Origin and Evolution of Viruses

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

1Department of Biology and Center for Molecular Genetics, University of California, San Diego, La Jolla, CA 92093±0116 USAE-Mail: [email protected].

2Centro de Biologia Molecular Severo Ochoa Universidad Autonoma de Madrid, Cantoblanco, 28049 Madrid, SpainE-Mail: [email protected].

Virus Origins

The origin(s) of viruses can not be known with

certainty. PCR and other sensitive molecular techni-

ques will reveal some viral genome sequences from

the relatively recent past, but very ancient viral

genomes will remain a matter for speculation.

Numerous theories have been advanced regarding

virus origins (reviewed in 1) and all necessarily

involve speculation. However, comparative sequence

analysis strongly suggests that both RNA (2) and

DNA (3) viruses have deep, archaic evolutionary

roots both for genome structural organization and as

regards certain genomic and protein domains. It is

also clear that both DNA and RNAviruses can emerge

and evolve by a variety of mechanisms including

mutation, recombination and reassortment. This can

involve point mutation, insertions and deletions,

acquisition or loss of genes (and gene domains, or

sets of genes), rearrangement of genomes and

utilization of alternate reading frames or inverted

reading frames (1±6).

Recombination can create new viruses by cap-

turing genes or gene segments or sets of genes either

from cellular nucleic acids or from other viruses. The

presence of cellular genes within virus genomes has

long been recognized (5). Likewise, the resemblance

of viruses to plasmids, episomes and other mobile

DNA or RNA replicons such as transposons or

retrotransposons is obvious (1,7±9). The only clear

distinction between many such mobile elements and

viruses is the maturation of the latter within capsids

(and envelopes) to affect ef®cient transmission and

target cell receptor speci®city. This is well-illustrated

in the cases of bacteriophage Mu (which is both a

virus and a transposon) and retroviruses (which are

retrotransposons containing a functional envelope

gene). As Temin pointed out (10), non-viral retroid

elements can become retroviruses only when such

retrotransposing protoviruses acquire envelope genes

from another viral or cellular source by recombina-

tion. DNA viruses and (non-retrovirus) RNA

riboviruses may also arise by recombinational (or

reassortment) reshuf¯ing of cellular and viral or

plasmid/episome/transposon mobile element genes.

Botstein (11) theory of modular evolution of DNA

viruses is quite plausible. It envisions virus evolution

by recombinational arrangement of interchangeable

genetic elements or modules. The advantage of such

modular evolution is obvious. It allows virus genes,

protein domains, regulatory systems, etc. to evolve

independently under a wide variety of selective

conditions. Thus, one module might have undergone

its most recent evolution as part of an integrated

episome, another as part of a transposon, a third as a

plasmid element, yet another as part of a cellular gene

or an integrated defective virus, etc. Such modular

mobility obviously can relax evolutionary constraints

which would prevail if all were required to co-evolve

within a single genomic unit. Of course, it will be an

extremely rare event which could bring about a

fortuitous compatible recombination of indepen-

dently-evolving modules to create a new virus

having good biological adaptive capacity. But

signi®cant virus emergences are likewise extremely

rare occurrences and the probabilities for emergence

of a drastically different virus solely by mutational

changes within a single genome are generally orders

of magnitude less probable. Sequence space as

elaborated by Eigen and colleagues (12) has incom-

prehensibly vast dimensions, and distant, previously

unexplored regions of sequence space can best be

reached (and mutationally explored) by the evolu-

tionary leaps which recombination or reassortment

afford. See Kauffman (13) for detailed discussion of

this point. Finally, it should be emphasized that

ordinary RNA viruses (riboviruses), in additional to

DNA viruses and retroviruses, can undergo such

modular evolution via RNA recombination (and

reassortment). The essence of all viruses is obligate

intracellular parasitism coupled with the capacity for

intimate genetic interactions with the DNA and RNA

of their hosts and of cohabiting mobile elements.

The nature of the earliest viruses can never be

determined, but it is likely that they arose very early

during the evolution of life on earth. It seems

extremely likely that elemental life forms involved

RNA replicons (14) and these might have borne

resemblance to present-day RNA replicons such as

viroids, virusoids or viruses. In fact, Robertson (15)

has suggested that very early, primitive autono-

mously-replicating, self-cleaving RNA replicons

akin to present-day viroids might have acquired

additional genes to form conjoined replicons which

later evolved into mosaic DNA-based entities.

Hepatitis D virus was suggested as a present day

example of such a conjoined viroid-like RNA

replicon. It contains an open reading frame encoding

the delta antigen protein joined to the viroid-like

domain. Recently (16), it was shown that liver cells

express a cellular homolog of the delta antigen,

suggesting that hepatitis D virus may have arisen by

the capture of a cellular RNA transcript by a viroid-

like RNA. A copy choice template transfer

mechanism was proposed for the recombinational

capture event. Robertson (15) suggested that such

events occurring early in the primitive RNA world

could later have given rise to mosaic DNA modules,

and might even be responsible for the present

widespread prevalence of split genes and introns and

RNA-catalyzed cleavage and ligation splicing sys-

tems (17). In general, it is quite plausible that not only

viroid-like, but plasmid-like transposon-like, retro-

transposon-like and virus-like autonomously-

replicating RNA and DNA elements (replicons)

have been intimately involved in nearly all evolution

of life on earthÐboth in precellular and cellular eras.

Therefore, it is probable that the origins of viruses

date from great antiquity and continue to the present.

Presently, of course, nearly all new viruses emerge via

evolution of old viruses. This is compatible with the

deep evolutionary trees deduced for both DNA and

RNA viruses (4) and with the long-recognized

capacity of viruses to acquire genetic elements from

host cell nucleic acids and form other mobile

replicons (1).

Virus Evolution

The evolution of existing viruses, as for all living

things, proceeds via a variety of mechanisms

including mutation, recombination, reassortment and

environmental selection. Space limitations prevent

extensive discussion of virus evolution in this short

review, so only major points will be discussed here.

For an excellent recent overview of the molecular

basis of virus evolution, see (4). More concise

coverage is provided in review articles (1,18±22).

RNA Virus Mutation Rates are Very High

It is now clear that all or nearly all RNA viruses have

extremely high mutation rates (18±23). Mutation rates

at individual base sites may vary considerably, but

average nucleotide base misincorporation rates are of

the order of 10ÿ 4 to 10ÿ 5 (reviewed in 21±23). This

results in the generation of quasispecies mutant

swarms even when the virus population has just

arisen from a clone (21,22,24). A clonal quasispecies

virus population is a diverse mixture of virus mutants

differing from each other at one or several genomic

sites, and can be envisioned as a cloud in sequence

space. A consensus sequence will represent the

average sequence at each genome site and the

master sequence(s) represent the most ®t member(s)

of the swarm in any particular de®ned selective

environment. When the selective environment

changes the master sequence(s) and the overall

composition of the quasispecies swarm will also

change.

Obviously, the generation of quasispecies mutant

swarms can provide RNA viruses with great adapt-

ability under conditions in which there is

environmental change, and in complex mammalian

hosts, viruses always encounter changing conditions

(e.g., different cell types, in¯ammatory responses,

14 Holland and Domingo

immune responses, fever temperatures, interferons,

etc.). It should be emphasized that, whereas the most-

adapted master sequence(s) and closely-related var-

iants will be the most abundant and most important

variants under rather constant environmental condi-

tions, the opposite will be true under rapidly changing

conditions (e.g., adaptation to a new mammalian host

or a new arthropod vector). Variants at the periphery

of the quasispecies mutant distribution (i.e., those

most distantly-related to the previous master

sequence) will usually offer the best opportunity for

rapid adaptation to the new conditions. Selected

peripheral variants from the previous mutant distribu-

tion will frequently also be peripheral variants in the

new distribution as the quasispecies moves through

sequence space to optimize adaptability in the new

environment and generate new master sequences. The

very essence of the quasispecies theory of Eigen,

Biebricher and colleagues (12,24±27) is the broad

reach through sequence space which is provided by

RNAvirus replicase error rates poised at the threshold

of error catastrophe. Finally, it should be noted that, as

in all evolution, rapid emergences of new RNA

variants are counterbalanced by rapid extinctions of

others.

Extremely High Mutation Rates Do NotNecessitate Rapid Evolution

Although, it is intuitively obvious that high rates of

RNA virus mutation facilitate rapid evolution, it

seems counterintuitive that RNA viruses sometimes

can exhibit rather long periods of relative evolu-

tionary stasis. In general, RNA viruses evolve rapidly

but rates can vary considerably, and relative stasis is

not uncommon. For example, evolutionary rates for

many RNA viruses can be as high as 10ÿ 2 to 10ÿ 3

base substitutions per nucleotide site per year

(1,6,18,20±23), but rates of evolution of arthropod-

borne viruses can be orders of magnitude slower.

Transovarial passage of the Phlebovirus toscana virus

in sand¯y vectors showed extreme genome stability

during 2 years transmission time and over 12 sand¯y

generations (28). Likewise, alphaviruses in the eastern

equine encephalitis complex evolved at rates nearly as

low as 10ÿ 4 base substitutions ®xed per site per year

(29,30). This low rate of evolution occurred despite

normally high rates of mutation and was attributed to

stabilizing selection for the ability to replicate

ef®ciently in two very disparate hosts; vertebrates

and invertebrate insects (31). Despite this relatively

slow rate of evolution, alphaviruses such as eastern

equine encephalitis virus (and other arboviruses)

undergo signi®cant evolutionary change over the

centuries. For example, it was estimated that the

North and South American antigenic varieties of

eastern equine encephalitis virus diverged about 1000

years ago and the two South American groups

diverged about 450 years ago (30). Venezuelan and

eastern equine encephalitis alphavirus complexes

diverged about 1400 years ago (30) while the Old

and New World alphavirus groups diverged roughly

2000 to 3000 years ago (32). Even today, new

epidemic/epizootic strains of Venezuelan encephalitis

emerge from enzootic strains in South America by

rather minor mutational change (33). Finally, the

western equine encephalitis group was estimated to

have emerged more than 1000 years ago (before the

North and South American equine encephalitis virus

divergence) by a very rare recombination event

between eastern equine encephalitis virus and a

sindbis virus-like progenitor (6,18,34).

Another example of slow versus rapid evolution

can be observed with vesicular stomatitis virus (VSV)

in both laboratory and natural settings. VSV Indiana

serotype has been observed to undergo extremely

rapid evolution under conditions of persistent infec-

tion in cell culture and relative genomic stasis under

conditions of repeated dilute passages in the same

cells (35). In nature, VSV in its enzootic focus in

Panama has undergone very little evolution over

recent decades (36). In contrast extensive evolution

was observed for strains isolated farther north. The

farther north the strains were isolated, the greater was

the sequence diversity from the genetically stable

( presumed ancestral) strains in enzootic foci in

Panama and Costa Rica (36). The greatest divergence

was found in strains from the extreme northern range

of VSV in the United States. Thus, there is a

geographic clock rather than a molecular clock as

would be expected from neutral evolutionary theory.

This apparent punctuated equilibrium evolution was

postulated to be due to different selective ecological

factors operating to drive virus evolution in diverse

geographic areas and different insect vector/hosts are

probably important among these (36). These extre-

mely unequal rates of evolution within a single virus

species and serotype dramatically con®rm the role of

selection in driving virus evolution. They also

Origin and Evolution of Viruses 15

dramatically emphasize the fact that high (and

probably rather constant) mutation rates can be

consistent with both rapid rates of evolution (mutation

®xation) or with evolutionary stasis. Relative stasis

(equilibrium) is favored under more constant selective

conditions in the environment-precisely as is pre-

dicted by quasispecies theory (see section above).

Another remarkable example of a single virus

species exhibiting either evolutionary stasis or

extremely rapid evolution is provided by the thorough

extensive studies of Webster and coworkers (20,37±

39) of in¯uenza virus in natural avian hosts or in

mammalian hosts including humans. In aquatic wild

birds, in¯uenza virus is apparently completely-

adapted to intestinal replication and shedding with

no signs of disease and with very little selection for

evolutionary change. Rapid evolution is the norm

when in¯uenza viruses emerge into mammalian hosts

(20,37,38). Immunity and other selective factors

apparently drive the extreme rates of in¯uenza virus

evolution exhibited during adaptation to mammals.

Overall, the work of Webster and colleagues indicates

that ducks and other waterfowl are the original hosts

for in¯uenza viruses. Rare genome segment reassort-

ment events or transfers of entire in¯uenza virus

genomes initiate emergence into mammalian species,

but it is the destabilizing selective forces in mammals

which drives the ensuing rapid evolution. This is

another good example of punctuated equilibrium in

virus evolution. There are theoretical reasons (based

upon expected movements between adaptive peaks in

adaptive landscapes) to expect that evolution will

frequently exhibit punctuated equilibrium (40). Plant

viruses, such as the tobamoviruses can also exhibit

genetic stability for long periods, again due to strong

environmental selective pressures restricting quasis-

pecies diversi®cation (41).

Effects of Virus Population Transmission Size onSelection, Fitness and Evolution

Whenever viruses become extremely-well-adapted to

host environments and evolutionary stasis is reached,

this equilibrium obviously can be upset readily

( punctuated) by host or vector switching or by drastic

environmental changes. Another, less obvious

mechanism involves changes in the size (dose) of

virus particle transmission. In 1964, Muller (42)

postulated that whenever mutation rates are high and

populations are small in asexual populations, there

can be an inexorable accumulation of deleterious

mutations leading to a ratchet-like decline in

replicative ®tness. Chao (43) convincingly demon-

strated the operation of Muller's ratchet in the

tripartite RNA bacteriophage f6. Chao et al. (44)

also showed that sexual crossing could often reverse

the effects of Muller's ratchet. Quantitation of ®tness

losses during repeated small population transfers

( plaque-to-plaque genetic bottleneck transmissions)

of VSV and food-and-mouth disease virus con®rmed

that variable, stochastic, often-profound ®tness

decreases occur rather regularly (45±49). Clearly,

genetic bottlenecks have the capacity to disturb virus

adaptive equilibrium and thereby to drive stochastic

evolutionary changes. The number of virus particles

which constitute an effective genetic bottleneck can

vary greatly from only several particles to tens of

particles depending upon initial virus population

®tness (50,51).

The opposite effect on ®tness occurs during

repeated transfers of very large numbers (105 to 106)

of infectious virus particles. Under these large dose

transmission conditions, regular exponential increases

in virus replicative ®tness occur and previous ®tness

decreases due to Muller's ratchet are reversed

(48,50,51). Unquestionably, virus population trans-

mission size can affect virus evolution very

profoundly, and this inevitably must also occur

during natural virus outbreaks. Transmission of large

doses of infectious virus particles often occurs during

transmissions involving close contact (e.g., sexual,

kissing); during transfusions or other medical/dental

blood/tissue transmission; intravenous drug abuse,

some insect vector or animal bite transmissions; and

during some very close respiratory droplet transmis-

sions, some fecal-oral transmissions, etc. Genetic

bottleneck transmissions inevitably occur during

many rather distant virus transmissions during

respiratory droplet inhalations. This is clear from

quantitative studies of both experimental and natural

virus aerosol transmissions (52±54). After sneezing

and coughing of infected people in a room, the volume

of room air which must be sampled to obtain a single

infectious particle can be very large (53,54). For

example, 15 men in bed infected with adenovirus type

4 and coughing frequently in a barracks room led to

recovery of one tissue culture infective unit per 2820

sq. ft. (54). Thus, genetic bottleneck transmissions

must be frequent and unavoidable for respiratory


viruses although large population transfers must also

occur often during close contact. Similar considera-

tions apply to fecal-oral spread, spread from

inanimate objects (fomites) and from insect vector

transmissions (55). Whenever large virus population

transmissions occur repeatedly, selection can operate

repeatedly to select the best of the best of the best . . .

in terms of virus ®tness in a constant host species.

Conversely, repetitive genetic bottleneck transmis-

sions interrupt selective forces and allow stochastic

changes in ®tness and in evolutionary directions.

These stochastic changes will usually be in the

direction of ®tness loss (and perhaps loss of virulence)

but rarely, by chance, the converse will be true.

Virulence is a multifactorial, multigenic trait which

may or may not correlate with replicative ®tness and

transmission ef®ciency. The major insight to be

gained from experimental studies of virus ®tness is

that RNA viruses are phenotypically quasispecies as

well as genetic quasispecies. Thus, all phenotypic

characteristics, including virulence, will be highly

variable among the numerous mutants present in a

complex quasispecies swarm. Therefore, chance

sampling events such as genetic bottlenecks may

profoundly affect virulence traits (and other traits)

during an epidemic. Likewise, repetitive large

population transmissions can preserve and enhance

virulence or other traits which had been sampled by

chance during earlier genetic bottleneck events. This

can, of course, in¯uence disease severity and outcome

in infected individuals and in small local host cohorts

infected by such sampling of the quasispecies swarm.

Recombination, Reassortment and GeneDuplication in Virus Evolution

As outlined in the chapters on virus origins,

recombination has long been recognized as a central

mechanism in the evolution of DNA bacteriophages

and/or DNA viruses and retroviruses of animals and

plants. Recombination with, and insertion into, and

excision from, cellular DNA allows intimate genetic

interactions with host genomes, episomes and

plasmids. Just as the frequent acquisition of cellular

genes can help shape virus evolution, so can the

frequent acquisition of virus and retroelement genes

help to shape host evolution. A remarkable example is

provided by maize in which many dozens or even

many hundreds of diverse retroelement families

account for 50% or more of the plants nuclear

DNA! Large blocks of reiterated retrotransposons

inserted within each other are found in gene-

containing regions as intergenic segments (56,57).

Clearly, these are involved in determining gene

expression, genome size and genome organization,

so that the distinction between host and sel®sh

parasitic genes is blurred beyond recognition.

Although RNA riboviruses cannot interact with

host DNA genomes as directly, nor as frequently as do

DNA viruses and retroviruses/retroelements, RNA

recombination is equally common and important

among them as evolutionary events. The mechanisms

and importance of recombination in riboviruses and

retroviruses are reviewed in (58,59). Riboviral RNA

recombines with both cellular and viral RNAs and

acquisition of genes and gene segments from both can

be very important in ribovirus evolution and

emergence. Homologous recombination occurs fre-

quently during every replication of most positive

sense riboviruses (58,60) but is extremely rare among

negative sense riboviruses in which non-homologous

recombination events usually generate defective

genomes (some quite bizarre), most of which are

dependent upon non-defective helper viruses for

replication (61). Very rarely, helper-dependent,

bizarre, defective RNA virus genomes are probably

involved in major virus evolutionary events via RNA

recombination with non-defective helper viruses.

Because their helper viruses provide all vital

replication functions, defective viral genomes are

largely unconstrained by selective forces and can

undergo extremely rapid, massive evolution. Thus,

they could (very rarely) donate extensively-mutated

and rearranged genome segments back to the helper

viruses from which they arose (61).

Among the segmented-genome viruses, reassort-

ment of segments provides a ready mechanism for

generating new viruses. The best-known examples, of

course, involve the periodic antigenic shifts of

in¯uenza A viruses which usually occur at multi-

decade intervals to initiate new human pandemics

(37±39). The gene segment reassortment events which

cause emergence of new in¯uenza A viruses are very

rare events because only certain permutations of avian

and mammalian gene segments will be highly

infectious and ®t, and because reassortment requires

rare dual infection (by appropriate progenitor viruses)

of appropriate mixing vessel hostsÐmost often

probably swine or humans (37±39). Once a ®t new


reassortant emerges into the human population very

rapid evolution ensues (37±39). Reassortment events

are important in the evolution of many other

segmented genome RNA viruses. For example,

bunyaviruses can evolve by reassortment in doubly-

infected mosquitoes (62) and naturally-occurring Sin

Nombre hantavirus reassortants have been observed

in Peromyscus deer mice in Nevada and Eastern

California (63). No reassortants were observed

between Sin Nombre and other hantaviruses indi-

genous to their region, suggesting that reassortants

between distantly-related hantaviruses are rare or non-

viable, and/or that host species speci®cities greatly

limit reassortment. Again, generation of ®t reassor-

tants between distantly-related and different-host-

adapted virus strains is generally a rare event in

nature.

A major mechanism observed in the evolution of

all life formsÐgene duplicationÐis sometimes

important in virus evolution. For example, beet

yellows virus, a ®lamentous RNA virus, contains a

coat protein gene duplication (64) and two rabies-

related rhabdoviruses, Adelaide river virus (65) and

bovine ephemeral fever virus (66) each contain two

consecutive glycoprotein genes of differing size and

sequence. Both glycoprotein genes are expressed in

each of these viruses; via monocistronic mRNAs in

the case of bovine ephemeral fever virus and

polycistronic mRNAs for Adelaide virus. Gene

duplication of this kind occurs by recombination

events, probably via intra- or inter-molecular copy

choice replicase leaps (58,67).

Finally, a very simple, effective mechanisms for

creation of new virus gene products involves acquired

usage of alternative reading frames of an existing gene

to create overlapping genes. This overprinting

mechanism is common in virus evolution as outlined

in the review of Gibbs and Kease (4).

Do DNA Viruses Evolve as Quasispecies?

DNA viruses generally do not form complex

quasispecies mutant swarms to the extent that RNA

viruses do because they generally have genomic

mutation rates about 300-fold lower than those of

RNA riboviruses and roughly 30-fold lower than

retroviruses (23,68). Proofreading and mismatch

repair (69) of DNA can provide ®delity for even

very large DNA virus genomes. Hence, DNA viruses

generally evolve to achieve and maintain optimal

function. They often tend to produce inapparent and

silent latent infections, to co-evolve slowly with their

hosts over geologic time periods, and even to

in¯uence the evolution of their host species (68,70±

73). Nevertheless, many DNA viruses can exhibit

considerable genetic plasticity (74) and this can be

manifested via antiviral drug resistance and other

clinical problems. This is not surprising because DNA

viruses can have host recombination systems avail-

able to them in addition to intrinsic viral mechanisms.

Also, DNA ®delity is limited for viruses which

replicate via single-stranded DNA because mismatch

repair/excision repair systems are unavailable for

single-stranded DNA genomes. But, even bacter-

iophage T7, a classic double-stranded DNA virus

exhibited rapid evolution of replicating ®tness in

single plaques (75). It should be noted that some DNA

viruses such as the canine/feline parvoviruses evolve

in nature at least as rapidly as the slower-evolving

RNA viruses and can change host species speci®cities

very readily as well (76). This is in marked contrast to

the primate papillomaviruses, for example. Van Ranst

et al. (77) estimated primate papillomavirus mutation

rates to be of the order of 3� 10ÿ 8 base substitutions

per site per year in the EG gene. This is only about 20±

30 times faster than the rate of evolution of their

primate host species and about a million-fold lower

than rates of evolution of the most rapidly evolving

RNA riboviruses (29±35,78).

Implications of RNA Virus Quasispecies forDisease and Disease Emergence

Some investigators have stated that quasispecies

mutant swarms are not really necessary for disease

processes during RNAvirus infections. Of course, this

could be argued quantitatively in terms of the minimal

mutation rate which quali®es to produce a quasis-

pecies. However, this misses the essence of RNAvirus

biology. RNA viruses have been the most abundant

and successful parasites since the appearance of

cellular life (see the ®rst chapter) and this has been

achieved by maintaining error rates very near the error

threshold (12). This allows maximal variability and

adaptability (21±27) This great adaptability allows

®tness to be increased rapidly in changing environ-

ments and the intact animal, human, plant or insect

vector organism always confronts invading microbes


with multiple, challenging and changing environ-

ments. RNA virus quasispecies frequently undergo

major or minor changes in composition in response to

in¯ammatory and immune responses (20,37,38,79) to

different host cell types within individual infected

organisms, to widely differing conditions in verte-

brate versus insect vector hosts (80), to antiviral drug

treatments (81,82), to inadequate vaccine programs,

etc. Different subsets of the lymphocytic choriome-

ningitis virus quasispecies swarm are involved in

lymphoid cell infection with immune suppression as

contrasted with neuronal cell infection with runting

syndrome (growth hormone de®ciency syndrome)

(83,84).

Perhaps the involvement of quasispecies in disease

is best illustrated by the propensity of polioviruses to

cause paralytic disease in a small percentage of

infected individuals (60), and for some Coxsackie

viruses to cause cardiomyopathy in a small percentage

of infected humans. Microevolution of the quasis-

pecies population present in the type 3 Sabin oral

poliovirus vaccine seed stocks (85) can cause

paralytic disease in a very small percentage of vaccine

recipients and can even initiate outbreaks of polio-

myelitis in unvaccinated populations (60,86). Clearly,

the quasispecies nature of the vaccine seed stocks and

of their progeny is responsible for this rare but

unfortunate disease complication of an otherwise

excellent vaccine. An endemic cardiomyopathy

affecting thousands in China has been associated

with both selenium de®ciency and isolation of

Coxsackieviruses from patients (87,88). Beck et al.

in a remarkable study of selenium de®cient mice (89)

showed that infection by a normally non-virulent

clone of Coxsackie B3 virus induced signi®cant

myocarditis in the Se-de®cient mice, and virus

recovered from the hearts of these myocarditic mice

regularly caused myocarditis in normal Se-adequate

mice! Complete sequence analysis of the recovered

myocarditic strain of Coxsackievirus B3 revealed six

speci®c nucleotide changes, all of which had appeared

in four separate isolates from the initial myocarditic

mouse examined and from 3 individual follow-up

mice (89). This study agrees with the sequence studies

of Chapman et al. (90) who also found that speci®c

changes at these six nucleotide sites are associated

with the cardiovirulent phenotype (89,90) of

Coxsackievirus B3. Nothing could more clearly

illustrate the importance of quasispecies mutant

swarms in RNA virus disease. It is important to

remember that myocarditis due to Coxsackie viruses

is rare relative to the number of human infections.

Virulence is a trait which is seldom selected except

when it correlates with replicative ®tness. Immune

de®cits arising from selenium de®ciency regularly

allowed expansion of the Coxsackievirus B3 quasis-

pecies mutant swarm and colonization of myocardial

cells to cause disease. The cardiovirulent variants

selected in heart muscle cells established a new

quasispecies distribution which was cardiovirulent for

normal mice after it was deliberately isolated from the

total whole animal wild type quasispecies swarm. It is

beyond question in this outstanding study that it is

those quasispecies subsets normally buried within the

total circulating quasispecies population which have

the potential to cause viral myocarditis (89,90). This is

likely a rather typical situation with regard to RNA

virus disease potential.

Finally, the role of RNA virus quasispecies in

future emerging diseases of humans, domestic

animals and crops is obvious. Most emerging human

diseases in recent years have been RNA virus

diseases; from AIDS to Ebola hemorrhagic fever to

hantavirus pulmonary syndrome. Most new or

emergent virus diseases in the future will also be

RNA viruses because of the rapid evolution potential

of their quasispecies. This trend will be accelerated so

long as the human population continues to expand

exponentially. For example, the recent rapid growth of

human populations in tropical areas has been matched

by an equally-rapid increase in dengue fever, dengue

hemorrhagic fever and dengue shock syndrome, and

by an increasing rate of evolution of dengue viruses

(91±93). All of us and our domestic animals (94) are

potential incubators for the rapid exploration of

previously-unexplored sequence space by evolving

RNA viruses. Sequence space is a v-dimensional

hypercube in which v is the genome length in bases or

base pairs (12). For a 10 kb RNA virus genome there

are 410,000 sequence permutations and combinations

which must be explored before all viable and adaptive

virus sequences have been testedÐeven if we assume

a constant genome size restriction (which never

happens). There is not enough space-time in countless

imaginable universes to test even a minuscule fraction

of such incomprehensibly vast dimensions. Hence,

new sequence spaces will be explored increasingly

rapidly in future decades. New RNA viruses and

new diseases will emerge with increasing rapidity as

long as human population growth remains in an


exponential phase. Planet earth has a ®nite human

carrying capacity, but its dimensions are uncertain and

will be affected by human choices concerning quality

of life, economics, environmental values, etc. (95).

But beyond human choice are the evolutionary paths

to be followed by exponentially-increasing quasispe-

cies swarms of RNA viruses exploring humans as

hosts. Virus evolution is stochastic and unpredictable

(35,96), but increasing numbers of human outbreaks

are inevitable. They will occur; they should be

anticipated and there should be reasonable preparation

for some unpleasant outbreaks. The human carrying

capacity of our planet may ultimately be determined,

not by human choice, but by RNA virus evolution.

Acknowledgments

Work in La Jolla, CA was supported by NIH Grant

AI14627 and in Madrid, Spain by Grants DGICYT PB

94-0034-C02-01, FIS 95/0034-1, Fundacion

Rodriguez Pascual, Communidad Autonima de

Madrid, and Fundacion Ramon Areces.

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Origin and Evolution of Viruses