Download Influenza A virus haemagglutinin polymorphism

Journal of General Virology (1993), 74, 1311-1316. Printed in Great Britain
1311
Influenza A virus haemagglutinin polymorphism: pleiotropic antigenic
variants of A[Shanghai[ll[87 (H3N2) virus selected as high yield
reassortants
E. D. Kilbourne, ~* B. E. Johansson, 2 T. Moran, 2 S. Wu, 4 B. A. Pokorny, 1 Xiyan Xu 3 and N. C o x 3
1Department of Microbiology and Immunology, New York Medical College, Valhalla, New York 10595, 2Mount
Sinai School of Medicine, New York, New York 10029, a W H O Influenza Center, Center for Infectious Diseases,
Atlanta, Georgia 30333 and 4Department of Medicine, University of Rochester, Rochester, New York 14642, U.S.A.
Genetic reassortment o f the A/Shanghai/11/87 (H3N2)
variant of influenza A virus with A/PR8/34 (H1N1) virus
[the standard donor of high yield (hy) genes for influenza
vaccine viruses] resulted in the isolation o f two reassortants with differing H3 haemagglutinin (HA) phenotypes, X-99 and X-99a. The two H A phenotypes were
derived from individual subpopulations of the H3N2
wild-type virus during the reassortment event. The H A
mutants and their respectively derived reassortants
(identical in RNA genotype) differed in antigenicity,
replication characteristics, yield in chick embryos and
haemagglutinin gene sequence. Despite antigenic differences in reactions to polyclonal rabbit antisera of
60 %, both X-99 and X-99a, the hy reassortants, were
equally immunogenic and protective in BALB/c mice to
challenge by parental wild-type virus. Differences in H A
phenotype were related to a Ser to Ile change at amino
acid position 186. These findings emphasize the polymorphism of influenza virus strains as well as the need
for caution in selection of vaccine strains from among
antigenicaUy distinct viral subpopulations.
Introduction
ants, although antigenically and biologically different,
were equally immunogenic and protective in a mouse
model system. Furthermore, the parental virus comprises
at least two haemagglutinin (HA) subpopulations from
which the reassortants, differing from each other by only
three coding changes in HA1, were derived.
Influenza viruses are an outstanding example of viral
polymorphism. The genetic non-homogeneity of standard laboratory strains (reviewed by Kilbourne, 1978,
1987), recent isolates (Robertson et al., 1991), or even
unpassaged clinical material (Katz & Robertson, 1992),
is increasingly recognized. For the most part, intrastrain
differences are minor and detectable only through
analysis with monoclonal antibody (MAb) panels. However, antigenic differences demonstrated using polyclonal
antibodies in hyperimmune sera have been described
(Kilbourne, 1978; Both et al., 1983; Johansson &
Kilbourne, 1992). Such differences have potential significance in the perennial fabrication of high yield (hy)
influenza virus reassortants (Kilbourne, 1969; Baez et
al., 1980) for use in vaccines against antigenic variants
emerging in nature.
We describe here two hy reassortants (X-99 and X99a) derived from different subpopulations of A /
S h a n g h a i / l l / 8 7 (H3N2) that differ in antigenicity,
binding affinity and yield. These differences introduced a
dilemma in vaccine choice in 1989 because X-99a, the
highest yielding reassortant, when subjected to initial
antigenic analysis with ferret antisera appeared to be less
broadly immunogenic. We shall show that both reassort0001-1488 © 1993 SGM
Methods
Viruses. The hy reassortant viruses X-99 and X-99a were produced
by genetic reassortment of A/Shanghai/l 1/87 (H3N2) with
A/PR/8/34 (H1N1) influenza A virus variants as described under
Results. Hy reassortants X-91, X-97 and X-101 were derived from
reassortment of A/PR/8/34 with A/Leningrad/360/86, A/Sichuan/
2/87 and A/Beijing 4/89 H3N2 viruses, respectively(Table 1).
Antisera. Antisera were produced by intravenous (i.v.) injection of
rabbits with 3000haemagglutinatingunits of purifiedvirus. The rabbits
were bled 42 days after initial injection and 7 days after a booster
injection of virus on day 42. Prior to use in neutralization and
haemagglutination inhibition (HI) tests, antisera were heated at 56 °C
for 30 min and treated with Vibriocholeraereceptor-destroyingenzyme
as previouslydescribed (Kilbourne et al., 1990).
Serological titrations and antigenic analyses. HI, neuraminidase
inhibition and neutralization tests and ELISA were carried out as
previously described (Kilbourne et al., 1990). HI tests for antigenic
analysis were performed in tubes using large volume transfers and
interpolated dilutions. This precise method has a mean S.D. of_+19%
(Horsfall & Tamm, 1953).Antibody titre ratios were calculated by the
method of Archetti & Horsfall (1950). Homologous and heterologous
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E. D. Kilbourne and others
goat antimouse IgG and the substrate 3-amino-9-ethyl carbazole.
Hybridomas positive on X-99a but not PR8 or X-99 were considered
X-99a-specific, and this was confirmed by HI testing.
T a b l e 1. Biological phenotype o f A / Shanghai/11/87
H A mutants and their reassortants
HA titre*
HI titter
Virus
Egg
MDCK
NHS$
A-9
A-56
A/Sh/87 (ly)
A/Sh/87 (hy)
X-99
X-99a
X-99I-§
X-99aEII
32
128
256
632
512
1264
16
45
8
128
-
11
7
11
7
8
5
< 1
7
< 1
8
< 1
< 1
< 1
7
< 1
8
< 1
< 1
* The reciprocal of endpoint dilution. Geometric mean of six
individual eggs.
t Expressed as logs of value. NHS, heated at 56 °C, as the source of
non-specific ct2-macroglobulin inhibitor.
§ Inhibitor resistant escape mutant from the passage of X-99 with
NHS.
II Escape mutant from the passage of X-99a with X-99a-specific MAb
A-9.
titre ratios were determined for each virus, Va and V2. The following
formula was used to calculate R (the coefficient of antigenic
relatedness): R = v ~ ,
where R 1 = homologous titre V1/heterologous titre V~; R~ = homologous titre VJheterologous titre V1. When
l / R = 2 , antigenic relatedness is 50%; if l / R = 4 , the antigenic
relatedness is 25 % etc. Antigenic differences of 50% or more (1/R >~
2) are significant (Archetti & Horsfall, 1950). All mathematical
calculations were performed on an IBM-XT using a BASIC program
written for these calculations (Kilbourne et al., 1990).
PAGE. The differential migration of isolated virion proteins was
studied by the method of Ritchey et al. (1977) using 7 to 14% gels
under reducing conditions.
RNA sequencing. RNA sequence analysis was performed by the
dideoxynucleotide chain termination method, using synthetic oligodeoxynucleotide primers and reverse transcriptase essentially as
described elsewhere (Cox et al., 1988) except that 10 gCi of [aSS]dATP
(Amersham; specific activity > 1000Ci/mmol) for each 5.5 ~tl of
reaction mixtures was used and reverse transcriptase incubations and
chase were done at 42 °C for 20 min. The HA1 domains of the HA
genes of the X-99aE and Sh/ly (low yield) viruses (Table 1) were
amplified by the PCR method (Saiki et al., 1988; X. Xu et al.,
unpublished) with primer 7, 5'd ACTATCATTGCTTTG as the
forward primer and reverse complement primer 1184, 5'd ATGGCTG
CTTGAGTGCTT as the reverse primer. Primers complementary
to the mRNA sense strand 5'd CCTGCGATTGCGCCGAAT, 5'd
CGATATGTCTCCCGGTTT, 5'd TGGCATAGTCACGTTCAG
and 5'd TAAGGGTAACAGTTGCTG beginning at nucleotides 1090,
809, 588 and 379, respectively, were used for sequencing the
asymmetrically amplified PCR products.
Production of X-99a HA-specific MAbs. Female BALB/c mice
(Charles River) were immunized with 25 gg of sucrose-gradient purified
X-99a virus by intraperitoneal injection. Animals were immunized on
two occasions, 3 weeks apart, rested for 10 weeks and boosted with 10 gg
X-99a intravenous injections, 3 days before fusion. The fusions were
performed by a standard method (Holmdahl et al., 1989) and positive
cultures were cloned by limiting dilution.
Screening of MABs by immunostaining. The method of Usuba et al.
(1990) was used. Briefly, MDCK cells were infected with virus and,
following fixation with paraformaldehyde, hybridoma supernatants
were added to each well. Binding was identified with peroxidase-linked
Infection of mice. Groups of 20 g female BALB/mice were infected
intranasally under light ether anaesthesia (Johansson & Kilbourne,
1991) and pulmonary samples of virus were measured as described
previously (Schulman & Kilbourne, 1963).
Results
Derivation o f X-99 and X-99a hy reassortants f r o m
A / S h a n g h a i / l l / 8 7 (H3N2) influenza virus
X-99 was p r o d u c e d by r e a s s o r t m e n t o f A / S h a n g h a i /
11/87 ( S h / 8 7 ) a n d A / P R / 8 / 3 4 (PR8) viruses b y the
usual p r o c e d u r e o f d u a l infection o f a chick e m b r y o
a l l a n t o i c sac ( K i l b o u r n e et al., 1971). Viruses c o n t a i n i n g
the H A a n d n e u r a m i n i d a s e ( N A ) antigens o f S h / 8 7 virus
were isolated b y p a s s a g e with P R 8 a n t i b o d y . W h e n X-99
p r o v e d relatively p o o r yielding in vaccine p r o d u c t i o n , we
screened 26 eggs i n o c u l a t e d with S h / 8 7 virus to o b t a i n
the highest yielding w i l d - t y p e virus, then c a r r i e d o u t a
second r e a s s o r t m e n t e x p e r i m e n t with the hy m u t a n t a n d
PR8. T h e r e a s s o r t a n t r e c o v e r e d (X-99a) consistently
p r o d u c e d two to three times m o r e H A t h a n X-99. Like
its w i l d - t y p e p a r e n t X-99a differed in o t h e r b i o l o g i c a l
p r o p e r t i e s f r o m X-99, i n c l u d i n g b i n d i n g affinity to
a n t i b o d y a n d non-specific i n h i b i t o r (see below). F u r t h e r m o r e , p r e l i m i n a r y testing o f X-99 a n d X - 9 9 a w i t h
specific ferret a n t i s e r a at the Centers for Disease C o n t r o l ,
A t l a n t a suggested t h a t there were significant antigenic
differences, with X-99a being less like c o n t e m p o r a r y
H 3 N 2 isolates a n d hence less suitable as a vaccine
c a n d i d a t e . R e p e t i t i o n o f r e c i p r o c a l H I t i t r a t i o n s with the
same ferret sera in the M o u n t Sinai l a b o r a t o r y c o n f i r m e d
the antigenic difference in the r e a s s o r t a n t s , which b y o u r
q u a n t i t a t i v e analysis ( K i l b o u r n e et al., 1990) was 65 %
( d a t a n o t shown).
Phenotypic characterization o f X-99 and X-99a
T a b l e 1 s u m m a r i z e s the b i o l o g i c a l p h e n o t y p e s o f X-99
a n d X - 9 9 a c o m p a r e d with the p h e n o t y p e s o f their H 3 N 2
p a r e n t a l viruses [Sh/87 a n d S h / 8 7 (hy)]. T h e p h e n o t y p i c
characteristics o f an escape m u t a n t X - 9 9 I - , d e r i v e d b y
p a s s a g e o f X-99 with n o r m a l horse s e r u m ( N H S ) a n d o f
a n o t h e r , X - 9 9 a E , derived b y p a s s a g e o f X - 9 9 a with the
X-99a-specific M A b , A-9, are also shown. I t is clear t h a t
the r e a s s o r t a n t s X-99 a n d X - 9 9 a have derived their
p l e i o t r o p i c serological a n d o t h e r characteristics f r o m
their respective S h / 8 7 a n d S h / 8 7 hy p a r e n t s a l o n g w i t h
a c q u i s i t i o n o f their surface g l y c o p r o t e i n s . A n a l y s i s o f the
escape m u t a n t s also suggests t h a t the H A s o f X-99 a n d
X - 9 9 a differ in at least two sites as defined b y r e a c t i o n s
with N H S a n d the X-99a-specific M A b s , A-9 a n d A - 5 6 ;
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Influenza virus haemagglutinin polymorphism
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Table 2. Antigenic relatedness of selected H3N2 strains as defined by
reciprocal HI tests with polyclonal rabbit antisera
(Ai/68)*
(Eng/72)
(Ln/86)
(Sich/87)
(Sh/87)
(Bj/89)
X-31
X-37a
X-91
X-975
X-99
X-99a
Sh/87
Sh/87(hy)
X-101
X-31
X-37a
X-91
lOOt
35
35
100
-
3
10
-
8
22
-
100
50
79
43
26
X-97
X-99
50
100
87
34
100
3
10
79
87
100
40
71
43
31
X-99a Sh/87 Sh/87 (hy)
8
22
43
34
40
100
43
89
24
71
43
100
40
-
(Bj/89)
X-101
43
89
40
100
-
26
100
31
24
100
* Strains from which HA and N A antigens were derived are in parentheses.
t Percentage antigenic relatedness as determined by fractional dilution HI (homologous
relatedness is 100 %).
$ Reassortant virus, X-97, and antiserum to Sich/87 were used in this analysis.
i.e. the antigenic and inhibitor phenotype are not linked
(co-varying).
100
~80
Antigenic characterization of X-99 and X-99a viruses
and antecedent and succeeding H3N2 variants
The same magnitude of antigenic difference between X99 and X-99a (60 %) is found for their respective wildtype antecedents Sh/87 and Sh/87 (hy) (Table 2).
Although not completely identical, Sh/87 and X/99 and
Sh/87 hy and X/99a were found to be related by 71%
and 89%, respectively, by HI analysis. In plaque
neutralization tests, X-99 and X-99a and Sh/98 and
Sh/87 (hy) share only 25 % identity.
Regarding the antigenic relatedness of recent H3N2
strains, summarized in Table 2, there is the significant
difference between X-99a and all other strains, and
similarity between X-99 and the Leningrad and Sichuan
viruses isolated in 1986 and 1987. The scant relationship
between both X-99 and X-99a to their antecedents of
earlier decades, HK/68 and Eng/72 is apparent.
The two Sh/87 HA variants, X-99 and X-99a, differ
almost as much from one another as does the first
epidemiologically significant H3N2 drift variant (Eng/
72) from the antecedent 1968 prototype virus (Fig. 1).
There are also differences between the reassortants with
respect to their reaction in serological (HI) tests using
sera of patients recently infected with Sh/87-1ike influenza virus. Geometric mean serum antibody titres was
fourfold higher when X-99 was the test antigen and
antibody increases after infection averaged 6.5-fold with
X-99, and 2.5-fold with X-99a virus (data not shown).
Whether indicative of differences amongst the reassortants in antibody binding affinity in the test system, or
reflective of preferential infection and/or antigenic
stimulation by the X-99-1ike phenotype in man, these
results constitute further evidence of significant HA-
i60
.R 40
g
20
0
Ai/68
Eng/72
Sh/87
Sh/87
(X-99) (X-99a)
Fig. 1. The antigenic relatedness of hy reassortants of A/Aichi/2/68
(Ai/68) and A / E n g l a n d / 4 2 / 7 2 (Eng/72) and of A / S h a n g h a i / l l / 8 7
(Sh/87) reassortants X-99 and X-99a are comparable in degree. (Each
pair has been separately compared.)
mediated differences in X-99 and X-99a. The differing
efficiency of MDCK-adapted and egg-adapted variants
in demonstrating human antibody response (Schild et al.,
1983) also may be relevant to the present observations.
Comparative immunogenicity of X-99 and X-99a viruses
in mice
We designed an experiment to test the relative efficacies
of X-99 and X-99a vaccines in protection of mice from
challenge infection by the uncloned Sh/87 virus from
which both reassortants had been derived. Groups of five
mice were injected intraperitoneally with one or two
doses of u.v.-inactivated X-99 or X-99a virus, then
infected intranasally with Sh/87 virus 28 or 41 days
later, respectively. Just prior to infection, serum HI
antibody was measured and 3 days after infection
infective murine pulmonary-derived virus was assayed in
chick embryos. The virus recovered from infected mice
was identical in phenotype (i.e. X-99-1ike) to the original
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1314
E. D. Kilbourne and others
Table 3. Amino acid differences among S h / 8 7 H A
mutants and reassortants
Amino
acid
Sh/hy X-99a X-99aE Sh/ly X-99 X-99I-
133
145
157
158
159
186
193
219
247
262
S
N
S
E
Y
S
K
S
E
Y
S
K
S
E
D
N
N
S
E
Y
S
N
S
E
Y
S
N
-*
Y
S
S
S
I
I
I
N
S
R
T
K
S
S
T
K
S
S
T
N
F
S
N
N
S
S
T
N
S
S
T
* Deletion.
infecting virus. The HI antibody responses to identical
viruses or to the Sh/87 challenge virus did not differ
significantly. When these mouse antisera were employed
in cross-HI tests of X-99 and X-99a viruses, as had been
done with rabbit and ferret antisera, they demonstrated
complete viral antigenic relatedness (103 %). Concordant
with this finding, both vaccines were found to be equally
protective against the dose of the parent Sh/87 virus used
in challenges following either primary or secondary
immunization.
Molecular basis o f the differences between X-99 and X99a
The envelope proteins of X-99 and X-99a were identified
by H1 and NI tests as being H3 and N2. The M 1 proteins
of both viruses, and by inference RNA 7, were identified
as derived from the PR8 virus through the use of PR8
M 1-specific MAb in ELISA (Johansson et al., 1989). The
remaining viral proteins were identified as derived from
the PR8 virus by PAGE of viral proteins (Ritchey et al.,
1977) (results not shown).
That the different reactivity of X-99 and X-99a in HI
tests with polyclonal antisera was not ascribable to
intrinsic differences in their N2 neuraminidases or to
different ratios of the HA in their virions was demonstrated by the antigenic identity of their NAs in cross-NI
tests and also by the preservation of typical X-99a-like
reactivity in an H3N1 (PR8) reassortant derived from X99a (data not shown). Therefore, attention turned to HA
as the determinant of the pleiotropic differences in X-99
and X-99a.
Sequencing of HA-coding RNA 4 demonstrated nonsynonymous (coding) changes reflected in the amino acid
differences shown in Table 3. Viruses reactive with X-99a
specific MAb (hy phenotype) differed from non-reactive
X-99 (ly phenotype) viruses by having serine rather than
isoleucine at amino acid 186 of HA1. X-99aE, an escape
mutant from the neutralization of X-99a with MAb, did
not, as expected, show the above substitution at position
186, but did differ from all other viruses by an aspartic
acid for tyrosine substitution at amino acid 159 which,
like 186, is located in the antigenic site B (see Discussion).
Discussion
The emphasis in these studies is not upon the frequently
reported minor antigenic variations detectable only with
MAbs, but rather on the antigenic variation that is
sufficiently extreme to be of potential epidemiological
and immunological significance. The present results add
to previous evidence of influenza virus strain heterogeneity (Kilbourne, 1987; de Jong et al., 1988; Robertson et al., 1991; Katz & Robertson, 1992) and confirm
that significantly different antigenic variants identifiable
with polyclonal serum as well as MAbs may coexist
within a given viral strain. Selection of antigenic variants
need not be immunological (Kilbourne, 1980; Erickson
& Kilbourne, 1980; Dietzschold et al., 1983). Rather,
antigenic change may be the consequence of selection for
the hy characteristic (Both et al., 1983), the result of host
adaptation (Schild et al., 1983), or may be entirely
fortuitous. The recovery of 'Czech/89-1ike' or
'Guangdong/89-1ike' antigenically distinct variants from
the same isolate has also been described (Johansson &
Kilbourne, 1992).
Recent studies suggesting a role for the host cultivation
system in the selection of HA antigenic variants (Wood
et al., 1989; de Jong et al., 1988) are relevant to the
suggestion that egg-grown viruses tend to have greater
antigenic diversity (Robertson et al., 1991 ; Wang et al.,
1989; Katz & Robertson, 1992) and to the identification
of certain sites on the HA molecule that appear to
characterize egg adaptation of the virus. One study,
however, demonstrated the identity of sequences obtained from an egg isolate and its corresponding clinical
specimen (Rajakumar et al., 1990). A mutation site
identified by Katz et aI. (1990) as a probable determinant
of host specificity is position 186, the site that apparently
is critical in the distinction of the X-99 and X-99a
antigenic phenotypes. Both variants, however, have been
cultivated only in the chick embryo host, in which
differences in viral yield are demonstrable with both the
wild-type (Sh/hy and Sh/ly) and reassortant (X-99 and
X-99a) pairs. It is worth noting that serine at position
186 was usual for most egg-grown and two MDCKgrown clones studied by Katz et al. with PCR, yet in the
present case, isoleucine at this position characterizes our
egg-grown high yield phenotype viruses. Although
Sh/hy, Sh/ly, X-99 and X-99a differ at other sites, these
pairs have no substitution differences in common other
than at position 186. With a panel of anti-H3 MAbs,
amino acid 186 maps to the antigenic site B at the top of
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Influenza virus haemagglutinin polymorphism
the HA molecule and at the interface of antigenic sites A
and B. This site has been associated with frequent
antigenic changes in nature (Underwood, 1984).
It is not clear whether or not the inhibitor phenotype
differences are defined also by changes at position 186.
Escape mutants X - 9 9 I - from X-99, and X-99aE from
X-99a, selected respectively with NHS and MAb A56,
demonstrated no reversion at site 186 coincident with
their changes in phenotype. Rather, a deletion of amino
acids 157 and 158 occurred with X - 9 9 I - , and a tyrosine
to aspartic acid substitution occurred at position 159
with X-99aE. These amino acids also lie within the
antigenic site B and might influence binding of either
antibody or inhibitor at position 186.
The control of influenza is dependent upon a global
(WHO) surveillance system that collects and categorizes
new viral isolates with respect to their antigenic identity.
At present antigenic characterization of new isolates is
based principally on HI tests with serum obtained from
ferrets 14 days after infection. However, the present
experiments demonstrate a lack of concordance of
antigenic analysis results when antisera from other
species (rabbit and ferret) are compared with mouse
antisera. Furthermore, whether or not the X-99 and X99a reassortants differ by 65 % (ferret), 60 % (rabbit) or
not at all (mice), vaccines from both viruses were equally
effective in protecting mice against infection with the
uncloned parental Sh/87 virus. This result suggests that
minor variation among vaccine candidate strains can be
tolerated, particularly because it is uncertain which
variant is truly representative of the original human
virus. In this connection, intraisolate antigenic heterogeneity has been shown to exist even in humans by
sequencing viral HAs directly from clinical specimens
(Katz et al., 1990) or from separate clones derived from
a single specimen (Robertson et al., 1991; Katz &
Robertson, 1992).
Prior studies have attempted to assess the significance
of minor HA antigenic variation with respect to crossprotection in animal models. Comparisons among these
studies and between these studies and our own are
confounded by differences in viruses, host species and
experimental design. Our finding that BALB/c mice
cannot detect antigenic differences in X-99 and X-99a as
measured either by HI antibody response or crossprotection are in accord with the studies in A/J mice by
Rota et al. (1989) with vaccinia virus recombinants
containing variant influenza B virus HAs. Katz et al.
(1987) also demonstrated cross-protection of ferrets
immunized by infection with antigenically distinguishable MDCK cell- and egg-grown variants. However,
Wood et al. (1989), studying similar H1N1 host-adapted
variants found a concordance of antibody response with
cross-protection in guinea-pigs, ferrets and hamsters.
1315
Although mapping of HA epitopes with MAbs in
combination with RNA sequencing has produced valuable information on virus structure, antigenic variation
and evolution, pragmatic consideration of the epidemiological significance of antigenic variation and vaccine
choice must depend also on the use of polyclonal
antibody for analysis of strain differences.
Ideally, whatever animal species is used for strain
antigenic characterization, immunization should be
carried out with non-replicating virus to forestall the
selection of HA antigenic mutants best suited for
replication in that particular host, but not necessarily
representative of the input immunizing virus. Further
research is needed on antigenic characterization as well
as correlated studies on protection in humans, using viral
replication quantification as the endpoint. Such experiments should aid in the perennial task of vaccine strain
selection.
This work was supported by grants from Connaught, Lederle, ParkeDavis, and Wyeth Laboratories, by contracts from the Center for
Biologics Evaluation and Research, Division of Virology, FDA, and by
the Aaron Diamond Foundation.
References
ARCHETTI, I. & HORSFALL, F.L., JR (1950). Persistent antigenic
variation of influenza A viruses after incomplete neutralization in ovo
with heterologous immune serum. Journal of Experimental Medicine
92, 441-446.
BAEZ, M., PALESE,P. & KILBOURNE, E. n . (1980). Gene composition of
high-yielding influenza vaccine strains obtained by recombination.
Journal of Infectious Diseases 141, 36~365.
BOTH, G. W., SHI, C. H. & KILBOURNE, E. D. (1983). Hemagglutinin of
swine influenza virus: a single amino acid change pleiotropically
affects viral antigenicity and replication. Proceedings of the National
Academy of Sciences, U.S.A. 80, 69967000.
Cox, N. J., KITAME, F., KENDAL,A. P., MAASSAB,H. F. & NAEVE, C.
(1988). Identification of sequence changes in the cold-adapted live
attenuated influenza vaccine strain, A / A n n Arbor/6/60 (H2N2).
Virology 167, 554-567.
DE JONG, J. C., DE RONDE-VERLOOP, F. M., VEENENDAAL-VANHERK,
T. M., WEIJERS,T. F., BIJLSMA,K. d~OSTERHAUS,A. D. M. E. (1988).
Antigenic heterogeneity within influenza A (H3N2) virus strains.
Bulletin of the Worm Health Organization 66, 4255.
DIETZSCHOLD, B., WUNNER, W.H., WIKTOR, T.J., LOPES, A.D.,
LAFON, M., SMITH, C. L. & KOPROWSKI, H. (1983). Characterization
of an antigenic determinant of the glycoprotein that correlates with
pathogenicity of rabies virus. Proceedings of the National Academy of
Sciences, U.S.A. 80, 70-74.
ERICKSON, A. H. & KILBOURNE, E. D. (1980). Comparative amino acid
analysis of influenza A viral proteins. Virology 100, 34-42.
HOLMDAHL, R., BAILEY, C., ENANDER, I., MAYER, R., KLARESKOG, L.,
MORAN, T. & BONA, C. (1989). Origin of the autoreactive anti-type
II collagen response. II. Specificities, antibody isotypes and usage of
V gene families of anti-type II collagen B cells. Journal of Immunology
142, 1881-1886.
HORSFALL, F. L., JR & TAMM, I. (1953). Fractional dilution procedure
for precise titration of hemagglutinating viruses and hemagglutinininhibiting antibodies. Journal of Immunology 70, 253-259.
JOHANSSON, B. E. & KILBOURNE, E. D. (1991). Comparison of intranasal and aerosol infection of mice in assessment of immunity to
influenza virus infection. Journal of Virological Methods 35, 109 114.
JOHANSSON, B. E. & KILBOURNE, E. n . (1992). Influenza vaccine strain
selection: equivalence of two antigenically distinct haemagglutinin
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sat, 17 Jun 2017 23:25:42
1316
E. D. Kilbourne and others
variants of 1989 H3N2 influenza A virus in protection of mice.
Vaccine 10, 603-606.
JOHANSSON, B.E., BUCHER, D., POKORNY, B.A., MIKHA1L, A. &
KILBOURNE, E.D. (1989). Identification of PR8 MI protein in
influenza virus high-yield reassortants by Ml-specific monoclonal
antibodies. Virology 171, 634-636.
KATZ, J. M. & ROBERTSON, J. S. (1992). WHO-NIH meeting on host
cell selection of influenza virus variants. Vaccine 10, 723 725.
KATZ, J.M., N ~ w , C.W. & WEBSTER, R.G. (1987). Host cellmediated variation in H3N2 influenza viruses. Virology 156, 386-395.
KATZ, J. M., WANG, M. & WEBSTER, R. G. (1990). Direct sequencing
of the HA gene of influenza (H3N2) virus in original clinical samples
reveals sequence identity with mammalian cell-grown virus. Journal
of Virology 64, 1808-1811.
KILBOURNE, E.D. (1969). Future influenza vaccines and the use of
genetic recombinants. Bulletin of the Worm Health Organization 41,
643-645.
KtLBOURNE, E. D. (1978). Genetic dimorphism in influenza viruses:
characterization of stably associated hemagglutinin mutants differing
in antigenicity and biological properties. Proceedings of the National
Academy of Sciences, U.S.A. 75, 6258-6262.
KILBOURYE, E.D. (1987). h~uenza, pp. 114-116. New York &
London: Plenum Press.
KILBOURNE, E. D. (1980). Influenza: viral determinants of the pathogenicity and epidemicity of an invariant disease of variable
occurrence. Philosophical Transactions of the Royal Society of London
Series B 288, 291 297.
KILBOURNE, E.D., SCHULMAN,J.L., SCHILD, G.C., SCHLOER, G.,
SWANSON, J. & BUCHER, D. (1971). Correlated studies of a
recombinant influenza-virus vaccine. I. Derivation and characterization of virus and vaccine. Journal of Infectious Diseases 124,
449-462.
KILBOURNE, E.D., JOHANSSON, B.E. & GRAJOW]~R, B. (1990). Independent and disparate evolution in nature of influenza A virus
hemagglutinin and neuraminidase. Proceedings of the National
Academy of Sciences, U.S.A. 87, 786-790.
RAJAKUMAR,A., SWlERKOSZ, E. M. & SCHULZE, I. T. (1990). Sequence
of an influenza virus hemagglutinin determined directly from a
clinical sample. Proceedings of the National Academy of Sciences,
U.S.A. 87, 4154-4158.
RITCrrEY, M. D., PALESE P. & SCHULMAN, J. L. (1977). Differences in
protein patterns of influenza A viruses. Virology 76, 122 128.
ROBERTSON, J. S., NICOLSON, C., BOOTMAN,J. S., MAJOR, D., ROBERTSON, E.W. & WOOD, J.M. (1991). Sequence analysis of the
haemagglutinin (HA) of influenza A (H1NI) viruses present in
clinical material and comparison with the HA of laboratory-derived
virus. Journal of General Virology 72, 2671 2677.
ROTA, P. A., SHAW, M. W. & KEN'OAr, A. P. (1989). Cross-protection
against microvariants of influenza virus type B by vaccinia viruses
expressing haemagglutinins from egg- or MDCK cell-derived
subpopulations of influenza virus type B/England/222/82. Journal
of General Virology 70, 1533 1537.
SAIKI, R. K., GELFAND, D. H., STOFFEL, S., SCARF, S. J., HIGUCHI, R.,
HORN, G. T., MULLIS, K. B. & ERLICH, H. A. (1988). Primer-directed
enzymatic amplification of DNA with a thermostable DNA
polymerase. Science 239, 487-494.
SCHILD, G. C., OXFORD, J. S., DE JONG, J. C. & WEBSTER, R. G. (1983).
Evidence for host-cell selection of influenza virus antigenic variants.
Nature, London 303, 706-709.
SCHULMAN, J.L. & KILBOURNE, E.D. 0963). Experimental transmission of influenza virus infection in mice. I. The period of
transmissibility. Journal of Experimental Medicine 118, 257-266.
UNDERWOOD,P. A. (1984). An antigenic map of the haemagglutinin of
the influenza Hong Kong subtype (H3N2), constructed using mouse
monoclonal antibodies. Molecular Immunology 21, 663 671.
USUBA, O., SCHULMAN,J. L., DEATLY, A. M., BONA, C. A. & MORAN,
T.M. (1990). New method for titration of virus infectivity by
immunostaining. Viral Immunology 3, 237-241.
WANG, M., KATZ, J.M. & WEBSTER, R.G. (1989). Extensive
heterogeneity in the hemagglutinin of egg-grown influenza viruses
from different patients. Journal of Virology 171,275-279.
WOOD, J. M., OXFORD, J. S., DUNEEAVY, U., NEWMAN, R. W., MAJOR,
D. & ROBERTSON, J. S. (1989). Influenza A (H1N1) vaccine efficacy
in animal models is influenced by two amino acid substitutions in the
hemagglutinin molecule. Virology 171, 214-221.
(Received 20 November 1992; Accepted 8 March 1993)
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