Molecular evolution of the major capsid protein VP1 of enterovirus 70.

JOURNALOFVIROLOGY, Feb.1994, p. 854-862 Vol. 68, No. 2 0022-538X/94/$04.00+0

Copyright C 1994,American SocietyforMicrobiology

Molecular

Evolution of the

Major Capsid

Protein VP1 of

Enterovirus 70

NAOKAZUTAKEDA,1* MASAKO TANIMURA,2ANDKIKUKO

MIYAMURA'

Departmentof Epidemiology, NationalInstituteof Health, Toyama 1-23-1, Shinjuku, Tokyo 162,1andDepartment of

Child Ecology, National Children's Medical Research Center, Taishido, Setagaya-ku, Tokyo 154,2Japan

Received 6July1993/Accepted 2 November 1993

Nucleotide sequencesofthe genome RNAencodingcapsid protein VP1 (918 nucleotides) of 18 enterovirus 70 (EV70) isolatescollectedfrom various partsoftheworld in1971to1981were determined,and nucleotide substitutions amongthem were studied. Thegeneticdistances between isolates were calculated by thepairwise comparisonofnucleotide difference.Regression

analysis

ofthegeneticdistancesagainsttime of isolation of the strains showed that the synonymous substitution rate was very high at 21.53 X 10-3 substitution per nucleotide per year, while the nonsynonymous rate was

extremely

low at 0.32 X 10-3 substitution per nucleotideper year.The rateestimated bythe average valueofsynonymousand nonsynonymoussubstitutions (W.-H. Li, C.-C. Wu, and C.-C. Luo, Mol. Biol. Evol. 2:150-174, 1985) was 5.00 X 10-3 substitution per nucleotide peryear. Taking the average value ofsynonymous and nonsynonymous substitutions as genetic distances between isolates, the phylogenetictree wasinferredbytheunweightedpairwisegroupingmethod of arithmetic average andbytheneighbor-joiningmethod. The tree indicated that the virus had evolved fromone

focal place, and the time ofemergence was estimated to beAugust 1967 ± 15 months, 2 yearsbefore first recognitionof the pandemic of acute hemorrhagicconjunctivitis. By superimposing everynucleotide substi-tution on the branches of thephylogenetictree, weanalyzednucleotidesubstitutionpatternsofEV70genome RNA. In synonymous substitutions,theproportionoftransitions, i.e., C<-->UandG*-->A,wasfoundtobe extremelyfrequent in comparison with that reportedonotherviruses orpseudogenes. In addition, parallel substitutions (independent substitutionsatthe same nucleotidepositionondifferentbranches,i.e., different isolates, ofthe tree) were frequently found in both synonymous and nonsynonymous substitutions. These frequent parallel substitutionsandthe low nonsynonymoussubstitutionratedespitethe veryhighsynonymous substitution rate described above imply a strong restriction on nonsynonymous substitution sites ofVP1, probably duetotherequirement formaintainingtherigid icosahedralconformation of the virus.

The epidemic of enterovirus 70(EV70),oneoftwo entero-viruses that cause acute hemorrhagic conjunctivitis (AHC),

wasfirst recognized in Ghana in 1969 and then spread rapidly in theEasternHemisphere inapandemic fashion withinafew years(16). Afteraquiescent period of severalyears,the virus again caused the second pandemic in the early 1980s inlarge parts of the world, including the Americas, which were not involved in the first pandemic. The viruswasclassifiedas a new type of enterovirus (26, 27). No genetically related virus has been foundamonghuman andnonhuman viruses(31, 32), and

seroepidemiologydidnotgive solid evidence of thehistory of the virus before its sudden appearance in humans(18).

EV70 is uniquely suited as a subject for studying the evolution ofRNAviruses innature,and itsrecent emergence enables us to trace genetic changes on the nucleotide level. Severe bleeding associated with conjunctivitis is the most prominent characteristic in the clinical presentations. The availability of isolates collectedover adecadesinceveryclose tothe time of itsappearanceand theprecise record of the date and place of virus isolation allowus to trace both the

evolu-tionary pathway of the isolates and the molecular events occurringin thevirus genomeduring transmission in nature.

Our previous study (28) on the evolution of EV70 by

oligonucleotide mapping using 16isolates obtained from

var-ious parts of the world revealed that all of the isolates had

*Correspondingauthor. Mailing address: Department of

Epidemi-ology,National Institute ofHealth, Toyama 1-23-1, Shinjuku, Tokyo 162, Japan. Phone: 3-5285-1111.Fax:3-5285-1177.

evolvedrapidlyataconstantevolutionaryratefromacommon ancestor which was estimated to have appeared in humans around 1967. In the presentstudy, we determined the nucle-otidesequencesof VP1 of 18 EV70isolates,including13of 16 isolates from theprevious study and 5 different isolates from theearly epidemicsin variousareas, so as toanalyzenucleotide

substitutions during evolution and estimatemore reliablythe time of emergence of the virus. For comparison, we have chosen VP1, themajor capsid protein, because of the region with least homology in both nucleotide and amino acid se-quences and therefore of less functional constraint among enteroviruses (31, 32). Comparison of the VP1 nucleotide sequences of the isolates showed a phylogenetic relationship

consistent with that previously reported by oligonucleotide mapping(29).

Thestudydoneby the method of Lietal.(23)enabledus to separately estimate the rate of synonymous and nonsynony-mous substitutions. The results revealed an extremely high synonymous ratebutanunexpectedlylownonsynonymous rate of nucleotide substitutionsduring 10-year evolution innature. The ratio ofnonsynonymous to synonymous substitutions in EV70 VP1 was shown to be remarkably lower than that reported for the envelope proteins of otherRNAviruses(11, 21).

Weestimated theputativeancestralsequenceof EV70 VP1 andsuperimposedeach nucleotide substitution oftheisolates

from the ancestralsequence on thebranches of the phyloge-netic tree. This method made it possible to analyze the nucleotide substitution patterns amongfour nucleotides, the proportion of transitional and transversional substitutions, and

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TABLE 1. EV70 isolates

Strain Place of collection _collectionDateof Reference

R6/71 Rabat, Morocco Jan. 1971 30

R20/71 Rabat, Morocco Jan. 1971 30

J648/71 Mie, Japan 16 Nov. 1971 19

J670/71 Hokkaido, Japan 25 Dec. 1971 19

SEC32/71 Singapore 1971 47

ENG/71 London, England 1971 12

M8/72 Mie, Japan 25 Sept. 1972 37

1/72 Indonesia 1972 20

G10/72 Gifu,Japan 27 Nov. 1972 14

T62/73 Tunisia 1973 45

FB/73 France July 1973 4

T260/74 Thailand 15 Dec. 1974 17

M51/76 Mie, Japan 16 Feb. 1976 36

HP85/78 Hokkaido, Japan 18 July 1978 1 TW266/81 Kaohsiung, Taiwan 14 Apr. 1981 3 HP185/81 Hokkaido,Japan 26 May 1981 3 V1635/81 Karachi,Pakistan 21July 1981 6

V1250/81 Honduras 6 Aug. 1981 15

the frequency of parallel nucleotide substitutions indepen-dently occurring at the same site of different evolutionary

lineages. Onthe basis oftheresults,thecharacteristics of the nucleotide substitution of the enterovirus genome RNA are

discussed.

MATERIALS AND METHODS

Viruses.Among the 18EV70isolates (Table 1)used inthis study, 13 had been previously analyzed by oligonucleotide fingerprinting (29). Onestrain, V1635/81 fromKarachi,

Paki-stan, hadafingerprintmapidenticaltothatof the previously analyzed V1604/81. Theother five strains, SEC32/71 isolated inSingaporein1971,ENG23356/71(ENG/71) from Londonin 1971, 113476/72 (1/72) from Indonesia in 1972, T62/73 from Tunisia in 1973, and FB/73 from France in July 1973, were

collected in 1971 to1973 from differentareasbutanalyzed for

the firsttime in this study.

Purification of virion RNA and RNA sequence analysis.

EV70 isolates were propagated in LLC-MK2 cells. Virus

particles and virion RNA were purified by sucrose density

gradient centrifugation aspreviously described (24). The

nu-cleotide sequence ofEV70VP1 (918 nucleotides)was deter-mined by the dideoxy-chain termination method (38), using

reverse transcriptase derived from avian myeloblastosis virus

(Seikagaku Kogyo, Tokyo, Japan) asdescribedpreviously (5), with slight modification.The sequencesfor theprimers,

resi-dues 2454to 2467,2627to 2643,2735 to2749, 2920to 2935, 3134 to 3150, 3279 to 3295, and 3437to 3452,were selected accordingtothecompletenucleotidesequenceof the standard strainofEV70, J670/71 (41). Ambiguousnucleotide sequence

wasconfirmedby direct sequence analysis usingDNA ampli-fiedby PCR (33). The syntheticDNA oligonucleotides were

prepared in an automated DNA synthesizer (model 391; Applied Biosystems, Foster City, Calif.), using

P-cyanoethyl

phosphoramidites.

Genetic distances between isolates. Four different nucle-otide differences between isolates were used to estimate the

genetic distances: the observed nucleotide difference, the number of synonymous substitutions per synonymous sites

(Ks),thenumber ofnonsynonymoussubstitutionsper

nonsyn-onymous sites (Ka), and the mean value (K,) ofKs and Ka

weighted by the respective number ofsynonymous and

non-synonymoussites. K,

Ka,

andK,werecalculated by using the computer program proposed by Li et al. (23), in which a nucleotide site in the codon is classified according to its degeneracy (i.e., nondegenerate, twofold degenerate, or four-fold degenerate), depending on how often nucleotide substi-tutions result in amino acid replacement. A site is fourfold degenerate if all possible changes at the site aresynonymous; the thirdpositions of 32 of the 61 sense codons (e.g., the third position of valine) are of this type. A site is twofold degenerate if one of the three possible changes is synonymous; the third position of 24 of the 61 sense codons (e.g., the third position of histidine) is of this type. A site is nondegenerate if all possible changes at this site are nonsynonymous or nonsense; the second positions of all sense codons belong to this type. Nucleotide changes are also classified as either transitional or transversional, and changes between codons are assumed to occurwith differentprobabilitieswhich aredetermined on the basis of the relative frequency of nucleotide changesin mam-malian genes.

Estimation ofevolutionary rate. The evolutionary rate was calculated by regression analysis. Genetic distances between the Rabat 6/71

(R6/71)

strain,oneof theearliest isolates, and other isolates were plotted along the vertical axis, and the isolation times of the isolates were plotted along the horizontal axis.

Neighbor-joining method. The method was described by

Saitou andNei (34).

UPGMA.Amodifiedunweightedpairwise groupingmethod of arithmetic average (UPGMA) phylogenetic tree was con-structed as described previously (28, 42), using adjusted ge-netic distances

(d'ij).

The average value (Kt) of synonymous and nonsynonymous substitutions was used as the genetic

distance

(dij)

between isolates. The value of

dij

obtained at different isolation times was converted to the

d'i

so as to represent the genetic distance between isolates at the same time(t).Calculationwasdoneontheassumptionthat the virus evolved ataconstantrate, accordingtotheequation

d'ij

=b(t

- t,) + b(t -

tj)

+

di,,

where bis theevolutionaryrateand

t1

and t, arethetimes

of

isolation of strainsi and

j,

respectively.

Thebranchingtime

(tij)

of strainsiand jwascalculatedas

tij=

t -

(d'j1/2b).

Relative substitution frequency. Relative frequency

(fij)

of substitutions among four nucleotides was calculated by

themethod ofGojoboriet al. (7) bytheequation

fi>=

> Pi x 100%,

i j*i

where

Pij

is theproportion of nucleotide changes from the ith typetothe

jth

type(i,

j

=A,U,C,orG),e.g.,PAU = 5/294in totalsubstitutions in Table 4.

Nucleotide sequences. The nucleotide sequence data

re-ported in this paper will appear in the DDBJ, EMBL, and

GenBank nucleotide sequence data bases under accession numbersD17595,D17596, D17597, D17598,D17599,D17600,

D17601, D17602,D17603, D17604,D17605,D17606,D17607,

D17608, D17609, D17610, D17611, and D17612.

RESULTS

Comparison of nucleotide and deduced amino acid se-quences among EV70isolates.The VP1 nucleotide sequences of 18 isolatesweredeterminedbythe

dideoxy-chain

termina-tionmethod,using

purified

virionRNAand

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856 TAKEDA ET AL.

TABLE 2. Numberof observednucleotidedifferences andestimatedpercentnucleotidesequencevariationsinVP1 regionof EV70

Difference'

Strain Strain

no. 1 2 3 4 5 6 7 8 9 10 11 12 14 15 16 17 18 19

1 R6/71 10 45 41 31 29 27 26 26 37 45 43 58 63 38 58 69 67

2 R20/71 1.11 45 41 35 27 29 28 28 39 45 43 56 61 40 62 71 69

3 FB/73 5.41 5.54 20 36 34 36 35 35 42 58 52 67 72 39 61 74 74

4 T62/73 4.94 5.07 2.26 35 27 31 28 28 39 55 49 66 71 38 58 69 71

5 ENG/71 3.61 4.11 4.30 4.15 14 12 11 11 20 36 30 47 52 23 43 58 60

6 SEC32/71 3.39 3.22 4.09 3.27 1.56 10 9 9 18 34 28 47 50 21 43 56 58

7 J670/71 3.13 3.43 4.30 3.71 1.33 1.12 5 5 16 32 24 43 46 23 43 54 56

8 J648/71 3.02 3.33 4.21 3.38 1.22 1.01 0.55 0 17 31 25 44 47 22 38 53 53

9 M8/72 3.02 3.33 4.21 3.38 1.22 1.01 0.55 0.00 17 31 25 44 47 22 38 53 53

10 G10/72 4.40 4.73 5.19 4.79 2.26 2.05 1.80 1.92 1.92 34 28 43 50 27 49 58 60

11 T260/74 5.50 5.65 7.47 7.03 4.19 4.02 3.71 3.61 3.61 4.00 15 40 39 45 63 68 70 12 M51/76 5.17 5.28 6.57 6.14 3.45 3.27 2.76 2.88 2.88 3.26 1.69 32 35 39 57 64 68 14 HP185/81 7.30 7.16 8.94 8.86 5.71 5.82 5.21 5.35 5.35 5.19 4.79 3.79 19 56 72 77 78 15 TW266/81 8.25 8.13 9.87 9.77 6.37 6.26 5.64 5.78 5.78 6.22 4.70 4.20 2.16 59 78 78 79 16 I/72 4.53 4.88 4.73 4.62 2.60 2.41 2.64 2.54 2.54 3.17 5.53 4.73 7.16 7.66 42 53 55 17 HP85/78 7.47 8.21 8.02 7.61 5.22 5.32 5.24 4.66 4.66 6.18 8.25 7.35 9.77 10.91 5.22 52 56 18 V1635/81 9.06 9.63 10.29 9.48 7.31 7.23 6.89 6.74 6.74 7.52 9.13 8.52 10.69 10.89 6.79 6.73 18 19 V1250/81 8.72 9.28 10.26 9.77 7.67 7.59 7.14 6.76 6.76 7.89 9.58 9.20 10.78 11.06 7.15 7.36 2.08

" Numberof observed nucleotide differences is shown right of the diagonal. Percent nucleotide sequence variation (the average value [K,] of synonymous and

nonsynonymous substitutions determinedby the method of Li et al.[23])is shownleftof the diagonal.

cleotide with reversetranscriptase.Wedidnotperform plaque cloning of the virus to avoid an arbitrary selection. Also, molecular cloning, which sometimes selects a minor popula-tion of cloned DNA,wasexcluded.

Alignmentof the 918-nucleotide sequences ofVP1 of the18 EV70 isolates demonstrated neither insertion nor deletion amongthe isolates collected over 11 yearsfrom various parts of the world (data not shown). The number of observed nucleotide differences between isolates by pairwise compari-sons is shown in the upper right half of Table 2. Frequent nucleotide substitutions between isolates were found; the maximumwas 79nucleotidesubstitutions (8.6%) betweenthe Taiwan isolate(TW266/81) andHonduras isolate (V1250/81)

obtained in the same year. Epidemiologically related strains had the same nucleotide substitutions. The proportion of commonnucleotide sequence decreased when earlier and later isolateswere compared. Eventually, 181 sites of 918 nucleo-tides (19.7% nucleotide sites) were found to have changed. The nucleotide substitutions occurredmostly(144of181 sites

[79.6%])in the thirdpositionofthe codon. On the otherhand, the amino acid sequences deduced from the nucleotide se-quencesweremuchmoreconserved; 28 of 306 amino acid sites had changed. The maximumamino acid substitution between isolates was 12 (3.9%), which was observed between strains

R6/71 and V1635/81, R20/71 and ENG/71, and ENG/71 and

T260/74.

Amongthe nucleotide substitutions, the number of

synony-mous substitutions (Ks), the number of nonsynonymous sub-stitutions

(K,),

and the mean value (K,) ofK,, and Ks were calculated by the method of Li et al. (23) as described in Materialsand Methods. Pairwise comparisons of K, expressed as percent nucleotide sequence variation are shown in the lower half of Table2.

Evolutionary relationship determined by the

neighbor-join-ingmethod. To explore the evolutionary relationship among

isolates,wefirstapplied the neighbor-joining method, using Kt

as a

genetic

distance(Fig. 1B).Theneighbor-joining method is based on the principle that the sum of the branch length

(shown

bythe numbersonthebranch) at each clustering stage is to be minimized (34). Thus, this method is suitable for

estimating the genetic relationship among isolates without taking account of the evolutionary rate or isolation time. The tree thusconstructedshowed that the 18 isolates divergedinto four mainlineages(I, II, III, and IV in Fig. 1). Lineage I of two Moroccan strains, R6/71 and R20/71, was considered to have diverged first from the other 1971 isolates; among the six early epidemic isolates from 1971, R6/71 and R20/71 were distinct from the other four 1971 isolates(SEC32/71, J670/71, J648/71, and ENG/71).

Estimation of nucleotide substitution rates. To confirm the constant accumulation of nucleotide substitutions on VP1 of EV70 isolates, the genetic distance (d) was plotted on the ordinate as the function of the isolation time (t) on the abscissa in Fig. 2. From the results drawn by the neighbor-joining method, which indicated that the lineage of R6/71 and R20/71 first diverged from other lineages, we performed regression analysis using the genetic distance of each isolate from the R6/71 strain (42). Four different genetic distances were used: the observednucleotide divergence,

K,

K,,,

and K, asdescribed in Materials and Methods. Isolates other than R20/71 were distributed on a single straight line,d =a + bt(O < a, 0 <b), indicating that these isolates evolved at a constant evolutionary rate from acommon ancestor.

The substitution rate for the observed nucleotide divergence was estimated to be 3.84 x 10-3 substitution per nucleotide per year(Fig. 2A). The rate calculated by K, increases to 5.00

X 10-3 substitution per nucleotide per year (Fig. 2B). This high rate is due to an extremely rapid synonymous rate (the regression coefficient of

Ks),

21.5 x

10'

substitution per synonymous nucleotide site per year (Fig.2C). In contrast, the nonsynonymous rate (regression coefficientof

Ka)

was remark-ably low at 0.32 x 10-3 substitution per nonsynonymous nucleotide site per year (Fig. 2C), representing almost 1/70 of thesynonymous rate.

Construction of a phylogenetic tree by UPGMA. By using the

K,

(Table 2, lower half) as the genetic distance among strains, a rooted phylogenetic tree was inferred by UPGMA as described in Materials and Methods. The phylogenetic tree of EV70 VP1 thus constructed (Fig. 1A) was basically consistent with that obtained by the neighbor-joining method (Fig. 1B)

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MOLECULAR EVOLUTION OF EV70 VP1 857

A Li RW171

I

.

R6/71

17 R

1969.11 1. B7

R20/71

S 17-9

19679

*sl

[-=

IE~~~~~~~~~

~~~~~~~~~T62/73

FB/73 L 2 64~ GlOn2 TO

C\ T260W4lM I

ENG/71

Msm6

1

970

SEC32/71

02

J670/71

IV

19683 1970.4 11.7J68777 H151

J6/197.S/6\V208

IV HP185/81

T2W266/81

-lwoz

~~~~~~~~~HP85/78

|

VlG35/81

1969S~~~~~~~~~~~~~~~~~~~~~~~~~~~17.

V1250/81

'70 '15 '80

FIG. 1. (A) Phylogenetic tree ofEV7Oinferred by UPGMA. The isolates are positioned at the time of virus isolation on the horizontalscale. The timeof branching between strains (shown by year and month in the tree) wascalculated by using the genetic distance between isolates shown in Table 2, together with the time of isolation of the strains and theevolutionary rate as described in Materials and Methods. The star indicates the hypothetical ancestral virus. (B) Phylogenetic tree ofEV7O inferred by the neighbor-joining method. Numbers represent estimated branch lengths in nucleotides.

andvery close to thetree previously constructed by oligonu-cleotide mapping (29). Thus,thetree indicated the following chronological and geometric epidemiology of EV70. (i)All of the isolates were derived from a common ancestor which appearedin August 1967 ± 15months (thestandard deviation

dividedby b), shortlybefore the first appearance of AHC in

Ghana in 1969. (ii) The branching timeswere concentrated

immediately before or during the two pandemics. (iii) The isolates diverged into four main lineages shortly after their progenitor emerged. Thetwo lineages (III and IV in Fig. 1) independently persisted, and the second epidemics were

causedbytheirprogenies (15, 29). (iv)Thethree Asianstrains (T260/74, M51/76, and HP85/78) from sporadic infections belongedtoeitherof thesetwosecondpandemic lineages. (v) The American strain (V1250/81) and the Pakistani isolates (V1635/81) arethought to have diverged from each otherin June1979,afewyearsbeforethesecondpandemic, suggesting thespreadofAHC from AsiatoSouth America(15, 29).

Nucleotide substitution pattern. Wesuperimposed the

nu-cleotidesubstitutionsoneach branch of thephylogenetictree according totheorder of branching (Fig. 3). Thisassignment made it possible to estimate the time when each strain's substitution occurred and, therefore, to estimate thepossible

number of nucleotide substitutionsoccurringonVP1 RNAof the particular isolates during transmission in nature. The putative ancestral sequence of EV70VP1 wasdeterminedon the assumption that the lineage of two Moroccan isolates,

R6/71 and R20/71, firstdiverged from those of other isolates and that thefrequencyof back substitutionsduring evolution was

minimal.

We observed 265 nucleotide changes in total at 181 sites

(Table 3)and analyzed 258 changes for nucleotidesubstitution

pattern

(Table 4);sevenchanges which occurredattheearliest

branching between the Moroccan strains and other isolates

(see Fig. 3)wereexcludedfromanalysisbecause it couldnotbe inferred whether the Moroccan strains' nucleotides had

changed to others or vice versa. We compared nucleotide

substitutions by the relative frequency

(fij)

(7)

calculated as

described in Materials and Methods.

Here,fij

x 100represents theexpectednumberof nucleotidechanges from the ith type to thejth type among every 100 substitutions in a random sequence,i.e.,inasequence inwhich the fournucleotidesare

equally frequent. Cumulative

fij

valuesfortransitionsare

given

inbrackets in therightuppercornerof the matrix in Table4. Amongthepossible12types ofsubstitutions,thetransitions

U*-

->Cand

G*-

-*A wereveryfrequent,

representing

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858 TAKEDA ET AL.

lor

A

5

3.84x10-3substitutions/nucleotide/year

0~~~~~~~0 0

B 1 5.00x 0-3 substitutions/nucleotide/year %

5 0 0

0 0

0-.

C

I1) 0 0

zo

0

21.53 x O-3substitutions/nucleotide/year

0

0 0

0.32x10-3substitutions/nucleotide/year

Time in months

FIG. 2. Estimation of the nucleotide substitution rateof theVP1 region of EV70. Isolateswereplotted by the nucleotide sequence variation from one of the early isolates, R6/71, against the time of isolation of eachstrain. The nucleotide substitutionrate wascalculatedfrom observed nucleotide differences (A), the meanvalueof synonymous and nonsynonymoussubstitutions estimated by the method of Lietal.(23) (B),and the number of substitutions per synonymousornonsynonymoussubstitution estimatedby the method of Lietal.(23) (C).Abroken lineisdrawnon theassumption that the virus has evolved at a constantsubstitution ratesince itemerged.

258 substitutions (91.1% inrelative frequency) intotal substi-tutions and 190 of 202 (94.8% in relative frequency) at the thirdpositionofcodons. The relative frequencyoftransitions at fourfold-degenerate sites, where all possible changes are synonymous, wasfound to be 90.1%. Since the frequency at a fourfold-degenerate site is likely to show the substitution frequency between nucleotides under no selective constraint (i.e., unbiased site), the result indicates that transitional sub-stitutions tend to occur at an extremely high rate in EV70VP1. The values were much higher than those reported in pseudo-genes (7), influenza viruses (34), or retroviruses (10) (see Discussion).

Parallel substitutions. On the basis of the substitutions on the branch of the phylogenetic tree in Fig. 3, it is possible to analyze parallelsubstitutions, namely, nucleotide substitutions at the same site on different branches of the tree. On the

assumption that there had been the least back substitution,

parallel nucleotide substitutionswerefrequentlyfound (Table 3); 12 of 28 nonsynonymous substitution sites and 47 of 154 synonymous substitution siteswere found tobesubstituted in parallel. Ten of twelve nonsynonymous parallel substitution sites and 40 of 47 synonymous parallel substitution sites had the same nucleotide pattern (e.g., U->C on different branch-es), while six of synonymous parallel substitution sites had different nucleotide patterns (e.g., U->C and U--A, respec-tively,atthe same siteondifferentbranches). In addition,the substitutions at one site (site 96 in Fig. 3) were parallel

between nonsynonymous and synonymous substitutions, and two sites (site 90 [nonsynonymous substitution] and site 215 [synonymoussubstitution])weresubstitutedtwotimes; in both sites, the U->Achangewasfollowed by A->G.Theseparallel

substitutions substantially reduced the observed nucleotide J. VIROL.

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[image:5.612.149.473.69.488.2]

AG GA CU

GAAU CU uc 2 R/71

3,306 193,265,276, CUucGAAG

2D6-24

C 0 o R20/71

CU UC GA UG CA

GAGI9~

FB

/73

'20,22,061421,59 cuGA UA UC T62/73 AG CU UC

97,277 93,169 1S 13 ENG/71

GA AAGUA

uc

*

°

*

SEC32/71

L 3 143 53 289

A G

J670/71

G (5UAG 1ss8277

_

3 2"53 CU

J648/71

UoC

293 M8/72

CU UC GA AG UA

cou

xooo

0

0oO

G10/72

IU,262, 50,261 114 27 12,256

UC t7 _ ~~~346 CUJUC GA AG AU

CU, GA CU UC AGGAU4

OxJu

>Xb~~~~17,13

1262K2

93

3"

UA CU UC AG M 17

0oco 0

M51/76

CG 258 50 23

CoU GA g ~~~~~C*8CU

GA CU UC GA AG_ UAAU 1

260 54,299 233,261 114,119,179 96 47 ~ U

AG CU UC GA AG 193

UA O 00 OC) OCO 172

o 133,148 261 119,231 39,169,227

215 CU UC GA AG AC

CU UC GA AG UA 61,128,176,186,222 15 47 IS 215 225

C cOID Dom *

268,293 93 CU UC GA AG AC

_cxxx

cxxxxxbo

'70

'75

UCGA AG CA

i o

o *

O HP'

31114 97 12S

UC_,AGAACCA_CA

TV

115,292

HP85/78

) 5xAG i

81,3 1,259 i58

_CU UC GA AG

265,270 47 179 39

FIG. 3. Nucleotide substitutionsoneachbranch of the phylogenetictree.Eachpossible nucleotidesubstitution is indicatedonthe branch of thephylogenetictreeshown inFig. 1. Acommonnucleotide substitutionamongthestrainsisindicatedon the branch shared with these strains.

Auniquesubstitutionfor astrain ison itsown branch. AGon abranch,for example,meansthe substitution fromadeninetoguanine. For the

seven substitutions observed between the Moroccanstrains(R6/71 andR20/71)andotherearlyisolates(upperleft;e.g.,GA/AG),the direction ofsubstitution, forexample G-A orG<-A, could not be inferred. One symbol corresponds toone substitution. 0, synonymous substitution occurring uniquelyinthetree;0,synonymoussubstitutionoccurringinparallelondifferentbranches; *,nonsynonymous uniquesubstitution;0,

nonsynonymousparallel substitution. Parallel nucleotide substitutionsareindicatedbythecorrespondingaminoacidscquence number.Examples of thesubstitutionsare asfollows. Aparallel substitution ofthesamenucleotidepattern,U-C,at site 138was onthebranchesofstrainsFB/73 andENG/71, respectively. Parallelsubstitutions ofadifferent nucleotidepattern,C-A and C-Uat site 128,were onthe branches ofHP185/81 andHP85/78, respectively. Twosubstitutionsatthesamesite,U-AfollowedbyA-*G,werefoundatsite215 ofHP85/78and alsoat site90on

the branch ofHP185/81 andTW266/81, respectively.

differences betweenisolates and therefore resulted in a

substi-tution rate lower than the actual rate(Fig. 1).

Amino acid substitutions. As shown by a low

nonsynony-mous substitutionrate,we observed averylimited number of amino acid substitutions; only 28 sites of 306 amino acid residues had been changed among the isolates. Amino acid alignments forVPI of other enteroviruses (31) indicated that theamino acidsequenceof 18EV70 isolatescorrespondingto the eight-stranded antiparallel 1-barrelconfiguration seemed to be well conserved among 18 EV70 isolates. Also well conserved was a segment corresponding to a loop structure (amino acid residues 202 to 219). In amino acid sequences

corresponding to the first corner (residues 82 to 95) and the thirdcorner (residues 132 to145), the respective two

replace-ments were observed in each region. Twelve (43%) of 28

substituted residueswereconcentrated in either the amino-or

carboxy-terminal region ofthe VP1 protein.

Among this small number of amino acid substitutions, we

observed frequent parallel amino acid substitutions; of 28 substituted residues were changed in parallel. This finding indicates a strong constraint for the amino acid sequence,

extremely limiting the number of changeable amino acid residues in VP1.

DISCUSSION

From the comparison of nucleotide sequences of capsid proteinVPl in 18 isolates ofEV70,weinferredaphylogenetic

tree which was quite consistent with that previously

con-structed by oligonucleotide map analysis of entire genome

* , GA/AG

2° GA/AG

0CT/TC

TC/CT

oCT/TC

oTC/CT

2G7 °GA/AG

[185/81

V266/81

V1635/81

V1250/81

'80

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860 TAKEDA ET AL.

TABLE 3. Nucleotide substitutions inVP1 of 18 EV70isolatesa

No.of sites with substitutions No. of nucleotide substitutions Type of substitution

Nonsyn. Synon. Total Nonsyn. Synon. Total

Total 28 154 181 47 218 265

Single 16 107 123 16 107 123

Parallel

Total 12 47b 58b 31 111 142

Same nucleotide pattern 10 40 50 28 96 124

Different nucleotide pattern 0 6 6 0 12 12

Nonsyn./synon.pair 1c 1c 1 1 1 2

Two times in one site 1 1 2 2 2 4

a Nonsyn., nonsynonymous; synon.,synonymous.

bIncludes one site(site47 inFig.3) whichhad both substitutionswiththe same andsubstitutionswithdifferent nucleotidepatternsinparallel.

cThe samesite (site96inFig. 3).

RNA(29). The involvement offive additional earlyepidemic (2), nucleoprotein gene (8, 40), and PB2 (9) of influenza A isolates indicated the emergence time of EV70tobe August virus and for the hemagglutinin

(13)

and NS genes (46) of 1967 ± 15 months, only 1 month earlier than previously influenzaBvirus; theywerein the range of 1.1 x 10-- to2.3 estimated. We noticed that divergent times between strains x

10'3

substitution per nucleotide per year for the observed wereconcentrated during or around the twopandemic periods nucleotidedifferences.Inaddition,afrequencyaslowas1.4 x

and that the second-pandemic strains (HP185/81, TW266/81, 10-4substitution per nucleotide per yearwasreported for the

V1635/81, and V1250/81) belonged to two different lineages, 26S structuralproteingeneofeasternequine encephalitisvirus which had already diverged from one another during the first (44). This high rate of EV70 VP1wasaccountedfor by itshigh epidemic. Three Asian isolates (T260/74, M51/76, and HP85/ synonymous rate (21.5 x 10-3 substitution per synonymous

78)from the nonepidemicperiod belongedtoeither of thetwo site peryear). Itwasapproximatel twotimeshigherthanthat second pandemic lineages. These observations indicate that of influenza HA at 14.1 x 10- and NA at 11.1 x

10-3

numerous genetically different strains have been produced synonymous site per year (11) and that of the entire human before or during a pandemic, and then the descendants have immunodeficiency virus type 1 (HIV-1) genome at 10.3 x

been endemically transmitted in various areas, accumulating 10 per synonymous site per year(23). A high synonymous nucleotide substitutions independently. This pattern of trans- substitution rate gives rise to frequent parallel substitutions. mission causes cocirculation of genetically and possibly anti- When two strains have aparallel substitution,this substitution genically different strains in different parts of the world. maynotbe countedas achange between the strains.Thus, the The nucleotide substitution rates of the EV70 VP1 gene accumulation of parallel substitutions for a longer periodmust obtained here(3.8 x 10-3substitutionpernucleotide per year eraseactually occurring substitutions detectable between

iso-forobserved nucleotide differences and 5.0 x

10'

substitu- lates. This might be the reason for the

lower

synonymous tion per nucleotide per year forthe weighted meanof synon- substitution rate of influenza viruses than of EV70, which was ymous and nonsynonymous substitutions [Kt]) were higher analyzed for approximately 10 years, whereas influenza viruses than those reported for the nonstructural protein (NS)gene were evaluatedfor nearly 50 years or more.

TABLE 4. Nucleotidesubstitutionpatternsandrelative substitution frequenciesinVP1 region of18 EV70 isolates

TypeofsubstitutionPreceding Total no. of No. in progenies Relative substitutionfrequency(fl)

Typeofsubstitution vrs_virus ncetdsl_{nucleotides A U C G Total' A U C G Total"}

Total A 294 5 3 43 51 1.47 0.88 12.62 15.02

U 236 10 66 1 77 3.67 24.22 0.36 28.25

C 210 3 82 3 88 1.24 33.81 1.24 36.29

G 178 42 0 0 42 20.43 0 0 20.43

Total 918 55 87 69 47 258(233) 25.34 35.28 25.10 14.27 100 (91.13)

At 3rdposition of codon A 91 1 2 33 36 0.40 0.80 13.26 14.46

U 94 6 56 0 62 2.33 21.79 0 24.12

C 66 3 67 0 70 1.66 37.13 0 38.79

G 55 34 0 0 34 22.61 0 0 22.61

Total 306 43 68 58 33 202 (190) 26.60 37.53 22.59 13.26 100 (94.78)

At4-fold-degeneratesite of

3rdposition of codon A 59 1 2 15 18 0.62 1.24 9.29 11.15

U 44 5 25 0 30 4.15 20.76 0 24.91

C 28 3 25 0 28 3.91 32.62 0 36.53

G 12 9 0 0 9 27.40 0 0 27.40

Total 143 17 26 27 15 85 (74) 35.46 33.24 22.00 9.29 100 (90.07)

aCumulativenumbers ofsubstitutionsaregiven in parentheses.

bCumulative

fii

values for transitions are given in parentheses.

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In addition, the remarkable feature of the nucleotide sub-stitution patterns of EV70 was the extremely high frequency of transitions, i.e., low transversions. The relative frequency of transitions (C*--iUand A*- ->G) was more than 90% in all three estimations, i.e., in total substitutions, at the third position of codons, and at fourfold-degenerate sites (Table 4). Also, a similar high frequency of transitions (87.0%) at the fourfold-degenerate site of the codons was obtained when parallelsubstitutions were excluded from analysis (not shown), indicating that the transitions were found throughout VP1 genome, not at the specified sites. Although transitions in naturally occurring mutations are known to be much more frequent than expected from random substitutions (7, 22), the frequency of transitions in EV70 VP1 was much higher than that reported for the pseudogene DNA of human origin (55.6% on average) (7), for the third codon position of the Mahoney and Sabin strains of type 1 poliovirus (77.1%) (our calculation), for the third codon position of influenza virus (77.7%) (35), and for fourfold-degenerate codon position of three coding regions (gag, pol, and env) of retroviruses (66.4%) (10). As noted above, it is probable that the analysis in the short evolutionary term, which made actually occurring substi-tutions more frequently detectable, accounted for the higher frequency of transitions in EV70 than other RNA viruses. We have found a similar high frequency of transitions as well as a high synonymous substitution rate in over 20 years of evolu-tionary study of coxsackievirus A24 variant, another enterovi-ruscausing AHC(43). At present, however, we do not know thefactor(s) affecting more frequent transitions observed on genomes of RNA viruses thanpseudogenes.

Shimizu et al. (39) studied the direction of substitution patterns of the HIV gag gene and reported that the relative frequency of nucleotide change between A*- --G at the third codon position was the highest among all types of nucleotide changes and much greater than that of the C*- -iU change. For influenza virus as reported by Saitou (34), on the other hand, the rate of nucleotide substitutions between A<- --G at thethird position is comparable to that between C*- --U. In our study of the EV70 VP1 genome (Table 4), the relative frequency of C<- --U was much higher than that of G*- ->A, both in total substitutions (58.0% versus 33.0%) and in four-fold-degenerate sites (53.4% versus 36.7%). However, the orderof relative substitution frequency atfourfold-degenerate sites was C-->U (32.6%), G->A (27.4%), U-->C (20.8%), and

A-*G

(9.3%). It is noteworthy that the value for G- A was higher than that for U->C. Therefore, we may not conclude at present that the frequency of theC*---U change was higher than that of the G*---A change for EV70 VP1 nucleotide substitutions.

Incontrast tothe highsynonymous rate, thenonsynonymous rate of EV70 VP1 was extremely low at 0.32 x

10-'

per nonsynonymous site per year. This value was almost 1/10 of that for influenzaAvirusHA or NA, 2.9 x 10-3 to 2.8 x

10-3pernucleotide per year (11), and 1/5 of those for HIV gag and env genes, 1.7 x

10-3

and 1.6 x 10-3pernucleotide per year, where the rates were the lowest among various coding

regionsofHIV(21). As a result, the ratio of nonsynonymous tosynonymous rates for EV70 VP1 is as low as 1/70, whereas the ratios for influenzaAvirus and HIV areapproximately 1/5. Suchalow ratio indicates a strongerrestriction in nonsynony-mousnucleotide changes for the EV70 VP1 gene than for HA andNAgenesof influenza virus or gag, pol, and env genes of HIV.

We chose theVP1 sequences for comparative study of the isolates because of the region with least amino acid homology among enteroviruses; the sequences could not be aligned

without presuming a certain insertion or deletion among them, particularly in both N and C termini. Our study revealed, however, that the rate of amino acid substitutions for EV70 VP1 among isolates was markedly lower than for the envelope proteins of other RNA viruses. The amino acid sequences of EV70 VP1 corresponding to the 1-barrel and loop structures known to constitute the internal framework of the viral capsid seem to be strictly conserved among the isolates. On the other hand, almost half (43%) of the amino acid substitutions were concentrated in the N and C termini of the VP1 protein. Thus, our study implies that the rigid icosahedral conformation of enteroviruses requires a much greater functional and confor-mational constraint against amino acid substitutions than do the outer surface proteins of enveloped viruses such as influ-enza viruses and HIV. The limited amino acid substitutions on VP1 and P1 of foot-and-mouth disease virus were described by Martinez et al. (25).

Despite such a low frequency, the different amino acid substitutions occurring on the EV70 genome of different evolutionary lineages persisted independently through en-demic transmission, giving rise to polymorphism among iso-lates in different parts of the world.

ACKNOWLEDGMENT

This work was supported in part by a grant-in-aid for scientific researchfromtheMinistryofEducation,ScienceandCulture, Japan.

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