R E V I E W Recombination among picornavirusesdownload.xuebalib.com/xuebalib.com.36665.pdf · Viral Encephalitides, p/o Institut Poliomielita, Moscow Region 142782, Russia. E-mail:

Rev. Med. Virol. 2010; 20: 327–337.Published online 14 July 2010 in Wiley Online Library

(wileyonlinelibrary.com).DOI: 10.1002/rmv.660

RR EE VV I E WWRecombination amA.N. Lukashev1,2*1M.P. Chumakov Institute of PoliomyelitisMoscow, Russia

Reviews in Medical Virology

�Corresponding authoViral Encephalitides142782, Russia.E-mail: alexander_luk

Abbreviations usedCVA, coxsackievirusphalomyocarditis viruhuman enterovirus; HCommittee on VirusNPEV, non-polio entnon-translated regionment length polymorplike cardioviruses; STheiler’s murine ence

Copyright # 20

ong picornaviruses

and Viral Encephalitides, Russian Academy of Medical Sciences,

2Institute 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 clinicalmanifestations in humans and animals. Many of these viruses are highly diversified and globally prevalent. Naturalrecombination has been reported in most picornavirus genera and is a key genetic feature of these infectious agents. Inseveral 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 geneticinformation. It can be suggested that on a short time scale recombination acts to promote virus diversity, and newrecombinant 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 andspeciation. This review covers existing evidence of recombination in most genera of the family Picornaviridae andpossible implications for diagnostics, epidemiology and classification. Copyright # 2010 John Wiley & Sons, Ltd.

Received: 18 March 2010; Revised: 20 April 2010; Accept
ed: 11 May 2010
INTRODUCTIONPicornaviruses are a diverse and ubiquitous familyof non-enveloped positive strand RNAviruses. Thegrowing family Picornaviridae currently comprises12 genera that infect mammals and birds. The mostclinically and economically significant picorna-virus genera are Enterovirus, Aphthovirus, Cardio-virus, Parechovirus, Hepatovirus and Kobuvirus.Among picornaviruses are such important patho-gens as poliovirus, enteroviruses, HAV, foot andmouth disease virus of cattle and many others. Thepicornavirus genome encodes a single polyproteinthat is cleaved into 3–4 structural proteins and 7–8

r: A.N. Lukashev, Institute of Poliomyelitis and, p/o Institut Poliomielita, Moscow Region

[email protected]

:A; E, echovirus; EV, enterovirus; EMCV, ence-s; FMDV, foot and mouth disease virus; HEV,PeV, human parechovirus; ICTV, InternationalTaxonomy; IRES, internal ribosome entry site;eroviruses; NSP, non-structural proteins; NTR,; RF, recombinant form; RFLP, restriction frag-hism; SEV, simian enterovirus; SLCV, Saffold-VDV, swine vesicular disease virus; TMEV,phalomyelitis virus.

10 John Wiley & Sons, Ltd.

non-structural proteins (NSP) (Figure 1). Con-served major NSP are 2C, 3A, 3B, 3C and 3D.The 2A, 2B and L proteins are highly diverse instructure and function among different picorna-virus genera and do not share a commonphylogenetic origin. The open reading frame ispreceded by a 50NTR that contains an internalribosome entry site (IRES) responsible for cap-independent initiation of translation. The 50NTRand 30NTR also encompass secondary structureelements responsible for genome replication.

Picornaviruses exhibit remarkable plasticity oftheir genomes. The RNA-dependent RNA poly-merase is very error-prone, resulting in roughlyone substitution per copied genome [1,2]. Most ofthese mutations are supposed to be deleterious,and recombination is a process capable of recreat-ing functional genomes from defective ones. Twodistinct recombination mechanisms have beensuggested. A classic copy-choice model suggeststhat recombination occurs when the viral poly-merase switches template strands in the course ofsynthesis of the negative RNA strand duringreplication of two viruses in a co-infected cell [3].Alternatively, it has been shown that recombina-tion can happen in the absence of replication

Figure 1. Schematic representation of genomes of the major picornavirus genera

328328 A. N. LukashevA. N. Lukashev

between RNA fragments that do not encode acomplete polymerase [4]. It was suggested that inthis case the recombination mechanism consistedof joining of the broken RNA molecules. Mostlikely, both mechanisms are valid; however, it isnot possible to conclude which of them ispredominant in nature.

Natural recombination is extremely frequent inpicornaviruses. Some time ago current knowledgeon recombination among enteroviruses was sum-marised [5]; however, many new works have beenpublished in the last few years, and several generaof picornaviruses have been recently acknowl-edged as important human pathogens. This reviewcovers present data on recombination in differentPicornaviridae genera and discusses possibleimplications for understanding the evolution ofthese viruses.

RECOMBINATION IN POLIOVIRUSESPoliovirus, a member of the genus Enterovirus, wasthe most significant picornavirus for practicalhealthcare in the 20th century because of pandemicpoliomyelitis. Recombination in poliovirus wasdiscovered as early as 1962. Cells were infectedwith two strains of poliovirus that carriedmutations providing resistance to either guanidi-nium or horse serum. Recovery of viruses withdouble resistance was 15–20 times higher thancould be expected to result only from mutation[6,7]. Recombination in polioviruses could be easilyreproduced in vitro and thus became a helpful toolto study the virus genome before the advent of

Copyright # 2010 John Wiley & Sons, Ltd.

reverse genetics [8]. The most valuable results onpoliovirus recombination in vivo were obtained inthe course of the polio eradication campaign. Livepolio vaccine is a mixture of three attenuated virusserotypes administered simultaneously, whichcreates ideal conditions for intertypic recombina-tion. As the parental vaccine viruses were known, itwas easy to identify recombinant viruses withinexpensive methods, such as PCR-RFLP, and tomap recombination points with high precision.Recombinants appeared soon after vaccination andcomprised up to 36% of the excreted viruses [9]. It isthus likely that recombination helps to restorefitness of the attenuated vaccine strains [10].Recombination occurred most frequently in theNSP genome part and less frequently between the50NTR and the coding genome region [11].Recombination within the capsid-encodingregion seems to be rather an exception than a rule[12]. Circulating wild and vaccine-derived polio-viruses readily recombine with other humanenterovirus C serotypes [13], which justifiesclassification of poliovirus within the HEV-Cspecies (see below).

RECOMBINATION IN NON-POLIOENTEROVIRUSESOver 100 serotypes of enteroviruses are currentlyknown and a few more are discovered each year.Human enteroviruses are classified into fourspecies, Human enterovirus A–D. Other membersof the genus are Human rhinovirus A–C, bovine,swine and simian enteroviruses and a number of

Rev. Med. Virol. 2010; 20: 327–337.DOI: 10.1002/rmv

Recombination among picornavirusesRecombination among picornaviruses 329329

unclassified enteroviruses. Human enterovirusesare ubiquitous and asymptomatic infection occursfrequently in infants [14]. Clinically prominentdisease is relatively rare, although enterovirusesare a significant cause of neurological disorders,meningitis, myocarditis, sepsis-like disease ofnewborns, etc. [15]. It can be also expected that awider use of modern screening methods wouldreveal a role of enteroviruses in a range ofsyndromes not currently associated with virusinfection. Non-polio enteroviruses (NPEV) havereceived much less attention until the last decadewhen apparent eradication of poliomyelitis inEurope and the Americas liberated the resourcesof surveillance laboratories. The first systematicstudy on recombination among non-polio enter-oviruses was performed in 1999 [16]. Conflictingphylogenies in genome regions encoding capsidand NSP were subsequently reported in a numberof works [17–21]. Common to all studies, allcirculating strains of human enteroviruses wererecombinant relative to the prototype strainsisolated in the 1950s. Phylogenetic grouping ofenteroviruses corresponded to the serotype only inthe genome region encoding the major capsidproteins—VP1, VP2 and VP3. All other genomefragments were highly shuffled by recombination,with the most prominent hotspots mapping tothe edges of the P1 genome region encodingproteins VP4 and 2A [22]. Reports of recombinationwithin the P1 genome region were rare andinvolved viruses of the same serotype [23–25].Recombination was most frequent within HEV-Bspecies [21]. This conclusion was based on bothdirect estimation of recombination rate over timeand analysis of recombination traces in thecompletely sequenced genomes. Prevalence ofrecombination within HEV-A species was signifi-cantly lower. Prototype strains of HEV-C specieswere shown to be recombinant relative to eachother [13], and circulating HEV-C strains wereshown to readily recombine with poliovirus;however, there has been no systematic study ofrecombination prevalence in circulating non-polioHEV-C.

One of the most prominent non-polio entero-viruses is enterovirus 71 (EV71), a member of theHEV-A species, which is distinguished as a causeof massive epidemics of hand, foot and mouthdisease in Asia and the Pacific. Apparent preva-lence of recombination among enterovirus 71


strains is lower than in HEV-B, and many groupsof strains are monophyletic in distant genomeregions [26]. This might reflect true recombinationfrequency, but could also be affected by biasedsampling. Indeed, there are over 100 contemporaryEV71 sequences available, but only one completesequence for each of the remaining HEV-Aprototype strains and no complete sequences ofthe modern HEV-A isolates. In addition, in manystudies EV71 strains are compared only to eachother, but not to the rest of the species. We noticedthat EV71 strains are recombinant relative to otherHEV-A species members and carry at least threedifferent variants of the 3CD genome region similarto the prototype strains of CVA5, CVA8 andCVA16 (unpublished observation).

Highly mosaic structure of enterovirus genomesrelative to each other brought an understandingthat classical enterovirus surveillance methodsbased on the serotype neglect nearly two thirdsof the genome. To address this problem, it wassuggested to study circulation of recombinantforms (RF), which are combinations of VP1 and3D without apparent signs of recombination, i.e.viruses that group together on phylogenetic treesfor both genome regions. Definition of RFs relies onabundant sequence information, so it is practicalonly in larger studies. This approach demonstratedthat new RFs of echovirus 30 emerge frequently,become dominant over Europe in a few years andthen often vanish completely from circulation[27]. It is unclear what allows a RF to spread overlarge territories and displace other viruses fromcirculation; it is still more intriguing what makesa RF vanish simultaneously over the wholecontinent.

Human rhinoviruses were only recently addedto the genus Enterovirus based on their geneticproperties [28]. There are three groups (putativespecies) of rhinoviruses, Human rhinovirus A–C.Unlike enteroviruses, rhinoviruses are acid liableand usually cause a mild respiratory infection.Rhinoviruses are commonly typed by sequencingthe VP4/VP2 genome region. There is evidence forfrequent recombination between group C andgroup A rhinoviruses that involves the adjacent50NTR and VP2/VP4 genome regions [29,30].Recombination between rhinoviruses of differentgroups was also observed in the NSP part of thegenome [30]; therefore, delineation of rhinovirusspecies requires further clarification.



ANIMAL ENTEROVIRUSESAnimal enteroviruses are probably as diverse andubiquitous as their human congeners, but they aremuch less studied. There is evidence of recombina-tion events among bovine and simian enteroviruses[31,32]; however, lack of sequence data preventsestimating recombination frequencies. A key ques-tion for practical healthcare is how frequentlyenteroviruses change host species and if geneticinformation can be exchanged between human andanimal viruses. There is at least one example ofcoxsackievirus B5 that crossed the species barrierand became prevalent as swine vesicular diseasevirus (SVDV) only 50 years ago [33]. Interestingly,the rate of recombination in SVDV has droppedmarkedly compared to its human progenitor afterchanging host species. Several simian entero-viruses phylogenetically belong to human enter-ovirus A species [34], but they are ancestral tohuman serotypes and there was no evidence ofrecombination between human and simian enter-oviruses [31]. Therefore, the possibility of geneticexchange between human and animal entero-viruses remains to be investigated. Highly preva-lent recombination was also reported in tescho-viruses, which were formerly classified as porcineenteroviruses [35,36]. As in enteroviruses, break-points (locations in the genomewhere phylogeneticaffiliation of a strain changed, indicating recombi-nation) mainly mapped to P1 region boundaries,but they could also be found all over the NSP partof the genome.

APHTHOVIRUSESThe major member of the genus Aphthovirus is foot-and-mouth disease virus (FMDV), which causessevere infection in cloven-hoofed animals and is amajor veterinary concern. There are seven sero-types of FMDV. Recombination in FMDV wasdiscovered in 1965 using the same experimentalapproach as for poliovirus [37]. Currently there areover 150 full genome FMDV sequences available,which allows profound recombination analysis.Recombination breakpoints map predominantly tothe borders of the P1 genome region, much like inother picornaviruses [35]. Recombination has alsobeen reported within the P1 genome region [38],but it seems to be uncommon as in otherpicornavirus genera [35]. Interestingly, threeFMDV serotypes that are endemic in Africa,SAT1-SAT3, show almost no evidence of recombi-


nation with the more common and globally spreadserotypes [39]. As several recombination eventscould still be observed, this is probably an exampleof spatial separation that has not yet resulted inreproductive incompatibility. It is tempting tohypothesise that evolutionary relations of theendemic SAT and common epidemic FMDVserotypes might have parallels in genetics ofcommon human pathogens, such as enteroviruses,which possibly exchange genetic information withanimal viruses.

PARECHOVIRUSESParechoviruses (HPeV) were first isolated in 1950sand were initially classified as echovirus 22 and 23.Analysis of the genome sequence justified theirreclassification as a new genus of picornaviruses[40]. Prevalence and diversity of parechoviruseswas not truly anticipated until very recently.Parechovirus infection proved to be highly preva-lent in humans, with the proportion of seroposi-tives reaching 88% at the age of 2 years [41].Currently, 14 types of parechoviruses are associ-ated with a wide range of infant diseases [42]including meningitis and sepsis-like disease ofnewborns [43,44]. In general, recent reports showthat epidemiological and clinical features ofparechoviruses are similar to human enteroviruses.Recombination is also highly prevalent in par-echoviruses [45–48], and recombination break-points map to the same genome regions as inenteroviruses. The hotspots are the boundaries ofthe P1 genome region, recombination in NSP isquite common, and there is no intertypic recombi-nation in most of the P1 genome part. Inenteroviruses a recombination hotspot maps tothe genome region encoding the minor capsidprotein VP4 [22]. In parechoviruses, unlike in mostother picornavirus genera, VP0 is not cleaved intoVP2 and VP4 proteins. Nevertheless, a recombina-tion hotspot in parechoviruses is found in the 50

part of VP0 genome region that corresponds to VP4in other picornaviruses. As in enteroviruses of thesame species, all parechoviruses of different typescan recombine. The only exclusion is HPeV type 3viruses that rarely recombine with any other type[46]. It is intriguing to see if this observation resultsfrom a sampling bias or reflects some biologicalbarrier to recombination between HPeV3 and otherparechoviruses. In the latter case we might be



witnessing emergence of a novel parechovirusspecies.

A distinctive feature of recombination in par-echoviruses is a clear geographic pattern of thenon-structural genome part. A study of partialgenome sequences in VP1 and 3D genome regionsrevealed that the latter genome fragment in strainsof different types isolated in Netherlands wasdistinct from British and Brazilian isolates [49]. Astudy of full-genome parechovirus sequencessuggested that a unique variant of 3ABC genomeregion was ‘endemic’ in Brazil for the last centuryand did not occur elsewhere in the world (Drexleret al., submitted).

CARDIOVIRUSESFor a long time two rodent cardioviruses, ence-phalomyocarditis virus (EMCV) and Theiler’smurine encephalomyelitis virus (TMEV), werethe only known members in this genus. Therewas no reliable evidence of cardiovirus infection inhumans until 2007 when a virus distantly related toTMEVwas identified in a faecal sample dating backto 1982 from an infant with fever of unknownorigin [50]. The virus was named Saffold virus. Inthe following 2 years eight types of Saffold-likecardioviruses were found mainly in patients withgastroenteritis [51–54]. Serologic studies indicatethat asymptomatic cardiovirus infection is highlyprevalent in infants below 2 years old [51].Evidence of widespread recombination in Saf-fold-like cardioviruses (SLCV) appeared almostimmediately upon their discovery. Most reportsindicated recombination occurring on the borderbetween structural and non-structural genomeregions and in the NSP genome region [47,53,55].Recombination quite often took place throughoutthe NPS genome region between viruses of anytype, and in the capsid-encoding part of thegenome between viruses of the same type [55].New strains of TMEV were recently isolated fromrodents on different continents, and recombinationwas shown to be common among murine TMEV[56], with patterns very similar to those amongSaffold-like cardioviruses.

HEPATITIS A VIRUSHAV is the most common cause of water-bornehepatitis. The virus has several features uniqueamong picornaviruses. First, the genus isrepresented by one sole serotype. Second, it was


hypothesised that the virus prefers rare codons,presumably to limit the translation rate and avoideliciting immune responses [57]. Recombinationhas been reported for HAV, but the pattern ofrecombination is strikingly different from that inother picornaviruses. Recombination points havebeen reported in the capsid-encoding genomeregion [58].

MEDICAL IMPLICATIONS OFRECOMBINATION IN PICORNAVIRUSESIt is common to human picornaviruses that mostinfection cases are asymptomatic, and only a fewresult in a clinically prominent disease. Symptomsof infection with enteroviruses and parechovirusescan be diverse; one virus type can produce differentmanifestations and the same syndrome can becaused by different virus types. It is also typical ofseveral human picornavirus genera, especiallyenteroviruses, HAV and Aichi virus, that sympto-matic disease occurs as outbreaks, which couldimply emergence of a more pathogenic virusvariant prior to an outbreak. Molecular determi-nants of picornavirus pathogenicity are generallypoorly understood, and almost all known onesmap to the capsid-encoding genome region that isused for typing. Due to frequent recombinationbetween P1 and other genome regions in enter-oviruses, parechoviruses and cardioviruses classictyping approaches, serological or molecular,characterise roughly one-third of the genome,while the rest is neglected. Therefore, any geneticfeatures encoded outside the P1 genome part havemuch lower chance to be discovered spon-taneously. It is tempting to suggest that recombina-tion plays a role in emergence of more pathogenicvirus strains, but it would not be possible to test thishypothesis while only one genome region is usedfor virus identification.

PRACTICAL ISSUES OF RECOMBINATIONSTUDIES IN PICORNAVIRUSESPhylogenetic methods are continuously improv-ing, allowing more sensitive and reliable detectionof recombination events. There are, however,several issues that complicate recombinationanalysis in picornaviruses.

First, asymptomatic infection with many picor-naviruses is common. If there is a 5% chance ofspontaneous isolation of a virus, about 5% of virusisolates can be expected to contain two strains. In



our experience, this prediction roughly corre-sponds to the actual rate of detection of enterovirusmixtures. Sequencing of such mixture fromPCR products can result in detection of a falserecombination if primer pairs to distinct genomeregions amplify different components of a virusmixture. Thus, mixed strains can introduce an errorin recombination studies, especially if two distinctnon-overlapping genome regions were sequenced.This issue can be alleviated by sequencing long(2–7Kb) PCR products and increasing an overlapbetween PCR products to at least 100 nt. Mistakesresulting from differential amplification of virusmixture components could not create a picture offrequent ubiquitous recombination we observe inmost picornaviruses, but any detection of a singleunusual (e.g. interspecies) recombinant should betreated with caution.

Careful selection of the sequence set is critical forreliable conclusions. A limited data set canjeopardise detection of recombination, as occurredin several early studies that reported absence ofrecombination in modern strains of enteroviruses.Detection of recombination within the capsid-encoding genome region of picornaviruses seemsto be also highly dependent on the sequence setused. There are over 100 serotypes of enteroviruses,and one or a few full genome sequences for anyparticular serotype, which in addition are rarelycompared to each other. This might explain whyreports of recombination in the P1 genome regionare relatively rare. In HAV, on the contrary, thereare multiple genomic sequences of the sameserotype, and recombination within the P1 genomeregion is apparently common. In cardioviruses,recombination in P1 could be reliably demon-strated after four sequences for type 2 becameavailable [55].

Most recombination detection methods arebased on a change of phylogenetic relations ofviruses over the genome. Only distinct recombina-tion events can be reliably detected this way, andreported recombination points can reflect bordersof preserved fragments with significantly differentphylogeny rather than the true breakpoints. Thispresents a problem when analysing sequences thatunderwent multiple recombination events arounda recombination hotspot. For example, phyloge-netic signal was extremely low in the 2AB genomeregion in HEV-B strains for no apparent reason.The phylogenetic tree for this part of the genome


was star-like, with few reliably supported groupsand long branches of individual strains originatingclose to the tree root. Such tree topology is mostlikely explained by extremely frequent recombina-tion in this region that destroyed the phylogeneticgrouping [22]. Similar regions of low phylogeneticsignal, likely due to multiple recombinationevents, can be seen in the 2AB genome region ofother enteroviruses, parechoviruses, cardiovirusesand aphthoviruses. Recombination breakpointsdetected by various methods were often at theedges, but not within, this low-signal region,leading to an apparently lower recombinationrate in this genome region. This can in part becompensated by investigating a huge number ofisolates, but analysis approaches that wouldaccount for this issue should greatly contributeto a more profound understanding of picornavirusrecombination.

GLOBAL MOLECULAR EPIDEMIOLOGYOF PICORNAVIRUSESNatural recombination in picornaviruses cannotbe discussed separately from other evolutionaryproperties, such as global prevalence and a verydynamic epidemiology.

Molecular epidemiology studies in most picor-naviruses did not reveal a clear geographic pattern.It is common for picornavirus genotypes to spreadglobally in short periods of time, usually on theorder of months. In many cases the spreadinggenotype would completely displace other similarviruses from circulation. Enterovirus 71 genotypesthat were circulating in the 1970s became com-pletely extinct after 1980 [59]. Early echovirus 30(E30) genotypes also became globally extinct after1978 [60], and global diversity (maximum nucleo-tide sequence difference) of E30 capsid-encodingregion dropped from 0.32 to 0.13 [61]. Similarly, allmodern isolates of E6, E12, E30 and CBV3 groupedtogether relative to their prototype strains isolatedin the 1950s [19]. This tendency could also beobserved for the majority of modern E13 isolates[62], but it was not apparent in a large-scale studyof E11 epidemiology [63]. A similar process wasobserved independently for the 3D encodingregion of HEV-B. Most ancestral polymerasevariants became extinct, and two groups of E30and E9-like polymerase became highly prevalent in1980s-1990s [19]. Therefore, global diversity ofmost NPEV has decreased over the last 50 years.



One could suggest that the modern viruses havehigher fitness, but this still requires solid proof. Theselection in enterovirus P1 genome region ispurifying. This is indicated by a low non-synon-ymous to synonymous mutation ratio [36], whichreflects pressure to conserve protein sequence andexclude most random substitutions that result inchanges of the amino acid sequence. However,purifying selection does not explain globallydecreasing diversity of the enterovirus RNAsequence, as most substitutions are synonymousand are unlikely to experience a significantselection pressure. The majority of cardiovirusesand parechoviruses were isolated only recently, sounfortunately it is not yet possible to track temporaldynamics of their global gene pools to confirmobservations done on enteroviruses.

RECOMBINATION ON A GLOBAL SCALEAS A MEANS OF SPECIES MAINTENANCERecombination plays the key role in the micro-evolution of picornaviruses and similar viruseswith high polymerase error rates. It can circumventconstant degradation of the genome due toaccumulation of deleteriousmutations and recreatea functional genome from several impaired ones.On a global scale, recombination allows entero-virus genome fragments to have distinct phyloge-netic history. New recombinant forms of picorna-viruses emerge very frequently [64], and eachisolate is just a snapshot from a global pool of

Figure 2. Frequencies of pairwise distances between enteroviruses. Co

enteroviruses 89–92) and all Enterovirus B strains available in GenBank

sequence. Nucleotide (Jukes–Cantor corrected) and amino acid (uncor

shows number of pairwise distances in every range on the x-axis


genetic information. Therefore, recombination wascommonly viewed as amechanism that contributesto the diversity of picornaviruses. While this is trueon a short time and space scale, over the long term,frequent recombination inmost picornaviruses actsrather as a stabilising force that preserves the globalspecies integrity by constantly shuffling divergingviral genomes. In the non-structural genomeregion, recombination averages the global consen-sus sequence of a species. This hypothesis impliesthat every virus in the world must recombine withanother virus of the same species over a limitedperiod of time (probably on the order of decades),otherwise high substitution rate would result inemergence of a new species or critical deteriorationof the virus sequence. Viruses of the same speciesregularly undergo recombination, and thus do notdiverge beyond a certain threshold, for example30% of nucleotide and 10% of amino acid sequencein the NSP genome region in enteroviruses(Figure 2). It can be suggested that such degreeof diversity corresponds to biologic compatibilityof genome fragments and thus allows emergence ofviable recombinants. Different species do notrecombine with each other in this genome region.They can, therefore, gradually separate from eachother and differ by over 50% of nt sequence andover 35% of aa sequence in this genome region.Owing to recombination within a species, there areno pairs of enteroviruses that would differ by 30–50% of nt sequence or 10–35% of aa sequence in this

mplete non-structural genome regions of Enterovirus A (excluding

(a total of 162 strains) were aligned based on the putative protein

rected) distances were calculated with MEGA 4.0 [68]. The y-axis



dataset, which provides clear delimitation of thespecies. In the capsid-encoding genome regionrecombination similarly conserves the (sero)typesequence by shuffling genomes of the same type,and prevalence of intratypic recombination in theP1 genome region might currently be under-estimated due to limited sampling of isolates ofthe same serotype. Therefore, in a two-way process,restrained RNA diversity within species permitsrecombination, which, in turn, limits diversifica-tion of the global (worldwide) gene pool of anestablished species. Therefore, recombination isboth a key property of picornavirus species and themeans to maintain them.

IMPACT OF RECOMBINATION ONCLASSIFICATION OF PICORNAVIRUSESThe International Committee on Virus Taxonomy(ICTV) terms picornavirus species as viruses with<70% amino acid identity in P1 and <70% aminoacid identity in the 2Cþ 3CD [28]. This definition isattractively robust, yet it carries limited biologicalsense. In general biology, species are generallytermed as ‘organisms able to interbreed andproduce fertile offspring’. This definition is uni-versal because the possibility of sexual procreationimplies a common gene pool and restricteddivergence within a species. Recombination inenteroviruses could be thought of as an analogue ofsuch reproductive strategy in higher organisms.Recombination in picornaviruses is strictly con-fined to groups that in most cases correspond to theestablished species. It was suggested previouslythat the possibility of natural recombination couldbe used as an additional species criterion inenteroviruses [19]. Based on absence of recombina-tion between mouse and human cardioviruses,human Saffold-like cardioviruses and TMEV couldbe identified as distinct species [55]. Similarly, mostknown human parechoviruses can be seen as a solespecies, as they freely recombine with each other.

There are, however, groups of picornavirusesthat obviously belong to a certain species but do notrecombine with other representatives of the samespecies or do so very rarely. For example,parechovirus type 3 is not remarkably distant fromother parechoviruses, but it does not recombinewith other types [45]. Such genetic isolation couldeventually lead to emergence of a new species.Bayesian phylogenies in different genome regionsshowed that the most recent common ancestor of


the known HPeV-3 isolates dates back only about30 years. It is likely that HPeV-3 is at an earlystage of a path to speciation. When viruses stoprecombining with the global gene pool, they beginto diverge and would eventually form a newspecies. Reasons for such events could be a changeof the cellular receptor used for viral entry, changeof host species, geographic isolation, etc. Anotherexample of picornaviruses in speciation could be anumber of simian enteroviruses that formallybelong toHEV-A species [31], but do not recombinewith human viruses andwould eventually separateinto distinct species. SVDV stopped recombiningwith HEV-B species after the host switch to pigs[33] and would most likely drift away from theparental virus and found a distinct species withinseveral decades. Three serotypes of HEV-C species,CVA1, CVA19, CVA22, are monophyletic through-out the genome with no evidence of recombinationwith other HEV-C members [13]. These virusesdiffer from other HEVs by inability to grow in cellculture, probably due to use of a different receptor.One could speculate that these viruses are also on apath of forming a new species.

The distribution of recombination events overthe genome inmost picornavirus genera has certainregularities. Recombination hotspots are found inall species on the borders of the capsid-encodinggenome region. These parts of the genome encodenon-structural proteins 2A, 2B and L, and struc-tural protein VP4. Proteins 2A, 2B and L are themost diverse proteins among picornaviruses. Theyhave variable size and function in differentpicornaviruses, and they are completely absentin some genera (Figure 1). VP4 is an internalstructural protein that is not tightly integrated inthe capsid structure, and it is absent in parecho-viruses and kobuviruses. It is likely that modularevolution of genome fragments encoding capsidand the replication complex proteins increasedevolutionary flexibility around these hotspots,which in turn could lead to emergence of diverse2A, 2B and L proteins.

CONCLUSIONThis review covers only recombination in thefamily Picornaviridae. There is extensive evidence ofrecombination in order Picornavirales (formerpicorna-like viruses) reviewed in Reference [5].Modular evolution and a global gene pool modelwere suggested for many other viruses. In small



DNA viruses, recombination patterns resemblethose in picornaviruses, with a preserved cassetteof capsid genes and recombination hotspotsbetween genome regions encoding structural andnon-structural genome regions [65]. Frequentrecombination and modular evolution weresuggested in adenoviruses [66], traditionallythought to be stable and conserved DNA viruses.It seems likely that frequent recombination and aglobal gene pool are common to almost all virusesthat feature global prevalence, notable diversityand common co-infection, which comprisesmost ofthe respiratory and gastrointestinal viruses. There-fore, most of the conclusions of this review can beextended to other virus families.

Frequent recombination in picornaviruses, com-bined with global and highly dynamic epidemiol-ogy, creates a global gene pool that can be observedas species. Such population dynamics of picorna-viruses might be confusing for an epidemiologist,but is well known in the world of phages that alsoexist as a global gene pool [67]. Unlike phages,human picornaviruses are commonly sampled andsequenced around the world for epidemiologicalstudies. Therefore, independent evolution of geneson a worldwide scale can be conveniently studiedon a time range of years and even months. Thismakes human picornaviruses an ideal model for amanageable analysis of the lateral drift of geneticinformation that played a major role in emergenceand development of life on Earth.The author is grateful to Dr J.F. Drexler (BonnUniversity), Prof. V.A. Lashkevich, Dr A.P. Gmyl(Chumakov Institute of Poliomyelitis, Moscow)and the anonymous reviewer for helpful remarksand suggestions on the manuscript.

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