination occurring on the border between structural and non-structural genome regions and in the NSP genome region [47,53,55]. Recombination quite often took place throughout the NPS genome region between viruses of any type, and in the capsid-encoding part of the genome between viruses of the same type [55]. New strains of TMEV were recently isolated from rodents on different continents, and recombination was shown to be common among murine TMEV [56], with patterns very similar to those among Saffold-like cardioviruses. HEPATITIS A VIRUS HAV is the most common cause of water-borne hepatitis. The virus has several features unique among picornaviruses. First, the genus is represented by one sole serotype. Second, it was hypothesised that the virus prefers rare codons, presumably to limit the translation rate and avoid eliciting immune responses [57]. Recombination has been reported for HAV, but the pattern of recombination is strikingly different from that in other picornaviruses. Recombination points have been reported in the capsid-encoding genome region [58]. MEDICAL IMPLICATIONS OF RECOMBINATION IN PICORNAVIRUSES It is common to human picornaviruses that most infection cases are asymptomatic, and only a few result in a clinically prominent disease. Symptoms of infection with enteroviruses and parechoviruses can be diverse; one virus type can produce different manifestations and the same syndrome can be caused by different virus types. It is also typical of several human picornavirus genera, especially enteroviruses, HAV and Aichi virus, that symptomatic disease occurs as outbreaks, which could imply emergence of a more pathogenic virus variant prior to an outbreak. Molecular determinants of picornavirus pathogenicity are generally poorly understood, and almost all known ones map to the capsid-encoding genome region that is used for typing. Due to frequent recombination between P1 and other genome regions in enteroviruses, parechoviruses and cardioviruses classic typing approaches, serological or molecular, characterise roughly one-third of the genome, while the rest is neglected. Therefore, any genetic features encoded outside the P1 genome part have much lower chance to be discovered spontaneously. It is tempting to suggest that recombination plays a role in emergence of more pathogenic virus strains, but it would not be possible to test this hypothesis while only one genome region is used for virus identification. PRACTICAL ISSUES OF RECOMBINATION STUDIES IN PICORNAVIRUSES Phylogenetic methods are continuously improving, allowing more sensitive and reliable detection of recombination events. There are, however, several issues that complicate recombination analysis in picornaviruses. First, asymptomatic infection with many picornaviruses is common. If there is a 5% chance of spontaneous isolation of a virus, about 5% of virus isolates can be expected to contain two strains. In

6 332 A. N. Lukashev our experience, this prediction roughly corresponds to the actual rate of detection of enterovirus mixtures. Sequencing of such mixture from PCR products can result in detection of a false recombination if primer pairs to distinct genome regions amplify different components of a virus mixture. Thus, mixed strains can introduce an error in recombination studies, especially if two distinct non-overlapping genome regions were sequenced. This issue can be alleviated by sequencing long (2 7 Kb) PCR products and increasing an overlap between PCR products to at least 100 nt. Mistakes resulting from differential amplification of virus mixture components could not create a picture of frequent ubiquitous recombination we observe in most picornaviruses, but any detection of a single unusual (e.g. interspecies) recombinant should be treated with caution. Careful selection of the sequence set is critical for reliable conclusions. A limited data set can jeopardise detection of recombination, as occurred in several early studies that reported absence of recombination in modern strains of enteroviruses. Detection of recombination within the capsidencoding genome region of picornaviruses seems to be also highly dependent on the sequence set used. There are over 100 serotypes of enteroviruses, and one or a few full genome sequences for any particular serotype, which in addition are rarely compared to each other. This might explain why reports of recombination in the P1 genome region are relatively rare. In HAV, on the contrary, there are multiple genomic sequences of the same serotype, and recombination within the P1 genome region is apparently common. In cardioviruses, recombination in P1 could be reliably demonstrated after four sequences for type 2 became available [55]. Most recombination detection methods are based on a change of phylogenetic relations of viruses over the genome. Only distinct recombination events can be reliably detected this way, and reported recombination points can reflect borders of preserved fragments with significantly different phylogeny rather than the true breakpoints. This presents a problem when analysing sequences that underwent multiple recombination events around a recombination hotspot. For example, phylogenetic signal was extremely low in the 2AB genome region in HEV-B strains for no apparent reason. The phylogenetic tree for this part of the genome was star-like, with few reliably supported groups and long branches of individual strains originating close to the tree root. Such tree topology is most likely explained by extremely frequent recombination in this region that destroyed the phylogenetic grouping [22]. Similar regions of low phylogenetic signal, likely due to multiple recombination events, can be seen in the 2AB genome region of other enteroviruses, parechoviruses, cardioviruses and aphthoviruses. Recombination breakpoints detected by various methods were often at the edges, but not within, this low-signal region, leading to an apparently lower recombination rate in this genome region. This can in part be compensated by investigating a huge number of isolates, but analysis approaches that would account for this issue should greatly contribute to a more profound understanding of picornavirus recombination. GLOBAL MOLECULAR EPIDEMIOLOGY OF PICORNAVIRUSES Natural recombination in picornaviruses cannot be discussed separately from other evolutionary properties, such as global prevalence and a very dynamic epidemiology. Molecular epidemiology studies in most picornaviruses did not reveal a clear geographic pattern. It is common for picornavirus genotypes to spread globally in short periods of time, usually on the order of months. In many cases the spreading genotype would completely displace other similar viruses from circulation. Enterovirus 71 genotypes that were circulating in the 1970s became completely extinct after 1980 [59]. Early echovirus 30 (E30) genotypes also became globally extinct after 1978 [60], and global diversity (maximum nucleotide sequence difference) of E30 capsid-encoding region dropped from 0.32 to 0.13 [61]. Similarly, all modern isolates of E6, E12, E30 and CBV3 grouped together relative to their prototype strains isolated in the 1950s [19]. This tendency could also be observed for the majority of modern E13 isolates [62], but it was not apparent in a large-scale study of E11 epidemiology [63]. A similar process was observed independently for the 3D encoding region of HEV-B. Most ancestral polymerase variants became extinct, and two groups of E30 and E9-like polymerase became highly prevalent in 1980s-1990s [19]. Therefore, global diversity of most NPEV has decreased over the last 50 years.

7 Recombination among picornaviruses 333 One could suggest that the modern viruses have higher fitness, but this still requires solid proof. The selection in enterovirus P1 genome region is purifying. This is indicated by a low non-synonymous to synonymous mutation ratio [36], which reflects pressure to conserve protein sequence and exclude most random substitutions that result in changes of the amino acid sequence. However, purifying selection does not explain globally decreasing diversity of the enterovirus RNA sequence, as most substitutions are synonymous and are unlikely to experience a significant selection pressure. The majority of cardioviruses and parechoviruses were isolated only recently, so unfortunately it is not yet possible to track temporal dynamics of their global gene pools to confirm observations done on enteroviruses. RECOMBINATION ON A GLOBAL SCALE AS A MEANS OF SPECIES MAINTENANCE Recombination plays the key role in the microevolution of picornaviruses and similar viruses with high polymerase error rates. It can circumvent constant degradation of the genome due to accumulation of deleterious mutations and recreate a functional genome from several impaired ones. On a global scale, recombination allows enterovirus genome fragments to have distinct phylogenetic history. New recombinant forms of picornaviruses emerge very frequently [64], and each isolate is just a snapshot from a global pool of genetic information. Therefore, recombination was commonly viewed as a mechanism that contributes to the diversity of picornaviruses. While this is true on a short time and space scale, over the long term, frequent recombination in most picornaviruses acts rather as a stabilising force that preserves the global species integrity by constantly shuffling diverging viral genomes. In the non-structural genome region, recombination averages the global consensus sequence of a species. This hypothesis implies that every virus in the world must recombine with another virus of the same species over a limited period of time (probably on the order of decades), otherwise high substitution rate would result in emergence of a new species or critical deterioration of the virus sequence. Viruses of the same species regularly undergo recombination, and thus do not diverge beyond a certain threshold, for example 30% of nucleotide and 10% of amino acid sequence in the NSP genome region in enteroviruses (Figure 2). It can be suggested that such degree of diversity corresponds to biologic compatibility of genome fragments and thus allows emergence of viable recombinants. Different species do not recombine with each other in this genome region. They can, therefore, gradually separate from each other and differ by over 50% of nt sequence and over 35% of aa sequence in this genome region. Owing to recombination within a species, there are no pairs of enteroviruses that would differ by 30 50% of nt sequence or 10 35% of aa sequence in this Figure 2. Frequencies of pairwise distances between enteroviruses. Complete non-structural genome regions of Enterovirus A (excluding enteroviruses 89 92) and all Enterovirus B strains available in GenBank (a total of 162 strains) were aligned based on the putative protein sequence. Nucleotide (Jukes Cantor corrected) and amino acid (uncorrected) distances were calculated with MEGA 4.0 [68]. The y-axis shows number of pairwise distances in every range on the x-axis

8 334 A. N. Lukashev dataset, which provides clear delimitation of the species. In the capsid-encoding genome region recombination similarly conserves the (sero)type sequence by shuffling genomes of the same type, and prevalence of intratypic recombination in the P1 genome region might currently be underestimated due to limited sampling of isolates of the same serotype. Therefore, in a two-way process, restrained RNA diversity within species permits recombination, which, in turn, limits diversification of the global (worldwide) gene pool of an established species. Therefore, recombination is both a key property of picornavirus species and the means to maintain them. IMPACT OF RECOMBINATION ON CLASSIFICATION OF PICORNAVIRUSES The International Committee on Virus Taxonomy (ICTV) terms picornavirus species as viruses with <70% amino acid identity in P1 and <70% amino acid identity in the 2C þ 3CD [28]. This definition is attractively robust, yet it carries limited biological sense. In general biology, species are generally termed as organisms able to interbreed and produce fertile offspring. This definition is universal because the possibility of sexual procreation implies a common gene pool and restricted divergence within a species. Recombination in enteroviruses could be thought of as an analogue of such reproductive strategy in higher organisms. Recombination in picornaviruses is strictly confined to groups that in most cases correspond to the established species. It was suggested previously that the possibility of natural recombination could be used as an additional species criterion in enteroviruses [19]. Based on absence of recombination between mouse and human cardioviruses, human Saffold-like cardioviruses and TMEV could be identified as distinct species [55]. Similarly, most known human parechoviruses can be seen as a sole species, as they freely recombine with each other. There are, however, groups of picornaviruses that obviously belong to a certain species but do not recombine with other representatives of the same species or do so very rarely. For example, parechovirus type 3 is not remarkably distant from other parechoviruses, but it does not recombine with other types [45]. Such genetic isolation could eventually lead to emergence of a new species. Bayesian phylogenies in different genome regions showed that the most recent common ancestor of the known HPeV-3 isolates dates back only about 30 years. It is likely that HPeV-3 is at an early stage of a path to speciation. When viruses stop recombining with the global gene pool, they begin to diverge and would eventually form a new species. Reasons for such events could be a change of the cellular receptor used for viral entry, change of host species, geographic isolation, etc. Another example of picornaviruses in speciation could be a number of simian enteroviruses that formally belong to HEV-A species [31], but do not recombine with human viruses and would eventually separate into distinct species. SVDV stopped recombining with HEV-B species after the host switch to pigs [33] and would most likely drift away from the parental virus and found a distinct species within several decades. Three serotypes of HEV-C species, CVA1, CVA19, CVA22, are monophyletic throughout the genome with no evidence of recombination with other HEV-C members [13]. These viruses differ from other HEVs by inability to grow in cell culture, probably due to use of a different receptor. One could speculate that these viruses are also on a path of forming a new species. The distribution of recombination events over the genome in most picornavirus genera has certain regularities. Recombination hotspots are found in all species on the borders of the capsid-encoding genome region. These parts of the genome encode non-structural proteins 2A, 2B and L, and structural protein VP4. Proteins 2A, 2B and L are the most diverse proteins among picornaviruses. They have variable size and function in different picornaviruses, and they are completely absent in some genera (Figure 1). VP4 is an internal structural protein that is not tightly integrated in the capsid structure, and it is absent in parechoviruses and kobuviruses. It is likely that modular evolution of genome fragments encoding capsid and the replication complex proteins increased evolutionary flexibility around these hotspots, which in turn could lead to emergence of diverse 2A, 2B and L proteins. CONCLUSION This review covers only recombination in the family Picornaviridae. There is extensive evidence of recombination in order Picornavirales (former picorna-like viruses) reviewed in Reference [5]. Modular evolution and a global gene pool model were suggested for many other viruses. In small

9 Recombination among picornaviruses 335 DNA viruses, recombination patterns resemble those in picornaviruses, with a preserved cassette of capsid genes and recombination hotspots between genome regions encoding structural and non-structural genome regions [65]. Frequent recombination and modular evolution were suggested in adenoviruses [66], traditionally thought to be stable and conserved DNA viruses. It seems likely that frequent recombination and a global gene pool are common to almost all viruses that feature global prevalence, notable diversity and common co-infection, which comprises most of the respiratory and gastrointestinal viruses. Therefore, most of the conclusions of this review can be extended to other virus families. Frequent recombination in picornaviruses, combined with global and highly dynamic epidemiology, creates a global gene pool that can be observed as species. Such population dynamics of picornaviruses might be confusing for an epidemiologist, but is well known in the world of phages that also exist as a global gene pool [67]. Unlike phages, human picornaviruses are commonly sampled and sequenced around the world for epidemiological studies. Therefore, independent evolution of genes on a worldwide scale can be conveniently studied on a time range of years and even months. This makes human picornaviruses an ideal model for a manageable analysis of the lateral drift of genetic information that played a major role in emergence and development of life on Earth. The author is grateful to Dr J.F. Drexler (Bonn University), Prof. V.A. Lashkevich, Dr A.P. Gmyl (Chumakov Institute of Poliomyelitis, Moscow) and the anonymous reviewer for helpful remarks and suggestions on the manuscript. REFERENCES 1. Ward CD, Flanegan JB. Determination of the poliovirus RNA polymerase error frequency at eight sites in the viral genome. Journal of Virology 1992; 66: Drake JW. Rates of spontaneous mutation among RNA viruses. Proceedings of National Academy of Science USA 1993; 90: Kirkegaard K, Baltimore D. The mechanism of RNA recombination in poliovirus. Cell 1986; 47: Gmyl AP, Korshenko SA, Belousov EV, et al. Nonreplicative homologous RNA recombination: promiscuous joining of RNA pieces? RNA 2003; 9: Lukashev AN. Role of recombination in evolution of enteroviruses. Review of Medical Virology 2005; 15: Ledinko N. Genetic recombination with poliovirus type 1: studies of crosses between a normal horseserum resistant mutant and several guanidine-resistant mutants of the same strain. Virology 1963; 20: Hirst GK. Genetic recombination with Newcastle disease virus, polioviruses and influenza. In Cold Spring Harbor Symposia on Quantitative Biology Agol VI, Grachev VP, Drosdov SG, et al. Construction and properties of intertypic poliovirus recombinants: first approximation mapping of the major determinants of neurovirulence. Virology 1984; 136: Georgopoulou A, Markoulatos P. Sabin type 2 polioviruses with intertypic vaccine/vaccine recombinant genomes. European Journal of Clinical Microbiology and Infectious Diseases 2001; 20: Macadam AJ, Arnold C, Howlett J, et al. Reversion of the attenuated and temperature-sensitive phenotypes of the Sabin type 3 strain of poliovirus in vaccinees. Virology 1989; 172: Georgescu M-M, Delpeyroux F, Crainic R. Tripartite organization of a natural type 2 vaccine/nonvaccine recombinant poliovirus. Journal of General Virology 1995; 76: Blomquist S, Bruu AL, Stenvik M, et al. Characterization of a recombinant type 3/type 2 poliovirus isolated from a healthy vaccinee and containing a chimeric capsid protein VP1. Journal of General Virology 2003; 84: Brown B, Oberste MS, Maher K, et al. Complete genomic sequencing shows that polioviruses and members of human enterovirus species C are closely related in the noncapsid coding region. Journal of Virology 2003; 77: Witso E, Palacios G, Cinek O, et al. High prevalence of human enterovirus a infections in natural circulation of human enteroviruses. Journal of Clinical Microbiology 2006; 44: Pallansch MA, Roos RP. Enteroviruses: polioviruses, coxsackieviruses, echoviruses, and newer enteroviruses. In Fields Virology (4th edn), Knipe DM, Howley PM (eds). Lippincott-Raven: Philadelphia, 2001; Santti J, Hyypia T, Kinnunen L, et al. Evidence of recombination among enteroviruses. Journal of Virology 1999; 73: Santti J, Harvala H, Kinnunen L, et al. Molecular epidemiology and evolution of coxsackievirus A9. Journal of General Virology 2000; 81: Lindberg MA, Andersson P, Savolainen C, et al. Evolution of the genome of Human enterovirus B: incongruence between phylogenies of the VP1 and

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