﻿Pattern of mutation in the genome of influenza A
virus on adaptation to increased virulence in the
mouse lung: Identification of functional themes
E. G. Brown*
, H. Liu*
, L. Chang Kit*, S. Baird*§
, and M. Nesrallah*
*Department of Biochemistry, Microbiology, and Immunology, University of Ottawa, 451 Smyth Road, Ottawa, ON, Canada K1H 8M5; Institute of
Poultry Science, Shandong Academy of Agricultural Science, 58 Huangtai Road North, Jinan, Shandong 250100, People's Republic of China;
and §Solange Gauthier Karsh Molecular Genetics Laboratory, Children's Hospital of Eastern Ontario Research Institute,
401 Smyth Road, Ottawa, ON, Canada K1H 8L1
Communicated by Edwin D. Kilbourne, New York Medical College, Valhalla, NY, April 3, 2001 (received for review February 27, 2001)
The genetic basis for virulence in influenza virus is largely un-
known. To explore the mutational basis for increased virulence in
the lung, the H3N2 prototype clinical isolate, AHK168, was
adapted to the mouse. Genomic sequencing provided the first
demonstration, to our knowledge, that a group of 11 mutations
can convert an avirulent virus to a virulent variant that can kill at
a minimal dose. Thirteen of the 14 amino acid substitutions (93%)
detected among clonal isolates were likely instrumental in adap-
tation because of their positive selection, location in functional
regions, andor independent occurrence in other virulent influenza
viruses. Mutations in virulent variants repeatedly involved nuclear
localization signals and sites of protein and RNA interaction,
implicating them as novel modulators of virulence. Mouse-adapted
variants with the same hemagglutinin mutations possessed dif-
ferent pH optima of fusion, indicating that fusion activity of
hemagglutinin can be modulated by other viral genes. Experimen-
tal adaptation resulted in the selection of three mutations that
were in common with the virulent human H5N1 isolate AHK
15697 and that may be instrumental in its extreme virulence.
Analysis of viral adaptation by serial passage appears to provide
the identification of biologically relevant mutations.
Virulence is the measure of the ability of a pathogen to
damage its host. Human influenza A virus infection typically
causes tracheobronchitis with a low incidence of fatal pneumo-
nia. In 1918, a virulent influenza A virus variant arose, causing
a devastating pandemic killing 50 million people (1). Although
this virus was not isolated, it must have possessed mutations that
increased its virulence. The genomic sequence of 1918 viruses
are being determined from archival tissues and, whereas the
sequence of the hemagglutinin (HA) and neuraminidase (NA)
genes are now available (2), we do not yet have the understand-
ing of the molecular basis for virulence needed to interpret this
information. The difficulty of discerning mutations that control
virulence among the background of unselected mutations has
been exemplified by sequence analysis of the highly virulent
AHong Kong15697-like (H5N1) virus that recently infected
humans directly from birds in Hong Kong (HK) (3), where even
the most closely related avian isolate, ACkHK22097, differs
from this virus at 28 amino acids (4). Subsequent human
infections by related avian H9N2 viruses indicate a continued
threat to the human population (1). There is thus a need to
understand the genetic basis for virulence in influenza virus
variants, with the hope that specific mutations will be indicators
and thus predictors of virulence.
No clinical isolates of human influenza virus are known to differ
in virulence (5), therefore necessitating the analysis of infection in
animals. Influenza virus is partially host restricted, where virus from
one host does not normally transmit or cause disease in other hosts.
The AHK15697 strain, however, is virulent for both chickens
and mice (6), indicating that a shared genetic basis for disease
production can exist among species. Adaptation of human influ-
enza virus to mice by serial passage results in the selection of highly
virulent variants that have acquired mutations in multiple genes
(7­9). Analyses of the genetic basis for virulence by using reassor-
tants that possess mixtures of genes from virulent and avirulent
strains have identified various groupings of genes, which in aggre-
gate implicate all eight genome segments (8). These data have led
to the untested assumption that virulence cannot be genetically
predicted, because there are too many degrees of freedom in the
control of virulence.
A goal of the study of influenza pathogenesis is to define the roles
of each viral gene in disease production. To begin to address this
aim, a complete sequence comparison was done between the
AFM147 (H1N1) parental strain (FM) and its mouse-adapted
variant, FM-MA, which had increased 104.6
-fold in virulence on the
basis of LD50. This process identified single amino acid substitutions
in 5 of its 10 genes (9). Reintroduction of each of these mutations
into the parental FM strain confirmed their roles not only in
increasing virulence but also in replicative fitness for the mouse (9).
These findings are compelling, because they show a clear relation-
ship between replicative fitness and the ability to damage the host.
It was also surprising that the MA variant did not possess unselected
mutations that typically accumulate in clinical isolates. This, how-
ever, would be predicted from studies of viral adaptation in cell
culture, where genetic variation is a function of virus population size
(10). Serial passage of large populations of virus under novel
conditions permits competition among all possible mutants with the
selection of optimal genotypes. In contrast, the transfer of small
populations, typical of normal disease transmission, leads to the
fixation of unselected and deleterious mutations because of sto-
chastic effects, a process termed Muller's ratchet (10).
The primary feature of organisms with adaptive mutations is that
they increase in prevalence in the population because of improved
replicative fitness. A strong indicator of adaptive change at the
molecular level is convergent evolution, characterized by the re-
peated and independent occurrence of common mutations in
adapted variants. The occurrence of identical mutations is termed
parallel evolution, and mutation at the same sites but with different
amino acids is termed directional evolution, both of which are
characteristic of instances of convergent evolution (11). This is the
standard criterion for identifying mutations responsible for drug
and inhibitor resistance, providing evidence for convergent evolu-
tion in many organisms, including influenza virus (12).
Abbreviations: MDCK, Madin­Darby canine kidney; MA, mouse adapted; PB1, PB2, and PA,
RNA polymerase subunits; HA, hemagglutinin; NP, nucleoprotein; NA, neuraminidase; NS1,
nonstructural protein; HK, Hong Kong; pfu, plaque-forming unit.
Data deposition: The sequences reported in this paper have been deposited in the GenBank
database (accession nos. AF348170­AF348206).
To whom reprint requests should be addressed. E-mail: ebrown@uottawa.ca.
The publication costs of this article were defrayed in part by page charge payment. This
article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C.
§1734 solely to indicate this fact.
www.pnas.orgcgidoi10.1073pnas.111165798 PNAS  June 5, 2001  vol. 98  no. 12  6883­6888
MICROBIOLOGY
Because the parental AFM147 strain was subjected to
mouse passage immediately on isolation in 1947 (13), it carries
evidence of preexisting mouse adaptive mutations detectable on
analysis by genetic reassortment (14, 15). In the present study,
the prototype H3N2 clinical isolate, AHK168, without a prior
history of mouse passage, was used to generate virulent variants
by serial mouse-lung passage. The objective of this analysis was
to identify and characterize the complexity and nature of the
mutations that control virulence. Genomic sequencing of a
highly virulent MA variant identified 11 mutations that were
acquired on mouse adaptation. Sequencing of clonal variants
showed that most of these mutations were positively selected in
the population and affected specific regions of individual genes
that identify functional themes for regulating virulence. Exper-
imental evolution may have recapitulated natural evolution, at
least in part, because the Hong Kong H5N1 lineage of viruses
possessed several mutations in common with the MA strains,
suggesting their instrumental operation in virulence.
Materials and Methods
Viruses. The prototype human H3N2 isolate, AHK168, was
obtained from the Laboratory Centre for Disease Control, Health
Canada, Ottawa. Viruses were clonally purified by two plaque
isolations in Madin­Darby canine kidney cell (MDCK) monolayer
followed by stock preparation in the allantoic cavity of 9-day-old
chicken embryos. Virus was passaged in mouse lungs by cycles of
intranasal infection with 50 l of 110 diluted lung extracts under
halothane anesthesia for groups of 3 mice for 3 days, as described
previously (14). MA viruses were clonally isolated and titrated by
plaque assays on MDCK cell monolayer, as described (14)
Nucleotide Sequencing. Viral RNA was purified by phenol extrac-
tion from stock virus, as previously described (15). Each genome
segment was amplified by reverse transcription­PCR (RT-PCR)
and purified in agarose gels or Sepahcryl S400 spin columns
(Amersham Pharmacia) before direct dideoxy-terminated cycle
sequencing by using the ABI Prism Dye Terminator Cycle
Sequencing Ready Reaction Kit (Perkin­Elmer) and an Applied
Biosystems automated sequencer, Model ABI 373. Genome
segment-specific primers for RT-PCR were complementary to
the first 16 nucleotides of each segment in combination with a
primer complementary to the 12 nucleotides at the 3 end of
vRNA, and all possessed a 5 terminal adapter sequence of
CCGC. Sequencing primers (sequences available on request)
were complementary to related H3N2 viruses (GenBank acces-
sion nos. J02135­40 and X59240, respectively).
SDSPAGE. MDCK cells were infected, [35
S]Met-labeled, and
analyzed, as described previously (14). Labeled virus was puri-
fied by adsorption to guinea pig erythrocytes. Trypsin was used
at 1 gml (15 min at 37°C) to cleave HA. Tunicamycin was used
at 10 gml from 5.5 h postinfection (pi) onward, with pulse
labeling at 6.5 h pi for 1.5 h. Immunoprecipitations were
performed as described previously, by using AHK168 specific
rabbit immune serum (16).
Virulence Assay. Virulence was measured as the lethal dose in
CD1 strain Swiss­Webster mice. The median lethal dose (LD50)
in plaque-forming units (pfu) was measured by intranasal infec-
tion of five groups of five mice each with serial 10-fold dilutions
of virus, as described previously (14). Mortality from influenza
virus infection occurs primarily before day 8 and was thus
monitored for 10 days.
Statistical Analysis. LD50 values were determined by using the Karber
method (14). The significance of differences in virulence and
growth values was determined by using the Z statistic for a standard
normal distribution. The probability of multiple independent mu-
tations in the same clonal isolate was predicted by the Poisson
distribution by using the observed mutation frequency.
Growth in MDCK Cells, Chicken Allantoic Cavity, and Mouse Lung.
MDCK cells were infected at a multiplicity of infection of 5
pfucell and incubated at 34°C in the presence of 1 gml trypsin
(15). Supernatants were collected over a 48-h period, and plaque
assays were performed in duplicate for each sample. Groups of
CD-1 mice were infected intranasally with 5  103
pfu of each
virus. Over a 10-day period, lungs were removed from groups of
three mice, pooled, and sonicated for quantification by plaque
assay on MDCK monolayers, as described above. The yield in
chicken egg allantoic cavity was determined by infecting 9- to
10-day-old chicken embryos with 104
pfu and incubation at 48 h
at 34°C. Pools of three eggs each (two to six poolsvirus) were
titrated by plaque assay on MDCK cells.
Hemolysis and Hemagglutination. Hemolysis assays were performed
by using human type O blood and citrate buffers in microtiter plates
at 21°C, as described (17). Hemagglutination and hemagglutination
inhibition assays were performed as described previously (18). 
inhibitor was prepared by sonicating mouse lung (10% suspension)
and centrifugation to prepare extracts, and mouse serum was used
as a source of  inhibitor (17).
Results
MA variants of the human isolate, AHK168 (HK), were
produced by serial lung-to-lung passage, beginning with intra-
nasal inoculation of 106
pfu of virus per mouse. Subsequent
passages involved the inoculation of diluted lung extracts, con-
stituting dosages of 104­5
pfumouse (data not shown). Viru-
lence was assayed by LD50 after 12 and 20 passages for both the
total population of virus obtained directly from titrated lungs
extracts without further culturing, as well as six clonal isolates
from each of these passage levels obtained by plaque purification
of 107
-fold diluted lung extracts in MDCK cells (Table 1). The
parental virus was totally avirulent for mice, LD50 107.7
pfu;
however, after 12 passages, the LD50 for the population of virus
in lung extracts had decreased 104
-fold and after 20 passages
105
-fold, indicating that these virus populations had acquired
mutations that profoundly affect virulence. Several HKMA
clonal isolates from each passage level were similar in virulence
to their respective populations and were thus representative of
these populations; however, some were less virulent, indicating
Table 1. Virulence of mouse-adapted isolates of AHK168
AHK168 viruses passaged in
mouse lung
LD50,
pfu
Change in LD50
(LOG10 decrease)
Unpassaged AHK168 parent clone HK 107.7 NA
Passage 12 population 103.7 4.0
Passage 12 clone HKMA12 104.0 3.7
Passage 12 clone HKMA12A 103.9 3.8
Passage 12 clone HKMA12B 103.6 4.1
Passage 12 clone HKMA12C 104.6 3.1
Passage 12 clone HKMA12D 104.7 ND
Passage 12 clone HKMA12E 104.9 ND
Passage 20 population 102.7* 5.0
Passage 20 clone HKMA20 104.2 3.5
Passage 20 clone HKMA20A 104.0 3.7
Passage 20 clone HKMA20B 103.0* 4.7
Passage 20 clone HKMA20C 102.60.3 5.1
Passage 20 clone HKMA20D 103.5 4.2
Passage 20 clone HKMA20E 103.6 4.1
*, viruses not significantly different in virulence relative to HKMA20C,
(Z value, P  0.05); NA, not applicable; ND, not done.
6884  www.pnas.orgcgidoi10.1073pnas.111165798 Brown et al.
genetic heterogeneity within these populations. HKMA20C was
the most virulent virus clone, LD50  102.5
pfu, similar to the
passage 20 population (Table 1).
Sequence Analysis. To identify the mutations responsible for the
increased ability to cause fatal lung infection, we initially se-
quenced the genome of the most virulent clonal isolate,
HKMA20C, as well as the HK parent for comparison. A total of
11 amino acid substitutions involving 8 of the 10 viral proteins
(Table 2) and 4 silent substitutions were detected, which pro-
duced 2 coding changes in each of PA, HA, and nucleoprotein
(NP), as well as single amino acid substitutions in PB2, NA, M1,
M2, and NS1, and no mutations in polymerase subunit PB1 and
nuclear export protein NS2. The noncoding changes (data not
shown) included an insertion of an extra A in the polyA coding
region of the NA gene (Table 2).
Although it is clear that these mutations as a group must
account for the difference in biology of the HKMA20C variant,
it is not clear that they are all instrumental in adaptation to
increased virulence. Because adaptive changes increase replica-
tive fitness, viruses that possess these changes will be present at
a greater frequency in the virus population than their rate of
formation predicted from the mutation frequency. To detect
mutations that were positively selected on mouse adaptation, we
tested for the null hypothesis that the mutations in individual
virus clones were randomly and thus independently generated.
Given the observed frequency of coding mutations in
HKMA20C of 2.2  103
amino acid, the probability of having
two viruses with the same mutation because of independent
mutational events is predicted by chance to be P  5  106
n 
1, where n is the number of isolates tested. The occurrence of the
same mutations in more than one isolate is thus significant
evidence of positive selection rather than the occurrence of
independent mutational events. The HKMA12 and HKMA20
variants were subjected to sequence analysis to determine
whether they shared mutations with HKMA20C. Genome seg-
ments four to eight were completely sequenced, as well as the
mutant loci detected in the PB2 and PA genes. The HKMA12
clone possessed 5 mutations in common with both of the passage
20 clones, and HKMA20 possessed 6 mutations in common with
HKMA20C, indicating positive selection of 6 mutations (Table
2). The low probability of pairs of viruses with five or six
mutations in common is also a clear indication of positive
selection, as this is extremely unlikely to occur by chance
(Poisson distribution P  1  1013
and P  1.4  1017
).
Because two more mutations were detected in the HKMA20
clone (M1-Asp-2323Asn and NS1-Val-233Ala), segments 7
and 8 (M1, M2, NS1, and NS2 genes) were sequenced for
HKMA-12, 12A, 12B, and the 5 remaining HKMA20 series
of clones (Table 1). This analysis identified a further mutation,
NS2-Lys-883Arg, and demonstrated positive selection for 4 of
6 mutations in the M and NS genes (Table 3).
Protein Changes. The HA2-Thr-1563Asn substitution is ex-
pected to result in the loss of a glycosylation site that would be
detectable by increased mobility on SDSPAGE. Electrophore-
sis of infected MDCK cell proteins showed that the uncleaved
form of HA protein (HA0) possessed higher electrophoretic
mobility than HK for 3 of 6 passage 12 clones and 6 of 6 passage
20 clones (Fig. 1A). The increased electrophoretic mobility was
caused by decreased glycosylation, because the unglycosylated
forms of their HA0 proteins resulting from tunicamycin treat-
ment were indistinguishable in electrophoretic mobility from the
parental virus (Fig. 1B), and the HA of HKMA12A was distinct
in its properties. The mutations affecting glycosylation were
confirmed to map to the HA2 subunit by trypsin cleavage of
Fig. 1. SDSPAGE of proteins of parental and MA clones of AHK168. (A)
35[S]met-labeled infected MDCK cell proteins were immunoprecipitated to
show the position of the HA0 precursor. (B) The unglycosylated form of HA0
protein synthesized in the presence of tunicamycin. (C) The position of the NP
proteins is shifted for HKMA20B, -C, and -D. (D) The shift in mobility of the HA2
subunit generated by trypsin in variants.
Table 3. Mutations in the M1, M2, NS1, and NS2 genes for
clones of mouse-adapted AHK168
Virus
Amino acid variation (nucleotide position)
M1 M2 NS1 NS2
167 (524) 232 (617) 44 (155) 23 (94) 103 (333) 88 (289)
HK Thr (A) Asp (G) Asp (G) Val (T) Phe (T) Lys (A)
HKMA12      
HKMA12A      
HKMA12B      
HKMA20  Asn (A)  Ala (C)  
HKMA20A  Asn (A)  Ala (C)  
HKMA20B   Asn (A)  Leu (C) Arg (G)
HKMA20C Ala (G)  Asn (A)  Leu (C) 
HKMA20D     Leu (C) 
HKMA20E   Asn (A)   
, indicates wild-type sequence.
Table 2. The nature and location of predicted amino acid changes for HK and HKMA viruses (nucleotides in parentheses)
PB2 PA HA1 HA2 NP NA M1 M2 NS1
701
(2128)
133
(422)
556
(1691)
218
(729)
156
(1531)
34
(145)
480
(1483)
468
(1422) (1457)
167
(524)
44
(155)
103
(333)
HK Asp (G) Glu (A) Gln (A) Gly (G) Thr (C) Asp (G) Asp (G) Pro (C)  Thr (A) Asp (G) Phe (T)
HKMA20C Asn (A) Gly (G) Arg (G) Trp (T) Asn (A) Asn (A) Asn (A) His (A) (Ade) Ala (G) Asn (A) Leu (C)
HKMA12 Asn (A)*   Trp (T) Asn (A) Asn (A)  His (A)    
HKMA20 Asn (A)*  Arg (G)* Trp (T) Asn (A) Asn (A)  His (A)    
*, from partial sequence; , indicates wild-type sequence; , indicates insertion of noncoding nucleotide.
Brown et al. PNAS  June 5, 2001  vol. 98  no. 12  6885
MICROBIOLOGY
purified virions, because this subunit was shifted to higher
relative electrophoretic mobility, corresponding to a 5.5-kDa
decrease in apparent size relative to HK HA2 (Fig. 1D).
On SDSPAGE analysis, it was observed that the NP proteins
of HKMA20-B, -C, and -D possessed increased mobility that
corresponded to a 1-kDa decrease in apparent size (Fig. 1C).
This correlated with the presence of the mutation at amino acid
480 of HKMA20C relative to HKMA20 and HKMA12 and
indicated positive selection of this mutation.
Changes in Biology of HA. The Gly-2183Trp mutation site in HA1
has also been shown to mutate in antibody escape mutants that
become altered in inhibitor sensitivity as well as pH of fusion
(19). We assessed the pH optimum of fusion of HK, HKMA-12,
20, and 20C (Fig. 2) and, as expected, the three MA variants
possessed different pH optima of fusion relative to the HK
parent, but surprisingly they differed from each other as well.
The HK parent possessed a median pH of fusion (1 SD) of 4.9 
.04 relative to 5.7  .02, 4.5  .02, and 4.6  .02 for HKMA-12, 20,
and 20C, respectively. This was not expected, because each of the
MA viruses had the same HA gene sequence. Consistent with the
conformational change triggered by low pH, the pH sensitivity of
hemagglutination activity was found to reflect the relationship
shown for pH of fusion (data not shown).
The susceptibility of MA variants to nonspecific inhibitors was
assessed by hemagglutination inhibition assay with mouse serum
or lung extract as a source of  and  inhibitor, respectively. The
three MA strains had all increased in resistance to  inhibitor,
seen as a 4-fold reduction in inhibition of hemagglutination (re-
duction in titer from 1,280 to 320). Only the HKMA20 clone had
become resistant to  inhibitor (reduction in titer from 8 to 2).
Growth in Different Hosts. The difference in virulence of the MA
variants was not caused by an increased ability to infect mice,
because the HK parent was already highly infectious (median
infectious dose of 13 pfu, as assessed by serological conversion).
The relative ability of HK and the HKMA-12, 20, and 20C
clones to replicate was assessed in mouse lung, MDCK cells, and
chicken allantoic cavity. All of the MA clones grew faster and to
higher titer than HK in mouse lung (Fig. 3A). Comparison of the
yield 1 day postinfection as well as the maximum yields of
infectious virus indicated that the HKMA20C virus had the
greatest increase in replicative fitness relative to HK in the
mouse, which was consistent with its relative virulence (Table 4).
The increases in fitness 1 day postinfection were dramatic,
demonstrating the replicative basis for selection of MA variants.
All of the MA clones tested also had an increased ability to
replicate in MDCK cells (Fig. 3B); however, both of the passage
20 MA clones yielded less infectious virus in chicken allantoic
cavity than the HK parent or the less passaged HKMA12 clone
(P  0.05) (Fig. 3C). Thus mouse adaptation selected variants
with increased replicative fitness in the mouse, but some of the
mutations conferring these properties were host specific.
Evidence of Parallel Evolution on Mouse Adaptation. To assess
whether the MA variants of HK had selected any mutations in
Fig. 3. Yield of HK and HKMA variants in different hosts. (A) Mouse lung; each point represents the average of three mice; values were not obtained for
HKMA20C after 4 days because of lethality. (B) Yield in MDCK cells. (C) The yield of infectious virus in chicken allantoic cavity; values are the average plus standard
deviation of two to six pools of three eggs.
Fig. 2. The pH dependence of hemolysis by AHK168 and the HKMA12,
HKMA20, HKMA20C virulent MA variants. The relative extent of hemolysis
was measured in duplicate for each of two experiments at 0.1 pH increments.
The hemolysis profiles of FM-MA and the J9 reassortant that possesses the
same HA gene (17) are shown as controls.
Table 4. Relative ability of HK and HKMA variants to replicate in
mouse lung
Virus
Yield in mouse lung 1
day postinfection
Maximum yield in
mouse lung
Titer,
pfuml
Relative
fitness
Titer,
pfuml
Relative
fitness
HK 1.1e5 1 4.8e6 1
HKMA12 2.0e7 180 3.6e7 7.5
HKMA20 5.6e7 510 5.6e7 12
HKMA20C 1.3e8 1,180 1.3e8 27
6886  www.pnas.orgcgidoi10.1073pnas.111165798 Brown et al.
common with FM-MA, their genome sequences were compared.
The PA-556, NP-34, and M1­232 mutations occurred at sites that
differed between HK and FM-MA virus. The probability of each
of these sites being mutated purely by chance was extremely
unlikely (P  0.0002), given the low number of ``target'' amino
acid substitutions in the FM-MA genes relative to HK (30 of 716,
24 of 498, and 4 of 252, respectively). These mutations existed in
the prototype AFM147 and may have been selected on
mouse adaptation of this virus in 1947. These sites are positioned
on the genetic map of influenza A virus by blue downward
arrows, relative to the unique mutations in FM-MA (black
downward arrows) and the mutations observed among HKMA
variants (black upward arrows) (Fig. 4).
Discussion
Adaptation is believed to be the driving force in evolution, where
organisms are selected in nature because of increased fitness
conferred by beneficial mutations. This paper extends the analysis
of the mutational basis for virulence by using mouse adaptation,
because this system provides the identification of mutations that
generate evolutionarily relevant phenotypic variation. The pattern
of mutations associated with HK-MA variants was consistent with
multiple events in the virus population followed by assembly of
mutations into genomes by reassortment and competition among
viruses with different mutations. Thus the passage 12 virus had
already acquired 5 mutations that were predominant in the popu-
lation at passage 20. Between passages 12 and 20, subsequent
mutations or combinations of mutations in individual genome
segments resulted in subpopulations as shown for segment 7 (M1
and M2 genes) and 8 (NS1 and NS2 genes). Such cycles of
competition among mutant viruses drive rapid evolution in influ-
enza virus to result in progressive selection of variants. The reason
for the replacement of the parental virus population by the adapted
variants is a function of increased replication rate (180- to 1,180-
fold) as well as peak yield (7.5- to 27-fold) that produced dominant
viruses that outcompete less fit viruses.
Positive Selection. A total of 14 coding mutations and one insertion
mutation were observed on mouse adaptation of HK. The positive
selection of 11 of 14 mutations indicates they confer a replicative
advantage; however, there is also reason to believe that the re-
maining three mutations observed in individual clones from the
passage 20 population were also adaptive. The Glu-1333Gly
mutation in the PA protein occurred in the middle of the first
nuclear localizations signal at amino acids 124­139 (Fig. 4; ref. 12)
and may affect this activity. The M1-Thr-1673Ala mutation was
independently selected in 1972 to become fixed in the H3N2 lineage
(data not shown), indicating that this mutation has been selected on
human passage. The NS2-Lys-883Arg mutation was found in
several virulent viruses, including the virulent Hong Kong H5N1
lineage of viruses, suggesting that this is an adaptive mutation that
plays a role in modulating virulence (data not shown). Thus 13 of
14 mutations detected on mouse passage of HK virus were deemed
to be instrumental in adaptation to increased virulence, because
they either were being positively selected, involved functionally
important regions of proteins (discussed below) andor were found
to have occurred independently in other virulent influenza viruses
(data not shown) (Figs. 2­4). From previous genetic analysis of the
AFM147-MA variant, all of the HKMA mutations would be
expected to be adaptive (9, 15).
Parallel Evolution Associated with Virulence. Three mutations ob-
served on mouse adaptation of HK occurred at sites of preexisting
variation in FM, which may represent adaptive changes selected on
mouse adaptation in 1947 and is consistent with the presence of
virulence determinants in this virus (9, 14, 15). Ten of 14 sites that
mutated in HKMA variants were also found to have mutated in
other naturally virulent or laboratory-adapted strains (data not
shown). With respect to the virulent AHK15697 strain, it is
interesting that this virus possessed three amino acid mutations,
PB2-Asp-7013Asn, NS1-Phe-1033Leu, and NS2-Lys-883Arg,
in parallel with HK MA variants, and suggests they may operate in
this virus to mediate its virulence. The PB2-Asp-7013Asn muta-
tion is located between the second nuclear localization signal
(NLS2) at 736­739 and the cap-binding motif at 634­650 (Fig. 4;
ref. 12), both of which could be modified in activity. The NS1 gene
is required for counteracting several antiviral effects of IFN (20),
and so the Phe-1033Leu mutation may be affecting this function,
because mice lacking an IFN response can develop a systemic
infection with neurovirulent influenza virus (20) that is similar to
AHK15697 infection of wild-type mice (6). The remaining
mutation, NS2-Lys-883Arg, may be affecting the function of this
protein in nuclear export of viral nucleocapsids (12). Sequence
analysis of AHK15697 clones that are attenuated in virulence
for mice was associated with the loss of the PB2-Asp-7013Asn
mutation as well as two mutations, NS1-Asp-1013Asn and HA1-
Pro-2113Thr, which are near the NS1­103 and HA1­218 muta-
tions observed on mouse adaptation (21), further supporting the
relevance of these mutations as determinants of virulence.
Fig. 4. Location of mutations selected on mouse adaptation on the func-
tional genetic map of Influenza A virus. The location of mutations seen in
FM-MA that have been confirmed to control virulence (9) are shown with
black downward arrows, and those preexisting in FM virus are indicated in
blue. Mutations observed in HKMA clones are shown with upward arrows. The
functional regions and binding sites are described elsewhere (12). NLS, nuclear
localization signal; POL, polymerase; NTP, nucleotide triphosphate; PKR,
dsRNA-dependent protein kinase; effector indicates a multiple protein-
binding region; NES, nuclear export signal; regions labeled with influenza
virus protein names indicate sites of interaction.
Brown et al. PNAS  June 5, 2001  vol. 98  no. 12  6887
MICROBIOLOGY
Parallel Mutation with Human Lineages of Virus. Two of the MA
mutations had also been independently selected in humans. The
NS1-Val-233Ala mutation is convergent with most influenza
viruses, including human H1N1 and H2N2 strains (data not
shown). The M1-Thr-1673Ala mutation was also established in
the human H3N2 lineage in 1972 (data not shown). Thus,
experimental evolution appears to recapitulate the selective
processes of natural evolution of influenza viruses.
MA Variants Possess Mutations in Specific Functional Regions. It is
apparent that increased virulence is because of mutations that
increase the ability of influenza virus to exploit the host envi-
ronment. Given the location of mutations selected on mouse
adaptation in FM-MA and HKMA variants (Fig. 4), they appear
to implicate the central processes of viral replication and sites of
interaction with host and viral components. Specific types of
functional regions that are repeatedly mutated include RNA-
binding sites, as seen for NS1­23, M1­139, and possibly NP-34;
nuclear localization signals of polymerase subunits, PB2­482,
and PA-133, as well as mutations at subunit interfaces of HA,
HA1­218 in HKMA variants, and HA2­47 in FM-MA (Table 4;
ref. 14). The NP-480 mutation is adjacent to amino acid 479,
which affects NP oligomerization (12), and the PB2-binding site
of the PB1 polymerase subunit is mutated in FM-MA (9, 12).
The Thr-1563Asn mutation of HA2 results in the loss of a
glycosylation site adjacent to the cleavage site of HA0 and may be
relevant to cleavage activation, because there is detectable cleavage
of HA without trypsin for the HKMA-12, 20, and 20C strains
produced in MDCK cells (Fig. 1D). This is analogous to the loss of
a glycosylation site near the cleavage site of the HA of ACK
Pennsylvania137083 (H5N2), which resulted in increased cleav-
ability and high virulence (22). The other HA mutation, Gly-
2183W of HA1, occurs at the trimer interface adjacent to the
receptor-binding site. Mutations at this site in monoclonal antibody
escape mutants have been shown not only to affect HA structure
such that antibody binding is maintained without neutralization but
concomitantly to result in alteration in receptor specificity and the
pH threshold for membrane fusion (19). Consistent with these data,
the HKMA-12, -20, and -20C variants were altered in pH of fusion,
resistance to -inhibitor (mouse serum lectin), and receptor binding
(data not shown). A common feature of other MA variants is
mutations in HA associated with resistance to inhibitors and in
some instances altered pH of fusion, as seen for FM-MA and
APhilippines282BSML10 (8, 17).
Extragenic Modulation of HA Function. The observation that the
HKMA-12, 20, and 20C strains had the same HA amino acid
sequence but were heterogeneous in their pH of fusion as well
as resistance to  inhibitor indicates that other mutations in the
genome presumably present on an interacting protein were
responsible for modulating the properties of HA (Fig. 4). The
M1 protein is a strong candidate for this novel role, because it
differs in both HKMA20 and HKMA20C relative to HKMA12
and has been implicated in the modulation of HA function.
Strains of human influenza H1N1 viruses with identical HA
molecules differ in their ability to agglutinate chick erythrocytes
because of the nature of their M1 proteins (23). Interestingly,
fixation of the M1­167 mutation in the human lineage in 1972
coincided with a change in receptor specificity (24) that did not
correlate with changes in HA sequence, again suggesting the
involvement of another protein in HA function. Because there
is direct evidence that the M1 protein interacts with the cyto-
plasmic projections of HA and NA (25), it is possible that the
nature of this interaction affects HA biology.
The M2­44 site, at the cytoplasmic border of the transmem-
brane domain (Fig. 4), is also mutated in two highly virulent
avian strains, ACkFPVRostock34 (FPV) (H7N7) and
ACKPenn137083 (H5N2). Previous studies have shown that
the FPV M2 protein is required to prevent premature acid-
induced conformational change of its HA in endosomes (26).
Reassortants of ACKPenn137083 that possess its HA gene
invariably possess its M1 and M2 genes (27), suggesting a
functional requirement for another gene interaction.
Host-Restrictive Mutation. The nature of the polymerase, NP, and NS
proteins has been implicated in host restriction (12). The
HKMA20C variant has become host restricted in its growth char-
acteristics, possibly because of the PA-133 mutation in the first
nuclear localization signal (NLS1). The FM-MA variant has the
same growth properties because of a combination of mutations in
the PB2 (NLS1) and PB1 (PB2-binding site) subunits (9). The
PA-556 mutation may also be relevant to host specificity, because
this site has mutated only in two other viruses, both with a history
of mouse adaptation (APR834 and AWS33). The NP gene
has evolved into host-specific lineages (12), where Asp-34 is found
in all human clinical isolates but has mutated in HKMA variants.
In conclusion, mouse adaptation is caused by selection of host-
dependent as well as host-independent mutations that result in
heightened exploitation of the host, seen clinically as increased
disease. The means of achieving optimal competitive advantage
appears to be controlled by acquisition of a relatively discrete subset
of mutations that involve specific types of functional regions of the
influenza virus genome. The location of these mutations identifies
novel modulators of virulence. The adaptive approach to analyzing
virulence should have general applicability to other viruses (11).
Sequence analysis was initiated in R. Levandowski's laboratory, Office of
Vaccines Research and Review, Center for Biologics Evaluation and
Research, Federal Drug Administration. Helpful criticism was provided by
E. D. Kilbourne and K. W. Wright. This work was funded by the Natural
Sciences and Engineering Research Council of Canada, Grant 0GP004177.
L.C.K. and M.N. were funded by Natural Sciences and Engineering Re-
search Council (Canada) Undergraduate Research Awards and H.L. by the
Chinese Scholarship Council, People's Republic of China.
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