Virus Genes 16:1, 13±21, 1998 # 1998 Kluwer Academic Publishers, Boston. Manufactured in The Netherlands. Origin and Evolution of Viruses JOHN HOLLAND1* & ESTEBAN DOMINGO2 1 Department of Biology and Center for Molecular Genetics, University of California, San Diego, La Jolla, CA 92093±0116 USA E-Mail: email@example.com. 2 Centro de Biologia Molecular Severo Ochoa Universidad Autonoma de Madrid, Cantoblanco, 28049 Madrid, Spain E-Mail: firstname.lastname@example.org.Virus Origins in the cases of bacteriophage Mu (which is both a virus and a transposon) and retroviruses (which areThe origin(s) of viruses can not be known with retrotransposons containing a functional envelopecertainty. PCR and other sensitive molecular techni- gene). As Temin pointed out (10), non-viral retroidques will reveal some viral genome sequences from elements can become retroviruses only when suchthe relatively recent past, but very ancient viral retrotransposing protoviruses acquire envelope genesgenomes will remain a matter for speculation. from another viral or cellular source by recombina-Numerous theories have been advanced regarding tion. DNA viruses and (non-retrovirus) RNAvirus origins (reviewed in 1) and all necessarily riboviruses may also arise by recombinational (orinvolve speculation. However, comparative sequence reassortment) reshuf¯ing of cellular and viral oranalysis strongly suggests that both RNA (2) and plasmid/episome/transposon mobile element genes.DNA (3) viruses have deep, archaic evolutionary Botstein (11) theory of modular evolution of DNAroots both for genome structural organization and as viruses is quite plausible. It envisions virus evolutionregards certain genomic and protein domains. It is by recombinational arrangement of interchangeablealso clear that both DNA and RNAviruses can emerge genetic elements or modules. The advantage of suchand evolve by a variety of mechanisms including modular evolution is obvious. It allows virus genes,mutation, recombination and reassortment. This can protein domains, regulatory systems, etc. to evolveinvolve point mutation, insertions and deletions, independently under a wide variety of selectiveacquisition or loss of genes (and gene domains, or conditions. Thus, one module might have undergonesets of genes), rearrangement of genomes and its most recent evolution as part of an integratedutilization of alternate reading frames or inverted episome, another as part of a transposon, a third as areading frames (1±6). plasmid element, yet another as part of a cellular gene Recombination can create new viruses by cap- or an integrated defective virus, etc. Such modularturing genes or gene segments or sets of genes either mobility obviously can relax evolutionary constraintsfrom cellular nucleic acids or from other viruses. The which would prevail if all were required to co-evolvepresence of cellular genes within virus genomes has within a single genomic unit. Of course, it will be anlong been recognized (5). Likewise, the resemblance extremely rare event which could bring about aof viruses to plasmids, episomes and other mobile fortuitous compatible recombination of indepen-DNA or RNA replicons such as transposons or dently-evolving modules to create a new virusretrotransposons is obvious (1,7±9). The only clear having good biological adaptive capacity. Butdistinction between many such mobile elements and signi®cant virus emergences are likewise extremelyviruses is the maturation of the latter within capsids rare occurrences and the probabilities for emergence(and envelopes) to affect ef®cient transmission and of a drastically different virus solely by mutationaltarget cell receptor speci®city. This is well-illustrated changes within a single genome are generally orders
14 Holland and Domingoof magnitude less probable. Sequence space as date from great antiquity and continue to the present.elaborated by Eigen and colleagues (12) has incom- Presently, of course, nearly all new viruses emerge viaprehensibly vast dimensions, and distant, previously evolution of old viruses. This is compatible with theunexplored regions of sequence space can best be deep evolutionary trees deduced for both DNA andreached (and mutationally explored) by the evolu- RNA viruses (4) and with the long-recognizedtionary leaps which recombination or reassortment capacity of viruses to acquire genetic elements fromafford. See Kauffman (13) for detailed discussion of host cell nucleic acids and form other mobilethis point. Finally, it should be emphasized that replicons (1).ordinary RNA viruses (riboviruses), in additional toDNA viruses and retroviruses, can undergo suchmodular evolution via RNA recombination (and Virus Evolutionreassortment). The essence of all viruses is obligateintracellular parasitism coupled with the capacity for The evolution of existing viruses, as for all livingintimate genetic interactions with the DNA and RNA things, proceeds via a variety of mechanismsof their hosts and of cohabiting mobile elements. including mutation, recombination, reassortment and The nature of the earliest viruses can never be environmental selection. Space limitations preventdetermined, but it is likely that they arose very early extensive discussion of virus evolution in this shortduring the evolution of life on earth. It seems review, so only major points will be discussed here.extremely likely that elemental life forms involved For an excellent recent overview of the molecularRNA replicons (14) and these might have borne basis of virus evolution, see (4). More conciseresemblance to present-day RNA replicons such as coverage is provided in review articles (1,18±22).viroids, virusoids or viruses. In fact, Robertson (15)has suggested that very early, primitive autono-mously-replicating, self-cleaving RNA replicons RNA Virus Mutation Rates are Very Highakin to present-day viroids might have acquiredadditional genes to form conjoined replicons which It is now clear that all or nearly all RNA viruses havelater evolved into mosaic DNA-based entities. extremely high mutation rates (18±23). Mutation ratesHepatitis D virus was suggested as a present day at individual base sites may vary considerably, butexample of such a conjoined viroid-like RNA average nucleotide base misincorporation rates are ofreplicon. It contains an open reading frame encoding the order of 10 À 4 to 10 À 5 (reviewed in 21±23). Thisthe delta antigen protein joined to the viroid-like results in the generation of quasispecies mutantdomain. Recently (16), it was shown that liver cells swarms even when the virus population has justexpress a cellular homolog of the delta antigen, arisen from a clone (21,22,24). A clonal quasispeciessuggesting that hepatitis D virus may have arisen by virus population is a diverse mixture of virus mutantsthe capture of a cellular RNA transcript by a viroid- differing from each other at one or several genomiclike RNA. A copy choice template transfer sites, and can be envisioned as a cloud in sequencemechanism was proposed for the recombinational space. A consensus sequence will represent thecapture event. Robertson (15) suggested that such average sequence at each genome site and theevents occurring early in the primitive RNA world master sequence(s) represent the most ®t member(s)could later have given rise to mosaic DNA modules, of the swarm in any particular de®ned selectiveand might even be responsible for the present environment. When the selective environmentwidespread prevalence of split genes and introns and changes the master sequence(s) and the overallRNA-catalyzed cleavage and ligation splicing sys- composition of the quasispecies swarm will alsotems (17). In general, it is quite plausible that not only change.viroid-like, but plasmid-like transposon-like, retro- Obviously, the generation of quasispecies mutanttransposon-like and virus-like autonomously- swarms can provide RNA viruses with great adapt-replicating RNA and DNA elements (replicons) ability under conditions in which there ishave been intimately involved in nearly all evolution environmental change, and in complex mammalianof life on earthÐboth in precellular and cellular eras. hosts, viruses always encounter changing conditionsTherefore, it is probable that the origins of viruses (e.g., different cell types, in¯ammatory responses,
Origin and Evolution of Viruses 15immune responses, fever temperatures, interferons, ef®ciently in two very disparate hosts; vertebratesetc.). It should be emphasized that, whereas the most- and invertebrate insects (31). Despite this relativelyadapted master sequence(s) and closely-related var- slow rate of evolution, alphaviruses such as easterniants will be the most abundant and most important equine encephalitis virus (and other arboviruses)variants under rather constant environmental condi- undergo signi®cant evolutionary change over thetions, the opposite will be true under rapidly changing centuries. For example, it was estimated that theconditions (e.g., adaptation to a new mammalian host North and South American antigenic varieties ofor a new arthropod vector). Variants at the periphery eastern equine encephalitis virus diverged about 1000of the quasispecies mutant distribution (i.e., those years ago and the two South American groupsmost distantly-related to the previous master diverged about 450 years ago (30). Venezuelan andsequence) will usually offer the best opportunity for eastern equine encephalitis alphavirus complexesrapid adaptation to the new conditions. Selected diverged about 1400 years ago (30) while the Oldperipheral variants from the previous mutant distribu- and New World alphavirus groups diverged roughlytion will frequently also be peripheral variants in the 2000 to 3000 years ago (32). Even today, newnew distribution as the quasispecies moves through epidemic/epizootic strains of Venezuelan encephalitissequence space to optimize adaptability in the new emerge from enzootic strains in South America byenvironment and generate new master sequences. The rather minor mutational change (33). Finally, thevery essence of the quasispecies theory of Eigen, western equine encephalitis group was estimated toBiebricher and colleagues (12,24±27) is the broad have emerged more than 1000 years ago (before thereach through sequence space which is provided by North and South American equine encephalitis virusRNA virus replicase error rates poised at the threshold divergence) by a very rare recombination eventof error catastrophe. Finally, it should be noted that, as between eastern equine encephalitis virus and ain all evolution, rapid emergences of new RNA sindbis virus-like progenitor (6,18,34).variants are counterbalanced by rapid extinctions of Another example of slow versus rapid evolutionothers. can be observed with vesicular stomatitis virus (VSV) in both laboratory and natural settings. VSV Indiana serotype has been observed to undergo extremelyExtremely High Mutation Rates Do Not rapid evolution under conditions of persistent infec-Necessitate Rapid Evolution tion in cell culture and relative genomic stasis under conditions of repeated dilute passages in the sameAlthough, it is intuitively obvious that high rates of cells (35). In nature, VSV in its enzootic focus inRNA virus mutation facilitate rapid evolution, it Panama has undergone very little evolution overseems counterintuitive that RNA viruses sometimes recent decades (36). In contrast extensive evolutioncan exhibit rather long periods of relative evolu- was observed for strains isolated farther north. Thetionary stasis. In general, RNA viruses evolve rapidly farther north the strains were isolated, the greater wasbut rates can vary considerably, and relative stasis is the sequence diversity from the genetically stablenot uncommon. For example, evolutionary rates for ( presumed ancestral) strains in enzootic foci inmany RNA viruses can be as high as 10 À 2 to 10 À 3 Panama and Costa Rica (36). The greatest divergencebase substitutions per nucleotide site per year was found in strains from the extreme northern range(1,6,18,20±23), but rates of evolution of arthropod- of VSV in the United States. Thus, there is aborne viruses can be orders of magnitude slower. geographic clock rather than a molecular clock asTransovarial passage of the Phlebovirus toscana virus would be expected from neutral evolutionary theory.in sand¯y vectors showed extreme genome stability This apparent punctuated equilibrium evolution wasduring 2 years transmission time and over 12 sand¯y postulated to be due to different selective ecologicalgenerations (28). Likewise, alphaviruses in the eastern factors operating to drive virus evolution in diverseequine encephalitis complex evolved at rates nearly as geographic areas and different insect vector/hosts arelow as 10 À 4 base substitutions ®xed per site per year probably important among these (36). These extre-(29,30). This low rate of evolution occurred despite mely unequal rates of evolution within a single virusnormally high rates of mutation and was attributed to species and serotype dramatically con®rm the role ofstabilizing selection for the ability to replicate selection in driving virus evolution. They also
16 Holland and Domingodramatically emphasize the fact that high (and populations are small in asexual populations, thereprobably rather constant) mutation rates can be can be an inexorable accumulation of deleteriousconsistent with both rapid rates of evolution (mutation mutations leading to a ratchet-like decline in®xation) or with evolutionary stasis. Relative stasis replicative ®tness. Chao (43) convincingly demon-(equilibrium) is favored under more constant selective strated the operation of Mullers ratchet in theconditions in the environment-precisely as is pre- tripartite RNA bacteriophage f6. Chao et al. (44)dicted by quasispecies theory (see section above). also showed that sexual crossing could often reverse Another remarkable example of a single virus the effects of Mullers ratchet. Quantitation of ®tnessspecies exhibiting either evolutionary stasis or losses during repeated small population transfersextremely rapid evolution is provided by the thorough ( plaque-to-plaque genetic bottleneck transmissions)extensive studies of Webster and coworkers (20,37± of VSV and food-and-mouth disease virus con®rmed39) of in¯uenza virus in natural avian hosts or in that variable, stochastic, often-profound ®tnessmammalian hosts including humans. In aquatic wild decreases occur rather regularly (45±49). Clearly,birds, in¯uenza virus is apparently completely- genetic bottlenecks have the capacity to disturb virusadapted to intestinal replication and shedding with adaptive equilibrium and thereby to drive stochasticno signs of disease and with very little selection for evolutionary changes. The number of virus particlesevolutionary change. Rapid evolution is the norm which constitute an effective genetic bottleneck canwhen in¯uenza viruses emerge into mammalian hosts vary greatly from only several particles to tens of(20,37,38). Immunity and other selective factors particles depending upon initial virus populationapparently drive the extreme rates of in¯uenza virus ®tness (50,51).evolution exhibited during adaptation to mammals. The opposite effect on ®tness occurs duringOverall, the work of Webster and colleagues indicates repeated transfers of very large numbers (105 to 106)that ducks and other waterfowl are the original hosts of infectious virus particles. Under these large dosefor in¯uenza viruses. Rare genome segment reassort- transmission conditions, regular exponential increasesment events or transfers of entire in¯uenza virus in virus replicative ®tness occur and previous ®tnessgenomes initiate emergence into mammalian species, decreases due to Mullers ratchet are reversedbut it is the destabilizing selective forces in mammals (48,50,51). Unquestionably, virus population trans-which drives the ensuing rapid evolution. This is mission size can affect virus evolution veryanother good example of punctuated equilibrium in profoundly, and this inevitably must also occurvirus evolution. There are theoretical reasons (based during natural virus outbreaks. Transmission of largeupon expected movements between adaptive peaks in doses of infectious virus particles often occurs duringadaptive landscapes) to expect that evolution will transmissions involving close contact (e.g., sexual,frequently exhibit punctuated equilibrium (40). Plant kissing); during transfusions or other medical/dentalviruses, such as the tobamoviruses can also exhibit blood/tissue transmission; intravenous drug abuse,genetic stability for long periods, again due to strong some insect vector or animal bite transmissions; andenvironmental selective pressures restricting quasis- during some very close respiratory droplet transmis-pecies diversi®cation (41). sions, some fecal-oral transmissions, etc. Genetic bottleneck transmissions inevitably occur during many rather distant virus transmissions duringEffects of Virus Population Transmission Size on respiratory droplet inhalations. This is clear fromSelection, Fitness and Evolution quantitative studies of both experimental and natural virus aerosol transmissions (52±54). After sneezingWhenever viruses become extremely-well-adapted to and coughing of infected people in a room, the volumehost environments and evolutionary stasis is reached, of room air which must be sampled to obtain a singlethis equilibrium obviously can be upset readily infectious particle can be very large (53,54). For( punctuated) by host or vector switching or by drastic example, 15 men in bed infected with adenovirus typeenvironmental changes. Another, less obvious 4 and coughing frequently in a barracks room led tomechanism involves changes in the size (dose) of recovery of one tissue culture infective unit per 2820virus particle transmission. In 1964, Muller (42) sq. ft. (54). Thus, genetic bottleneck transmissionspostulated that whenever mutation rates are high and must be frequent and unavoidable for respiratory
Origin and Evolution of Viruses 17viruses although large population transfers must also account for 50% or more of the plants nuclearoccur often during close contact. Similar considera- DNA! Large blocks of reiterated retrotransposonstions apply to fecal-oral spread, spread from inserted within each other are found in gene-inanimate objects (fomites) and from insect vector containing regions as intergenic segments (56,57).transmissions (55). Whenever large virus population Clearly, these are involved in determining genetransmissions occur repeatedly, selection can operate expression, genome size and genome organization,repeatedly to select the best of the best of the best . . . so that the distinction between host and sel®shin terms of virus ®tness in a constant host species. parasitic genes is blurred beyond recognition.Conversely, repetitive genetic bottleneck transmis- Although RNA riboviruses cannot interact withsions interrupt selective forces and allow stochastic host DNA genomes as directly, nor as frequently as dochanges in ®tness and in evolutionary directions. DNA viruses and retroviruses/retroelements, RNAThese stochastic changes will usually be in the recombination is equally common and importantdirection of ®tness loss (and perhaps loss of virulence) among them as evolutionary events. The mechanismsbut rarely, by chance, the converse will be true. and importance of recombination in riboviruses andVirulence is a multifactorial, multigenic trait which retroviruses are reviewed in (58,59). Riboviral RNAmay or may not correlate with replicative ®tness and recombines with both cellular and viral RNAs andtransmission ef®ciency. The major insight to be acquisition of genes and gene segments from both cangained from experimental studies of virus ®tness is be very important in ribovirus evolution andthat RNA viruses are phenotypically quasispecies as emergence. Homologous recombination occurs fre-well as genetic quasispecies. Thus, all phenotypic quently during every replication of most positivecharacteristics, including virulence, will be highly sense riboviruses (58,60) but is extremely rare amongvariable among the numerous mutants present in a negative sense riboviruses in which non-homologouscomplex quasispecies swarm. Therefore, chance recombination events usually generate defectivesampling events such as genetic bottlenecks may genomes (some quite bizarre), most of which areprofoundly affect virulence traits (and other traits) dependent upon non-defective helper viruses forduring an epidemic. Likewise, repetitive large replication (61). Very rarely, helper-dependent,population transmissions can preserve and enhance bizarre, defective RNA virus genomes are probablyvirulence or other traits which had been sampled by involved in major virus evolutionary events via RNAchance during earlier genetic bottleneck events. This recombination with non-defective helper viruses.can, of course, in¯uence disease severity and outcome Because their helper viruses provide all vitalin infected individuals and in small local host cohorts replication functions, defective viral genomes areinfected by such sampling of the quasispecies swarm. largely unconstrained by selective forces and can undergo extremely rapid, massive evolution. Thus, they could (very rarely) donate extensively-mutatedRecombination, Reassortment and Gene and rearranged genome segments back to the helperDuplication in Virus Evolution viruses from which they arose (61). Among the segmented-genome viruses, reassort-As outlined in the chapters on virus origins, ment of segments provides a ready mechanism forrecombination has long been recognized as a central generating new viruses. The best-known examples, ofmechanism in the evolution of DNA bacteriophages course, involve the periodic antigenic shifts ofand/or DNA viruses and retroviruses of animals and in¯uenza A viruses which usually occur at multi-plants. Recombination with, and insertion into, and decade intervals to initiate new human pandemicsexcision from, cellular DNA allows intimate genetic (37±39). The gene segment reassortment events whichinteractions with host genomes, episomes and cause emergence of new in¯uenza A viruses are veryplasmids. Just as the frequent acquisition of cellular rare events because only certain permutations of aviangenes can help shape virus evolution, so can the and mammalian gene segments will be highlyfrequent acquisition of virus and retroelement genes infectious and ®t, and because reassortment requireshelp to shape host evolution. A remarkable example is rare dual infection (by appropriate progenitor viruses)provided by maize in which many dozens or even of appropriate mixing vessel hostsÐmost oftenmany hundreds of diverse retroelement families probably swine or humans (37±39). Once a ®t new
18 Holland and Domingoreassortant emerges into the human population very generally evolve to achieve and maintain optimalrapid evolution ensues (37±39). Reassortment events function. They often tend to produce inapparent andare important in the evolution of many other silent latent infections, to co-evolve slowly with theirsegmented genome RNA viruses. For example, hosts over geologic time periods, and even tobunyaviruses can evolve by reassortment in doubly- in¯uence the evolution of their host species (68,70±infected mosquitoes (62) and naturally-occurring Sin 73). Nevertheless, many DNA viruses can exhibitNombre hantavirus reassortants have been observed considerable genetic plasticity (74) and this can bein Peromyscus deer mice in Nevada and Eastern manifested via antiviral drug resistance and otherCalifornia (63). No reassortants were observed clinical problems. This is not surprising because DNAbetween Sin Nombre and other hantaviruses indi- viruses can have host recombination systems avail-genous to their region, suggesting that reassortants able to them in addition to intrinsic viral mechanisms.between distantly-related hantaviruses are rare or non- Also, DNA ®delity is limited for viruses whichviable, and/or that host species speci®cities greatly replicate via single-stranded DNA because mismatchlimit reassortment. Again, generation of ®t reassor- repair/excision repair systems are unavailable fortants between distantly-related and different-host- single-stranded DNA genomes. But, even bacter-adapted virus strains is generally a rare event in iophage T7, a classic double-stranded DNA virusnature. exhibited rapid evolution of replicating ®tness in A major mechanism observed in the evolution of single plaques (75). It should be noted that some DNAall life formsÐgene duplicationÐis sometimes viruses such as the canine/feline parvoviruses evolveimportant in virus evolution. For example, beet in nature at least as rapidly as the slower-evolvingyellows virus, a ®lamentous RNA virus, contains a RNA viruses and can change host species speci®citiescoat protein gene duplication (64) and two rabies- very readily as well (76). This is in marked contrast torelated rhabdoviruses, Adelaide river virus (65) and the primate papillomaviruses, for example. Van Ranstbovine ephemeral fever virus (66) each contain two et al. (77) estimated primate papillomavirus mutationconsecutive glycoprotein genes of differing size and rates to be of the order of 3 Â 10 À 8 base substitutionssequence. Both glycoprotein genes are expressed in per site per year in the EG gene. This is only about 20±each of these viruses; via monocistronic mRNAs in 30 times faster than the rate of evolution of theirthe case of bovine ephemeral fever virus and primate host species and about a million-fold lowerpolycistronic mRNAs for Adelaide virus. Gene than rates of evolution of the most rapidly evolvingduplication of this kind occurs by recombination RNA riboviruses (29±35,78).events, probably via intra- or inter-molecular copychoice replicase leaps (58,67). Finally, a very simple, effective mechanisms for Implications of RNA Virus Quasispecies forcreation of new virus gene products involves acquired Disease and Disease Emergenceusage of alternative reading frames of an existing geneto create overlapping genes. This overprinting Some investigators have stated that quasispeciesmechanism is common in virus evolution as outlined mutant swarms are not really necessary for diseasein the review of Gibbs and Kease (4). processes during RNA virus infections. Of course, this could be argued quantitatively in terms of the minimal mutation rate which quali®es to produce a quasis-Do DNA Viruses Evolve as Quasispecies? pecies. However, this misses the essence of RNAvirus biology. RNA viruses have been the most abundantDNA viruses generally do not form complex and successful parasites since the appearance ofquasispecies mutant swarms to the extent that RNA cellular life (see the ®rst chapter) and this has beenviruses do because they generally have genomic achieved by maintaining error rates very near the errormutation rates about 300-fold lower than those of threshold (12). This allows maximal variability andRNA riboviruses and roughly 30-fold lower than adaptability (21±27) This great adaptability allowsretroviruses (23,68). Proofreading and mismatch ®tness to be increased rapidly in changing environ-repair (69) of DNA can provide ®delity for even ments and the intact animal, human, plant or insectvery large DNA virus genomes. Hence, DNA viruses vector organism always confronts invading microbes
Origin and Evolution of Viruses 19with multiple, challenging and changing environ- remember that myocarditis due to Coxsackie virusesments. RNA virus quasispecies frequently undergo is rare relative to the number of human infections.major or minor changes in composition in response to Virulence is a trait which is seldom selected exceptin¯ammatory and immune responses (20,37,38,79) to when it correlates with replicative ®tness. Immunedifferent host cell types within individual infected de®cits arising from selenium de®ciency regularlyorganisms, to widely differing conditions in verte- allowed expansion of the Coxsackievirus B3 quasis-brate versus insect vector hosts (80), to antiviral drug pecies mutant swarm and colonization of myocardialtreatments (81,82), to inadequate vaccine programs, cells to cause disease. The cardiovirulent variantsetc. Different subsets of the lymphocytic choriome- selected in heart muscle cells established a newningitis virus quasispecies swarm are involved in quasispecies distribution which was cardiovirulent forlymphoid cell infection with immune suppression as normal mice after it was deliberately isolated from thecontrasted with neuronal cell infection with runting total whole animal wild type quasispecies swarm. It issyndrome (growth hormone de®ciency syndrome) beyond question in this outstanding study that it is(83,84). those quasispecies subsets normally buried within the Perhaps the involvement of quasispecies in disease total circulating quasispecies population which haveis best illustrated by the propensity of polioviruses to the potential to cause viral myocarditis (89,90). This iscause paralytic disease in a small percentage of likely a rather typical situation with regard to RNAinfected individuals (60), and for some Coxsackie virus disease potential.viruses to cause cardiomyopathy in a small percentage Finally, the role of RNA virus quasispecies inof infected humans. Microevolution of the quasis- future emerging diseases of humans, domesticpecies population present in the type 3 Sabin oral animals and crops is obvious. Most emerging humanpoliovirus vaccine seed stocks (85) can cause diseases in recent years have been RNA virusparalytic disease in a very small percentage of vaccine diseases; from AIDS to Ebola hemorrhagic fever torecipients and can even initiate outbreaks of polio- hantavirus pulmonary syndrome. Most new ormyelitis in unvaccinated populations (60,86). Clearly, emergent virus diseases in the future will also bethe quasispecies nature of the vaccine seed stocks and RNA viruses because of the rapid evolution potentialof their progeny is responsible for this rare but of their quasispecies. This trend will be accelerated sounfortunate disease complication of an otherwise long as the human population continues to expandexcellent vaccine. An endemic cardiomyopathy exponentially. For example, the recent rapid growth ofaffecting thousands in China has been associated human populations in tropical areas has been matchedwith both selenium de®ciency and isolation of by an equally-rapid increase in dengue fever, dengueCoxsackieviruses from patients (87,88). Beck et al. hemorrhagic fever and dengue shock syndrome, andin a remarkable study of selenium de®cient mice (89) by an increasing rate of evolution of dengue virusesshowed that infection by a normally non-virulent (91±93). All of us and our domestic animals (94) areclone of Coxsackie B3 virus induced signi®cant potential incubators for the rapid exploration ofmyocarditis in the Se-de®cient mice, and virus previously-unexplored sequence space by evolvingrecovered from the hearts of these myocarditic mice RNA viruses. Sequence space is a v-dimensionalregularly caused myocarditis in normal Se-adequate hypercube in which v is the genome length in bases ormice! Complete sequence analysis of the recovered base pairs (12). For a 10 kb RNA virus genome theremyocarditic strain of Coxsackievirus B3 revealed six are 410,000 sequence permutations and combinationsspeci®c nucleotide changes, all of which had appeared which must be explored before all viable and adaptivein four separate isolates from the initial myocarditic virus sequences have been testedÐeven if we assumemouse examined and from 3 individual follow-up a constant genome size restriction (which nevermice (89). This study agrees with the sequence studies happens). There is not enough space-time in countlessof Chapman et al. (90) who also found that speci®c imaginable universes to test even a minuscule fractionchanges at these six nucleotide sites are associated of such incomprehensibly vast dimensions. Hence,with the cardiovirulent phenotype (89,90) of new sequence spaces will be explored increasinglyCoxsackievirus B3. Nothing could more clearly rapidly in future decades. New RNA viruses andillustrate the importance of quasispecies mutant new diseases will emerge with increasing rapidity asswarms in RNA virus disease. It is important to long as human population growth remains in an
20 Holland and Domingoexponential phase. Planet earth has a ®nite human Animal Virus Genetics. Academic Press, New York, 1981,carrying capacity, but its dimensions are uncertain and pp. 363±384. 12. Eigen M. and Biebricher C.K. in Domingo E., Holland J.J., andwill be affected by human choices concerning quality Ahlquist P. (eds), RNA Genetics. CRC Press, Inc., Boca Raton,of life, economics, environmental values, etc. (95). 1988, pp. 211±245.But beyond human choice are the evolutionary paths 13. Kaufmann S.A., The Origins of Order. Self Organization andto be followed by exponentially-increasing quasispe- Selection in Evolution. Oxford University Press, New York, 1993.cies swarms of RNA viruses exploring humans as 14. Gesteland R.F. and Atkins J.F. (ed.), The RNA World, Coldhosts. Virus evolution is stochastic and unpredictable Spring Harbor Lab Press, Plainview, NY, 1993.(35,96), but increasing numbers of human outbreaks 15. Robertson H.D. in Holland J.J. (ed.), Genetic Diversity of RNAare inevitable. They will occur; they should be Viruses. Curr. Top. Microbiol. Immunol., 1992.anticipated and there should be reasonable preparation 16. Brazas R. and Ganem D., Science 274, 90±94, 1996.for some unpleasant outbreaks. The human carrying 17. Robertson H.D., Science 274, 66±67, 1996. 18. Strauss J.H. and Strauss E.G., Annu Rev Microbiol 42, 657±capacity of our planet may ultimately be determined, 683, 1988.not by human choice, but by RNA virus evolution. 19. Zimmern D. in Domingo E., Holland J.J., and Ahlquist P. (eds), RNA Genetics. CRC Press, Boca Raton, 1988, pp. 211±240. 20. Webster R.G., Bean W.J., Gorman O.T., Chambers T.M., and Kawaoka Y., Microbiol Rev 56, 152±179, 1992.Acknowledgments 21. Domingo E. and Holland J.J. in Morse S. (ed.), Emerging Viruses. Oxford University Press, 1994, pp. 203±218.Work in La Jolla, CA was supported by NIH Grant 22. 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