Polymorphism and Evolution of Influenza A Virus Genes’
Naruya Saitou and Masatoshi Nei
Center for Demographic

and Population

Genetics,

Universityof Texas at Houston

The nucleotide sequences of four genes of the influenza A virus (nonstructuralprotein, matrix protein, and a few subtypes of hemagglutinin
and neuraminidase)are compiled for a large number of strains isolated from various locations andyears, and the evolutionary relationship of the sequences is investigated. It is shownthat all of these genes or subtypes are highly polymorphic and that the polymorphic
sequences (alleles) are subject to rapid turnover in the population, their averageage being much less than that of higher organisms. Phylogenetic analysis suggeststhat most polymorphic
sequences within a subtype or a gene appeared during thelast 80 years and that the divergence among the subtypes of hemagglutinin
genes
might have occurred during the last 300 years. The high degree of polymorphismin this RNA virus is caused by an extremely high rate of mutation, estimated tobe 0.0 l/nucleotidesite/year. Despite the high rate of mutation, most influenza
virus genes are apparently subject to purifying selection, and the rate of nucleotide
substitution
is substantiallylower than the mutation rate. There is considerable
variation in the substitution rate among different genes, and the rate seems to belower in nonhumanviral strains than in human strains. This difference might beresponsible for the so-called freezing effect in some viral strains.

Introduction

The influenza virus is a single-stranded RNA virus and is classified into three
types, A, B, and C, according to immunological differences (Webster et al. 1982). The
type A virus is the major cause of influenza epidemics not only in humans but inother mammals and birds. As do other RNA viruses, this virus has a high mutation
rate, the mutation rate per year being approximately one million times higher thanthat of DNA organisms (Air 198 1; Holland et al. 1982). Because of this high mutation
rate, the influenza A virus becomes progressively resistant to antibodies made against
older viruses. The high mutation rate is also responsible for the high degree of polymorphism observed in the genes in this organism (Nei 1983).
In recent years, the nucleotide sequences of several influenza virus genes havebeen determined for various strains, including those that have been kept in refrigeratorsfor many years. These data provide a unique opportunity for studying the evolutionary
history of polymorphic genes as well as for estimating the rate of nucleotide substitution
in evolution.
Previously, Krystal et al. (1983) and Martinez et al. (1983) estimated the rate ofnucleotide substitution for some of the genes of this virus, but their estimates are notvery reliable because they simply compared genes from two strains isolated in different
years. Nei (1983) also estimated the substitution rate by fitting a regression equation
1. Key words: influenza A virus, phylogenetic tree, polymorphism,

mutation rate, rate of nucleotide

substitution.
Address for correspondence and reprints: Dr. Masatoshi Nei, Center for Demographic and Population
Genetics, University of Texas at Houston, P.O. Box 20334, Houston, Texas 77225.
Mol. Biol. Evol. 3(1):57-74. 1986.

0 1986 by The University of Chicago. All rights reserved.
0737-4038/86/0301-3101$02.00

58

Saitou and Nei

to data from various strains isolated in different years. In the presence of polymorphism,
however, his method is expected to give an overestimate. A better method of estimating
the rate of nucleotide substitution for this case is first to examine the evolutionary
relationship of genes obtained from different strains and then use only strains that areclosely related by descent.
The main purpose of this paper is to conduct phylogenetic analyses of nucleotide
sequences obtained from various strains and to estimate the rate of nucleotide substitution by using the method mentioned above.
Nucleotide Sequences Used

The genome of the influenza A virus consists of eight RNA segments coding for
10 different proteins (e.g., Lamb 1983). In the present study, we used four different
genes, i.e., the hemagglutinin gene on segment 4, the neuraminidase gene on segment
6, the matrix protein 1 gene on segment 7, and the nonstructural protein 1 gene on
segment 8. The first two genes are known to be responsible for the antigenic variationof this virus. We used only the coding regions of these genes, excluding the initiation
codon. In the regions studied, there were no deletions or insertions. We used 46 strainsin this study, and they are presented in table 1. Each strain is designated by the abbreviation of the location and the year in which it was isolated (see the legend totable 1).
The hemagglutinin gene is highly variable and can be classified into 13 subtypes(HI-H13) according to the immunological differences in hemagglutinin (Webster etal. 1982). The average nucleotide difference among these subtypes is 5 1% per nucleotidesite (Nei 1983). We have therefore treated these subtypes separately, as though they
were different genes. Sequence data useful for our analysis were available only for four
subtypes, i.e., HI, H2, H3, and Hll. The viral strains used for these four subtypes
are given in table 1. The hemagglutinin gene is composed of three functional regions,
i.e., signal peptide (SP), hemagglutinin 1 (HAl), and hemagglutinin 2 (HA2), andsequence data for these regions were combined unless otherwise mentioned. The nucleotide sequences available were not always complete, and the number of nucleotides
used are presented in table 1.
The neuraminidase gene is also highly variable and can be divided into nine
subtypes (NI-N9) (Webster et al. 1982). Two of them (NI and N2) were used here.
Since sequence data for the Nl subtype were available only for the first 168 bases inmost strains, our analysis was restricted to this region. There were complete nucleotide
sequences (1,404 bases) available for five N2 subtype strains. The coding region forthe matrix protein 1 gene (MXI) overlaps with that for the matrix protein 2 gene(M.2) on segment 7, and, similarly, the coding region for the nonstructural protein
genes 1 (NSI) and 2 (NS2) overlap with each other. Therefore, we used only the genes
for MX 1 and NS 1. For gene NSI, there were two sets of data; partial sequences andcomplete sequences (see table 1). In the construction of phylogenetic trees, all sequence
data were used, excluding unshared nucleotides. In the estimation of the rate of nucleotide substitution, however, only complete sequences were used.
Phylogenetic Trees

Phylogenetic trees were constructed by using the maximum parsimony method(see Fitch 1977). The trees obtained for the four genes are presented in figures 1 andInfluenza

Virus Genes

59

Table 1
Influenza A Virus Gene Sequences Used in the Present Study
Strains b,cGenes and Subtypes*
Hemagglutinind
HI subtype:Sp(48), HAI(228)......

.

H2 subtype: Sp(42), H-41(207)... .....

H.3 subtype:SZ’(45), HA1(983),

HA2(656)”

HZ1 subtype:SZ’(45), HA1(201)..

WIS/3O(S), PR/34, NWSf33, BEL/42, FW/50, LO/
57, NJ/76 [ 11; WSN/33 [2]; USSR/77 [3]
RI/57, TOK/67, NED/68, BER/68, GDR/72(D),ALB/77(D), ALB77(P) [4]
UKR/63(D)[5]; NT/68 [6, 71; ENG/69, QU/70 [7];MEM/7 1 [8]; MEM/72 [9]; HK/7 1, ENG/72, PC/73,
VIC/76, AC/76, TX/77, BA2/79 [lo]: VIC/75 [ 111;
ENG/77 [ 121; BA1/79 [ 131
ENG/56(D), UKR/60(D),AU/75(T), MEM/76(D),W/78(D)

Neuraminidase:N1 subtype (168)
N2 subtype
Matrix protein.........

.

....

(1404).......

..

....

1 (210)..........

Nonstructuralprotein 1:
Partial sequences ( 192) . . . . . . . .
Complete sequences (687) f . . .

1141

WIS/3O(S), PR/34, BEL/42, FW/50, LO/57, NJ/76,
ON/77(D), USSR/77, MEM/78 [ 151; WSN/33 [ 161. . RI/57 [ 171; NT/68 [ 181; UD/72 [ 191; VIC/75 [20];BA1/79 [21]PR/34 [22]; FPV/34(F) [23]; FW/50, LO/57,
CG/77 [4]; UD/72 [24]; BA1/79 [25]

RI/57,

RI/57, CG/77, AU/78(B) [4]
PR/34 [26]; FPV/34(F) [27]; FM/47, FW/50, USSR/
77 [28]; UD/72 [29]; ALB/76 [30]; AL/77 [31]’Numbers in parentheses are the number of nucleotides.
b Numbers in brackets are references: 1 = Air et al. 1981; 2 = Hiti et al. 1981; 3 = Concannon et al. 1984; 4 = Air andHall 1981; 5 = Fang et al. 1981; 6 = Both and Sleigh 1980; 7 = Sleigh et al. 1981; 8 = Newton et al. 1983; 9 = Sleigh et
al. 1980; 10 = Both et al. 1983; 11 = Min Jou et al. 1980; 12 = Hauptman et al. 1983; 13 = Both and Sleigh 1981; 14= Air 198 1; 15 = Blok and Air 1982; 16 = Hiti and Nayak 1982; 17 = Elleman et al. 1982; 18 = Bentley and Brownlee
1982; 19 = Markoff and Lai 1982; 20 = Van Rompuy et al. 1982; 21 = Martinez et al. 1983; 22 = Allen et al. 1980; 23= McCauley et al. 1982; 24 = Lamb and Lai 1981; 25 = Ortin et al. 1983; 26 = Baez et al. 1980; 27 = Porter et al. 1980;
28 = Krystal et al. 1983; 29 = Lamb and Lai 1980; 30 = Baez et al. 1981; 31 = Buonagurio et al. 1984.
c The letter in parentheses denotes the organism from which the virus strain was isolated. B = black duck, D = duck, P
pintail (these three belong to Anatinae); T = tern (Steminae); F = fowl (includmg chicken and duck); and S = swine;s USscrofa). Strains without parentheses are from humans.
d SP = signal peptide; HAI = hemagglutinin 1; and HA2 = hemagglutinin 2.
’Ref. 13 does not have SP sequences, and Refs. 7 and 10 have only HA1 sequences, except for HA2 sequence of theMEM/72 strain. For NT/68, SP and HA2 are from ref. 6, and HA1 is from ref. 7.
‘Since the length of open reading frame for three strains (PR/34, FPVJ34, and ALB/76) is shorter than that for others,
seven unshared codons are not included.

2. When two or more trees with nearly the same total number of substitutions (oneor two differences) were obtained for the same set of data, a concensus tree withcollapsed node denoted by C was produced (e.g., fig. 1B). The root of a tree was locatedby using an outside strain. For hemagglutinin subtypes (fig. 1A- 1D), Webster et al.‘s(1982) dendrogram was used to identify outside strains. In the case of neuraminidase
subtypes (fig. 2A and 2B), there were no such dendrograms available, but for the Nlsubtype the root could be located by using information on the HI subtype, because
most of the strains studied were the same for the HI and Nl subtypes. For the N2
subtype, the oldest strain (RI/57) was assumed to be the ancestor (root). For matrix
protein 1, FPV/34(F) was used as the outside strain, and for nonstructural protein 1,
ALB/76(D) was assumed to be the outside strain. Both strains were isolated from

60

Saitou and Nei
BA1/790A2/79

ALB/77(P)AU/75(T)
UKR/jO(D)/

NY’78(D)/ME;/76(D)FIG. I.-Phylogenetic
trees for the HI, H2, H.?, and HZ1 subtypes of the hemagglutinin gene reconstructed by the maximum parsimony method. An unresolved node of a consensus tree is denoted by a circle
enclosing a C and the root by a circle enclosing an 0. For the determination of roots, see text. The letter inparentheses denotes the initial of the organism from which the strain was isolated. Strains without this initialare from humans. For the names of organisms, see table 1. Branch lengths are approximately proportional
to the number of nucleotide substitutions. (A) = the HZ subtype (both hemagglutinin 1 [ HAZ ] and signal
peptide [SP] used); (B) = the H2 subtype (HAZ and SP used); (C) = the H3 subtype (only HAZ used); and(II) = the HZZ subtype (HA1 and SP used).

birds and were quite different from other human strains. Since the number of nucleotide
substitutions for each branch cannot be estimated uniquely by the maximum parsimony method, only approximate branch lengths are given in figures 1 and 2.
The phylogenetic trees in figures 1 and 2 are quite different from ordinary trees

Influenza Virus Genes

61

ON/

NJ/76
BAl/79LOYKi7

NT/68

BA1/79
.
UDl72

RI/57

LOY/57"Fu/SO

m/34

FPV/34(F)FIG. 2.-Phylogenetic
trees for the NI and N2 subtypes of the neuraminidase gene, for the matrix
protein 1 gene and for the nonstructural protein 1 gene. (A) = the NZ subtype; (B) = the N2 subtype;(C) = matrix protein 1;and (D) = nonstructural protein 1. See fig. 1 for explanation of symbols.

because the strains used here were isolated in different years and mutations accumulated
rapidly. The differences in evolutionary pattern among the phylogenetic trees in figures
1 and 2 largely depend on whether there was polymorphism in the past. If a gene werealways monomorphic, one would expect all strains isolated in different years to belocated on a line without branching, as in the case of FW/50 and RI/57 in figure 2(C)(matrix protein 1). On the other hand, if a gene is highly polymorphic and polymorphic
alleles stay in the population for a long time, we would expect the type of trees represented by figures 1A and 1D (hemagglutinin).

62

Saitou and Nei

Figures l(A), l(B), l(C), and (D) show the phylogenetic trees for the HI, H2,
H3, and HI1 subtypes of the hemagglutinin gene, respectively. A substantial amountof polymorphism exists in all subtypes, and, in general, mutations accumulate moreor less linearly with time. However, figure l(A) shows one anomaly. That is, strain
USSR/77 did not accumulate mutations very much after it separated from FW/50.
This anomaly has been previously noted by Nakajima et al. ( 1978) and will be discussed
later. Subtypes H&H13 have been found only in nonhuman species, but the otherthree subtypes infect humans as well as other organisms. Except for NJ/76 of the HI
subtype, strains of subtypes HI, H2, and H3 isolated from humans are monophyleticand form one cluster (fig. 1A). NJ/76 is known to be close to swine strains (Blok andAir 1982), though it was isolated from humans, and our result, showing this strain
clustered with a swine strain (WIS/30), confirms Blok and Air’s earlier conclusion.
The recent H3 subtype strains in humans (Hong Kong flu) are all derived from NT/
68, which in turn shares a common ancestor with UKR/63 isolated from duck. A
main trunk is noticed in the phylogenetic tree, suggesting that in each year there wasa dominant strain. The topology of the tree of H3 subtype is similar to that of Bothet al. ( 1983) if we compare the same set of 12 strains. The HI 1 subtype infects only
nonhuman species and seems to be highly polymorphic.
Figures 2(A) and 2(B) present the phylogenetic trees for the Nl and N2 subtypesof the neuraminidase gene, respectively. The tree for the Nl subtype shares sevenstrains with that for the hemagglutinin HI subtype (fig. 1A). This is because the subtypes
that were dominant in humans until 1957 are HI for the hemagglutinin gene and Nl
for the neuraminidase gene. Consequently, the topologies of these two trees are similar.
In the tree of the N2 subtype (fig. 2B), a trunk is identified, as in the case of the H3
subtype of the hemagglutinin gene.
Figures 2(C) and 2(D) represent phylogenetic trees for the matrix protein 1 (M-XI)
and nonstructural protein 1 (NSI) genes, respectively. The tree for NSl is similar tothat of Buonagurio et al. (1984) if we consider the same set of strains. The trees forM.1 and NSI share six strains, and the topological relationships of these six strains
are identical, if the branch linking strains FW/50 and RI/57 is eliminated. This similarity of topology suggests that these two sequences have evolved together, although
they are located on two different RNA segments. Note that the strains used for constructing a tree for the matrix protein 1 and nonstructural protein 1 do not have thesame subtypes of the hemagglutinin and neuraminidase genes. For example, in figure2(C) the subtypes of the hemagglutinin and neuraminidase genes for strains PR/34,
RI/57, and BA1/79 are HlNl, H2N2, and H3N2, respectively. This is because thegenes of influenza A virus are segmented and occasionally reassorted (Webster et al.
1982).
Pattern of Accumulation of Nucleotide Substitutions
We studied the pattern of nucleotide substitution using the main evolutionarylines identified by the above phylogenetic analysis. The oldest strains, which were at
the root (RI/57 for the N2 subtype) or near the root (PR/34 for HI, Nl, MXI, andNSl; RI/57 for H2; and NT/68 for H3), were treated as the ancestral strains, andstrains that diverged earlier than the appearance of the ancestral strains were excluded
from the analysis to minimize the effect of polymorphism. Most of the strains excluded
were those isolated from nonhuman vertebrates, and the strains used for the analysis

Influenza Virus Genes

63

were all from humans. The HI1 subtype was not included in the analysis, since nomain evolutionary line was identified. The number of nucleotide substitutions per site
between two strains was estimated by means of Jukes and Cantor’s (1969) formula.
Figure 3 presents the pattern of accumulation of nucleotide substitutions for thegenes or subtypes (HI, Nl, MXI, and NSI) in which the ancestral strain was identified
as PR/34. The results for the other subtypes (H2, H.?, and N2) are given in figures 4
and 5. In the HI subtype, data for SP and HA1 were combined, since they weresimilar. There are two sets (partial and complete sequences) of data for NSl, and theresults from the comparison of complete sequences (687 bases) are shown, the data
for strains RI/57, CG/77, and AU/78 being excluded.
The accumulation of nucleotide substitutions is approximately linear for all genes
and subtypes examined. Particularly, in the H3 subtype of the HA1 gene, where thelargest number of strains is used, the linearity is quite satisfactory (fig. 5). However,there are exceptional strains that do not follow the pattern of linear accumulation.
They are USSR/77 and its close relative MEM/78 (marked by open circles in fig. 3).
The HI, Nl, and NSI genes in these strains show a small number of nucleotide
substitutions after they branched off about 1950. A slowdown of nucleotide substitution

t USSR/77 (NSl)

0

t
1934

I

I

I

I

I

10

20

30

40

50

t
1950

t
1977

Years

FIG. 3.-Accumulationof nucleotide substitutions in influenza A virus genes. Signal peptide (SP) andhemagglutinin 1 (HAI) of the HI subtype, neuraminidase (NA) of the NZ subtype, nonstructural protein 1(NSZ),and matrix protein 1 (MXI) are presented. The year of isolation of the ancestral strains is I934 for
all four genes. Two strains (USSR/77 and MEM/78) that were excluded from the regression analysis aredenoted by open circles, and they are connected by dashed lines with strains isolated in 1950. All nucleotidesof codons were used.

64

Saitou and Nei

SP(H3)
NA(N2)G

HA2(H3)

0

10

20
Years

FIG. 4.-Accumulationof nucleotide substitutions in influenza A virus genes. SP and HA1 of the H2
subtype, SP and HA2 of the H3 subtype, and NA of the N2 subtype are presented. The years of isolation
for the ancestral strains are 1957 for the H2 and N2 subtypes and 1968 for the H3 subtype.

is also observed for the MXl gene of strain CG/77, though it could arise from stochastic
errors. All genes of the USSR/77 strain have been shown to be quite similar to thosestrains circulating about 1950 (Nakajima et al. 1978), and the phylogenetic treesfor HI, Nl, and NSl confirm this finding (see Discussion). Since USSR/77 andMEM/78 are clearly abnormal, they were excluded from the following analysis.
Rate of Nucleotide Substitution

We applied two types of regression analyses to estimate the rate of nucleotide
substitution. One was the regression through the origin as used by Nei (1983). Thismethod seems to be suitable for the H2 and H3 subtypes of the hemagglutinin gene,
the N2 subtype of the neuraminidase gene, and the MXI gene, where the ancestral
strains of these genes are at the root or quite close to the root. On the other hand,when the ancestral strain branched off the root and if the branch length is not negligibly
small as in the case of the NSI gene from strain PR/34, this method may give an
overestimate of the substitution rate. Therefore, the usual regression method, which
would alleviate this problem, was also used for the HI, Nl, and NSI genes. The
regression coefficients for the former and the latter methods are given by

bl = C Xi.YJCXfInfluenza Virus Genes

65

0.00
0

5

t

10
Years

1968

FIG. 5.-Accumulationof nucleotide substitutions in the H3 subtype of the hemagglutinin 1 gene of
the influenza A virus genes. The patterns of the accumulation of nucleotide substitutions for the first andsecond positions (open circles) and for the third position (black dots) are shown. The year of isolation of
the ancestral strain is 1968. For each case, the regression lines are superimposed. The regression coefficientis O.O050/site/year for the first and second position and O.O104/site/year for the third position.

and
b2 =

[C

XiYi -<Cxi

CYi)lnl/[C xf - <C -d2/n19

respectively, where Xiis the time of isolation of the ith strain measured from the origin,
yi is the nu m b er of nucleotide substitutions between the ith strain and the ancestral
strain, and n is the number of strains used excluding the ancestral one. (Note that b,
usually has a smaller sampling error than does b2 because the number of degrees of
freedom for bl is larger than that for b2.) The regression coefficients thus obtained
were used as estimates of the rate of substitution. These estimates were obtained foreach of the three nucleotide positions of codons separately. The results obtained areshown in table 2. In this table, the average rates for the first and second positions are
combined, since these two positions show similar rates.
Table 2 shows that the rates of nucleotide substitution obtained from b2 are
generally smaller than those obtained from bl, as expected. The rate of nucleotide
substitution varies considerably with the gene examined. The highest rate (0.0 124) for“all positions” is observed for the signal peptide of the H.3 subtype, and the lowest

66

Saitou and Nei

Table 2Rates of Nucleotide Substitution

in Influenza A Virus Genes
MEAN +- SE SUBSTITUTION
RATE/SITE/
YEARS

GENE
Signal peptide: . . .

N

1st + 2d

3d

All

HZ

(4)
(4)
(4)

1.14 + 0.16
(1.16 + 0.23)
1.48 f 0.05
0.95 zk 0.27
1.18

0.55 f 0.08
(0.47 rfI0.11)
0.71 f 0.14
1.24 + 0.23
0.83

0.26

H2
H3

0.29 + 0.11(-0.01 I!I0.15)
0.35 Ik 0.20
1.39 +- 0.21
0.68

HI

(4)

H2
H3

(4)
(15)

0.29 + 0.06
(0.10 + 0.08)
0.12 Ik 0.02
0.50 I!z0.02
0.41

0.91 f 0.13
(0.54 + 0.18)
1.08 f 0.23
1.04 -e 0.03
1.02

0.48 -I- 0.08
(0.23 f 0.11)
0.42 + 0.07
0.68 + 0.01
0.61

0.32
(0.19)
0.11
0.48
0.40

Mean b . . . . . . .
Hemagglutinin 1:

RATIO(1st + 2d/3d)

SUBTYPE

Mean b . . . . . .

&h
1.46
0.58

Hemagglutinin 2

.

H3

(6)

0.10 + 0.02

0.70 + 0.07

0.30 + 0.03

0.14

Neuraminidase:

..

Nl

(4)

N2

(5)

0.38 f 0.01
(0.39 f 0.02)
0.29 f 0.02
0.30

0.61 + 0.09
(0.80 +- 0.12)
0.63 f 0.03
0.63

0.45 f 0.04
(0.52 f 0.05)
0.40 + 0.02
0.41

0.62
(0.49)
0.46
0.48

(5)

0.17 -t 0.02
(0.10 + 0.02)

0.44 +- 0.04
(0.31 + 0.05)

0.26 f 0.03
(0.17 + 0.03)

0.39(0.32)(7)

0.05 + 0.01

0.37 f 0.04

0.15 + 0.01

0.14

0.31

0.71

0.41

0.44

Mean b .

. ...

Nonstructuralprotein I...

Matrix protein 1.

Grand meanb.

’Values of X and its SE are multiplied by 100. Figures in parentheses are bz and its SE. lst, 2d, and 3d denote the first,
second, and third nucleotide positions of codons, respectively.
b Mean of b, weighted by the number of nucleotides.

rate (0.00 15) is observed in the gene for matrix protein 1, the former being about eighttimes higher than the latter when all nucleotide positions were compared. The rate
for the signal peptide of the H.? subtype is exceptionally high, and the other subtypes
for the signal peptide show lower rates. This difference results mainly from the highrate in the first and second positions for the H3 subtype. Similar difference among
subtypes is observed in the hemagglutinin 1 gene. However, even the average rate
(O.O083/site/year) for three subtypes of the signal peptide is more than five times
higher than that of the matrix protein 1 gene. The average rate for all genes examined
is 0.004 1/site/year.
Martinez et al. (1983) estimated the rate of nucleotide substitution for the N2
subtype of the neuraminidase gene to be 0.0033-0.0070. Our estimate for this subtype(0.0040) is closer to their minimum estimate. Krystal et al.‘s (1983) estimates for
nonstructural proteins and for the H3 subtype of hemagglutinins 1 and 2 are 0.00220.0034 and 0.0045-0.0065, respectively. Our estimates for the NSI gene (0.0026 bybl) and for the hemagglutinin gene (the average of estimates for HAI and HA2 of theH3 subtype weighted by the number of nucleotides = 0.0053) are again closer to thelower ends of the range of their estimates. This indicates that the estimate obtainedInfluenza Virus Genes

67

from a simple comparison of the two strains tends to give an overestimate. Nei’s(1983) estimate for MXl (0.0013) and NSl (0.0027) are both close to the estimatesobtained in the present study. In the case of NSl, Nei (1983) used Air and Hall’s( 198 1) shorter ( 192 nucleotides) sequence data for four strains, whereas we used longer(687 nucleotides) sequence data (see table 1) for five strains. The agreement between
the two estimates suggests that the rate of nucleotide substitution for NSI is rather
uniform for all gene regions.
It is seen from table 2 that the average rate of nucleotide substitution for the firstand second nucleotide positions of codons is considerably lower than that for the third
position except for the H3 signal peptide. Indeed, the ratio of the former to the latter
is 0.62 or less. This is because certain amino acids in proteins are highly conserved in
the evolutionary process. This is known to be the case even with this rapidly evolving
virus (Nei 1983).
The above estimates of the rate of nucleotide substitution were obtained using
human strains only. It is difficult to obtain reliable estimates of the substitution rate
for strains derived from nonhuman vertebrates, but there are some indications thatthe rate for nonhuman strains is lower than that for human strains. For example,
strain NJ/76 and a swine strain WIS/30 share a common ancestor in both the HI
subtype of HAI (fig. 1A) and the Nl subtype of neuraminidase (fig. 2A). (NJ/76 is
known to be essentially a swine strain, though it was isolated from humans; Blok andAir 1982.) Comparison of these two strains is expected to give an overestimate of therate, but the estimates obtained (0.0024 and 0.003 1 for HI and NI, respectively) arelower than those obtained from human strains. All five strains of the H1I subtype ofhemagglutinin were derived from birds. Because there was no main evolutionary line
for this gene (fig. 1D), these strains were not used for estimating substitution rate.
However, if we use MEM/76 as a reference strain, the rate can be estimated by the
regression analysis (b2) mentioned earlier. This gives an estimate of 0.00 19 (the signal
peptide and HAI genes combined), which is approximately one-third of the averagerate for three HAI genes. Comparison of two bird strains, FPV/34(F) and AU/78(B),in the NSI gene (fig. 2D) also gives a rate (0.0008) that is approximately one-third to
one-half of the rate (0.0026 by bl or 0.0017 by b2) for human strains (table 2). It is
not clear why the rate of nucleotide substitution is lower in nonhuman viral strains
than in human strains, if this difference is real.
Discussion

Rate of Nucleotide Substitution in RNA Genomes
We have seen that the rate of nucleotide substitution is of the order of 10w3/site/
year for most influenza A virus genes studied. Using the oligonucleotide mapping
technique, Takeda et al. ( 1984) estimated the substitution rate for the enterovirus type
70 (single-stranded RNA virus) to be 4 X 10m3/site/year. Gojobori and Yokoyama( 1985) estimated that the substitution rates for the gag gene (gene for internal proteins)and for the v-mos gene (oncogene) in a retrovirus (single-stranded RNA virus) are
6.3 X 10T4 and 1.3 1 X 10B3, respectively. These high rates of nucleotide substitution
in RNA viruses are believed to arise from the absence of proofreading exonucleases
for correcting replication error (Holland et al. 1982). In DNA viruses such as thepapovavirus, the rate of nucleotide substitution is much lower and of the order oflo-’ (Soeda and Maruyama 1982). The same order of substitution rate [(2-4) X lO_‘]

68

Saitou and Nei

has been observed for many mammalian genes (Li et al. 1985). Since DNA viruses
use the replication system of the host, the mutation rate is expected to be similar tothat of the host.
We have seen that the rate of nucleotide substitution at the first and second
positions of codons is much lower than that at the third position except in the genefor the H3 signal peptide of hemagglutinin. In this exceptional gene, all three nucleotide
positions have essentially the same substitution rate, and the rate is -O.Ol/site/year.
These values suggest that for some reason there is little purifying selection operating
for this gene and that nucleotide substitution occurs at the same rate as does themutation rate. They also suggest that the relatively high substitution rates for HAI
and neuraminidase are not caused by positive selection pressure owing to the hostimmune system against this virus but are simply the result of a high mutation rate.
The lower rate for HA2 than for HAI apparently occurs because hemagglutinin 2
constitutes the supporting leg for hemagglutinin 1 (Wilson et al. 198 1) and thus issubject to stronger purifying selection than hemagglutinin 1. The lowest rate observed
for the matrix protein 1 gene is also understandable from the function of this protein,since this protein underlies the lipid bilayer of the viral core and is important for viralconstruction and budding (Lamb 1983). Generally speaking, there seems to be a goodcorrelation between the rate of nucleotide substitution and the level of functional
constraint in the influenza A virus genes, as is true in the case of DNA genomes(Kimura 1983).
Deceleration of Substitution Rate
Since Nakajima et al. (1978) reported the striking similarity between USSR/77
and the strains that were circulating during the 195Os, the problem of deceleration of
nucleotide substitution has attracted attention from many authors. Krystal et al. (1983)
sequenced NSl genes from two strains isolated about 1950 and compared these sequences with that of USSR/77. They showed that only five nucleotide substitutions
occurred during more than 20 years. Our lower estimate (via b2) of the substitution
rate for the NSI gene is 0.00 17 (table 2). Therefore, if nucleotide substitution occurredat this rate, there should have been approximately 30 substitutions from 1950 to 1977
in the sequence of 687 nucleotides. This indicates how slow the nucleotide substitution
was in the USSR/77 lineage. The same slow rate of substitution is also observed for
the MEM/78 strain of the NI subtype of neuraminidase (see fig. 3). The reason forthis unusual deceleration is not known at present. The deceleration of the rate does
not seem to be an inherent property of these strains, since after their reappearance
they started to evolve quickly (Young et al. 1979). Palese and Young ( 1983) speculated
that USSR/77 might have originated from a laboratory stock that had been kept frozen
for a long time. Another possibility is that about 1950 the ancestor of this strain
infected some nonhuman organism, one in which the replication rate of this virus was
reduced, and that it later reinfected humans.
Age of Sequence Polymorphism
The phylogenetic trees in figures 1 and 2 indicate that human and nonhumanstrains can coexist for a long time showing sequence polymorphism. For example, thepolymorphism of strains (or lineages) NJ/76 (a “swine” strain) and LOY/57 in HI(fig. 1A) apparently existed for more than 27 years (i.e., from 1930 to 1957). It isInfluenza Virus Genes

69

possible to estimate the time of divergence between these two strains under the assumption of a constant rate of evolution. Let tl and t2 be the years of isolation ofstrains 1 and 2 respectively, with tl > t2. Then, the expected number of nucleotide
substitutions between the two strains (d) may be expressed as d = (2 T + tl - t2)h,where T is the time of duration of polymorphism, i.e., the time between t2 and theyear of divergence (the root denoted by 0), and h is the rate of nucleotide substitution
per site per year. The d value may be computed by Jukes and Cantor’s ( 1969) formula.
Therefore, T can be estimated by the equation
T = d/(2X) - (t, - t&2.

In the case of the HI subtype (fig. lA), we do not know the h value for nonhumanstrains, but if we use h = 0.005 (the average for SP and HAI) from table 2, we obtain2’= 16 years. This value is certainly an underestimate and apparently caused by thefact that h is smaller in nonhuman vertebrates than in humans. In the case of nonstructural protein 1 in figure 2(D), a more reasonable result is obtained even if we use
the h value from human viral strains. In this case the polymorphism of AL/77 (humanstrain) and ALB/76 (duck strains) existed for at least 42 years (i.e., from 1934 to 1976),
and the d value between the two strains is 0.383. If we use h = 0.0026, we obtainT = 72. This suggests that the two strains have coexisted since 1904 or earlier.
Polymorphismof Hemagglutinin

Genes

As mentioned earlier, 13 different subtypes have been identified in the hemagglutinin gene, and the extent of nucleotide divergence is very large. Nevertheless, they
are polymorphic “alleles” in classical genetics and have the same biological function.
It is known that they are usually host specific, and three of them (HI, H2, and H3)
are carried by strains infecting humans. However, host specificity is not absolute, andswitching of hosts occurs occasionally (e.g., Webster et al. 1982). The high degree ofgenetic diversity among the subtypes seems to result largely from the high rate of
mutation in this virus in association with host specificity (Nei 1983). It is therefore
interesting to know the evolutionary history of these subtypes. For this purpose weconstructed a dendrogram for 13 subtypes of this gene by using the unweighted pairgrouping method (UPGMA). Previously, Hinshaw et al. (1982) and Webster et al.
( 1982) constructed similar dendrograms, but they used various strains that were isolated
in different years. In our study we used only one strain for each subtype and chosethe one that was isolated in the same or nearly the same year. In this study we usedamino acid sequences rather than nucleotide sequences because the former give morereliable results when genetic divergence is large. We estimated the number of aminoacid replacements per site for each pair of subtypes by the equation d = -lo& 1 - p),
where p is the proportion of different amino acids. In this computation we used aregion of 78 amino acids (the shared segment of hemagglutinin 1 excluding deletions/insertions; see Air 198 1 and Hinshaw et al. 1982) but excluded 10 amino acids thatare apparently invariable. Thus, a total of 68 amino acids were used. The proportionof different amino acids (p) and the number of amino acid replacements per site (d)for all pairs of subtypes are given in table 3, and the dendrogram obtained by UPGMA
from this d matrix is presented in figure 6. This dendrogram need not represent the70

Saitou and Nei

Table 3
Amino Acid Differences among 13 Hemagglutinin
Hl
Hl . . . . .
H2 . . . . .
H5.
Hll . . . .
H6 . . . . .
H13 . . . .
H8 . . . . .
H9 . . . . .
H12 . . . .
H7 . . . . .
.
HI0
H4 . . .
H3 . . .

0.33
0.41
0.64
0.5 1
0.85
0.96
0.79
0.92
1.45
1.33
1.74
1.66

Subtypes

H2

H5

Hll

H6

H13

H8

H9

H12

H7

HlO

H4

H3

0.28

0.34
0.22

0.47
0.44
0.44

0.40
0.44
0.46
0.44

0.57
0.54
0.57
0.43
0.65

0.62
0.60
0.62
0.56
0.57
0.54

0.54
0.53
0.56
0.53
0.56
0.54
0.35

0.60
0.59
0.57
0.56
0.54
0.60
0.40
0.35

0.76
0.72
0.74
0.78
0.75
0.78
0.78
0.78
0.78

0.74
0.75
0.74
0.82
0.76
0.82
0.76
0.79
0.78
0.50

0.82
0.82
0.81
0.82
0.76
0.82
0.81
0.75
0.81
0.68
0.62

0.81
0.78
0.78
0.81
0.76
0.90
0.88
0.84
0.81
0.62
0.59
0.59

0.25
0.58
0.58
0.79
0.92
0.75
0.89
1.28
1.39
1.74
1.51

0.58
0.61
0.85
0.96
0.82
0.85
1.33
1.33
1.66
1.51

0.58
0.56
0.82
0.75
0.82
1.51
1.74
1.74
1.66

1.04
0.85
0.82
0.79
1.39
1.45
1.45
1.45

0.79
0.79
0.92
1.51
1.74
1.74
2.27

0.44
0.51
1.51
1.45
1.66
2.14

0.44
1.51
1.58
1.39
1.82

1.51
1.51
1.66
1.66

0.69
1.13
0.96

0.96
0.89

0.89

NOTE.-Figures above the diagonal are the observed proportions of amino acid differences (p)in a sequence of 68 aminoacids compared. Figures below the diagonal are the estimated number (d) of amino acid replacements per site. Subtypes
are arranged in the order of appearance in the dendrogram of fig. 6.

true evolutionary tree, since the rate of amino acid replacements might vary with hostand host switching occasionally occurs. Nevertheless, it gives a rough idea of the evolutionary divergence of the 13 subtypes of the hemagglutinin gene. Essentially thesame tree topology was obtained by using Far-As’s (1972) distance Wagner method(tree not shown).
The topology of the dendrogram obtained is quite similar to those of Hinshaw
et al. (1982) and Webster et al. (1982), though these authors used several strains forHl (1976)
HZ (1977)
H5 (1976)
H6 (1972)
Hll( 1976)
H13(1977)
H8 (1977)
H9 (1966)
H12(1976)
H7 (1971)
HlO(1965)
H4 (1976)
H3 (1977)
I

I

.

I

t

1.6

1.2

(1.8

0.4

0.0

d=-log(l-p)
FIG. 6.-Phylogenetic
tree of 13 hemagglutinin
subtypes of the influenza A virus made by UPGMA.
Sixty-eight amino acid sequences are used. HZ = New Jersey/l l/76 (Air et al. 198 1); H2 = duck/Alberta/
77/77 (Air and Hall 1981); H3 = Texas/l/77(Webster et al. 1982); H4 = duck/Alberta/28/76(Air 1981);
H5 = shearwater/Australia/28/76(Air 198 1); H6 = shearwater/AustraIia/72(Air 198 1); H7 = turkey/Oregon/
7 1 (Air 198 1); H8 = duck/Alberta/283/77(G. M. Air, unpublished data); H9 = turkey/Wisconsin/l/66(Air
198 I); HI0 = quail/Italy/l
117/65 (G. M. Air, unpublished data); HZ1 = duck/Memphis/546/76(Air 198 1);
HZ2 = duck/Alberta/60/76(Air 1981); and HZ3 = gull/Md/704/77(Hinshaw et al. 1982).

Influenza Virus Genes

71

most subtypes. This is apparently because the intrasubtype divergence is generally
much smaller than the intersubtype divergence (Air 198 1). However, the branch lengthsof our dendrogram are considerably longer than those of the dendrograms of theseauthors. Air (198 1) obtained a similar dendrogram for 12 subtypes of the hemagglutinin gene.
How long did it take for these subtypes to diverge from each other? This is adifficult question to answer, since the rate of amino acid replacement seems to varywith host and the extent of amino acid difference is so high. It is possible that somesubtypes have existed for a long time and that the remaining similarity between them
results from functional constraint rather than from lack of time to diverge. Nevertheless,
it is interesting to know how long it would take for the observed level of divergenceto occur under the assumption of a constant rate of amino acid replacement. For thispurpose we estimated the rate of amino acid replacement for the HI, H2, H.3, andHII subtypes using the same method (&) as that used to estimate nucleotide substitution. The estimates obtained were 0.00 10, 0.0005, 0.0102, and O.O009/amino acidsite/year for subtypes HI, H2, H.?, and HII, respectively, the average being 0.0035.
If we accept this replacement rate, the divergence between the two largest clusters ofhemagglutinins in figure 6 corresponds to 200-300 years. One may compare this resultwith the age of polymorphic alleles at the alcohol dehydrogenase locus in Drosophilamelanogaster. In this case the average nucleotide difference between two randomly
chosen alleles (DNA sequences) is only 0.007, but the coalescence time of polymorphic
alleles (time of the earliest divergence) has been estimated to be - 1 Myr (Stephensand Nei 1986). This indicates how rapidly gene divergence has occurred in the influenza
A virus.
Note added in proof-Afterthe submission of this paper, Hayashida et al. (Mol.
Biol. Evol. 2:289-303, 1985) reported an analysis of influenza A virus genes. Theirgeneral conclusions (extremely high mutation rate and constant rate of evolution) arevirtually the same as ours as well as Nei’s (1983). There is, however, one notabledifference. Hayashida et al. located the Sage (A/duck/Ontario/77)
strain as a direct
descendant of a 1950 strain (FW) in their figure 4. In our analysis, however, FW[=FW/50] is not the ancestor of the Sage [=ON/77(D)] strain but both are derived
from a common ancestor (fig. 2A). Therefore, Hayashida et al.‘s conclusion (9 yearsof frozen period) for the Sage strain is not supported by our analysis.
Acknowledgments

We thank Dr. G. M. Air for sending us two unpublished sequences of HA I subtypes.
We also thank Drs. A. K. Roychoudhury, J. C. Stephens, D. Graur, T. Gojobori, P.
Pamilo, and W. M. Fitch for their comments. This study was supported by research
grants from the National Institutes of Health and the National Science Foundation
to M. Nei.
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Received

April

editor18, 1985; revision received August 1, 1985.