Antigenic and genetic characterization of the ... - Semantic Scholar
Journal of General Virology (1992), 73, 2737-2742.
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Antigenic and genetic characterization of the haemagglutinins of recent cocirculating strains of influenza B virus Paul A. Rota,* Mark L. Hemphill, Toni Whistler, Helen L. Regnery and Alan P. Kendal Influenza Branch, Division of Viral and Rickettsial Diseases, National Center for Infectious Diseases, MS-G17, 1600 Clifton Road, Department of Health and Human Services, Centers for Disease Control, Atlanta, Georgia 30333, U.S.A.
The antigenic and genetic characteristics of the haemagglutinins of influenza type B viruses isolated since 1988 during periods of both widespread activity (1990/1991) and sporadic activity (1989/1990) were examined using microneutralization tests and direct RNA sequencing. During 1989/1990, influenza B viruses representative of two distinct lineages antigenically and genetically related to either B/Victoria/2/87 or B/Yamagata/16/88 were isolated, and a minor drift variant of B/Yamagata/16/88, B/Hong Kong/22/89, was identified. In 1990/1991,
B/Hong Kong/22/89- or B/Yamagata/16/88-1ike viruses accounted for the majority of the influenza virus isolates in most countries. Sequence analysis of the HA 1 domains of representative viruses confirmed the continued existence of two mainlineages among recent strains of influenza B virus and identified uniqueamino acid changes that could account for the altered antigenic reactivity of some variants. Sequence analysis of the HA2 domains of some of the recent influenza B viruses allowed for a comparison of the evolutionary rates and patterns between the HA1 and HA2 domains.
Influenza type B viruses have been isolated during periods of widespread influenza activity during five of the last 10 influenza seasons in the United States. During this time, each major peak of influenza B virus activity has been associated with the emergence of a new antigenic variant of the virus. The antigenic sites on the haemagglutinin (HA) of influenza type B viruses have been delineated by sequence analysis of both circulating viruses and laboratory-derived variants (Verhoeyen et al., 1983; Webster & Berton, 1981; Krystal et al., 1982, 1983; Berton et al., 1984; Berton & Webster, 1985; Hovanec & Air, 1984; Bootman & Robertson, 1988). For recent influenza B viruses, at least two discrete lineages have co-existed since at least 1983 (Rota et al., 1990; Kanegae et al., 1990). Viruses from each of these two lineages, represented by either B/Yamagata/16/88 (B/YM/88) or B/Victoria/2/87 (B/VI/87), had as many as 27 amino acid differences between their HA 1 proteins by 1988 and were distinct antigenically. During 1989/1990, influenza B viruses antigenically related to both B/VI/87 and B/YM/88 were isolated sporadically throughout the world and altogether, influenza B viruses represented less than 1% of the total number of influenza virus isolates reported in the United
States (Centers for Disease Control, 1990). Despite this limited circulation, antigenic drift variants were identified from each lineage of influenza B virus. During 1990/1991, the majority of influenza virus isolates in many countries were of type B (World Health Organization, 1991c). Although viruses closely related to both B/VI/87 and B/YM/88 were identified, 65% of these isolates were antigenically related to a drift variant of B/YM/88, B/Hong Kong/22/89 (Centers for Disease Control, 1991; World Health Organization, 1991a). In 1991/1992, influenza type A (H3N2) viruses were the predominant strain, but infrequent isolations of B/YM/88-, B/Hong Kong/22/89- and B/VI/87-1ike viruses have been reported (World Health Organization, 1992). In this report, influenza B viruses representative of viruses isolated during the 1989/1990 and 1990/1991 influenza seasons were further characterized by molecular and serological methods. Recently isolated influenza B viruses were initially identified as being most closely related to B/YM/88 or B/VI/87 using haemagglutination inhibition assays (data not shown) (Palmer et al., 1976), and strains representative of recently circulating influenza B viruses were chosen for further analysis. The results of microneutralization tests indicated that drift variants had arisen among the B/YM/88-1ike viruses (Table 1). B/HK289, B/PN/90 and B/BK/91 showed the
New sequence data reported in this paper are available from GenBank under accession numbers M65165 to M65177. 0001-1000 © 1992 SGM
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Table 1. Antigenic drift of influenza type B viruses: 1989 to 1991 Neutralization titre* with post-infection ferret antiserum to: Strain B/Yamagata/16/88 B/Hong Kong/9/89 B/Guandong/55/89 B/Texas/l/91 B/South Dakota/5/89 B/Texas/4/90 B/New York/3/90 B/Bangkok/163/91 B/Victoria/103/89 B/Hong Kong/22/89 B/Panama/45/90 B/Victoria/2/87 B/Beijing/2/87 B/Texas/37/88 B/Victoria/19/89 B/Paris/329/90 B/India]3/89
(B/YM/88) (B/HK/89) (B/GD/89) (B/TX/91) (B/SD/89) (B/TX/90) (B/NY/90) (B/BK/91) (B/VII03) (B/HK289) (B/PN/90) (B/VI/87) (B/BJ/87) (B/TX/88) (B/VI/89) (B/PS/90) (B/IN/89)
B/YM/88
B/BJ/87
1280 1280 640 1920 480 960 480 160 640 160 160 10 10 10 10 10 10
40 80 40 80 30 80 40 40 20 10 10 640 32,0 160 160 240 60
B/IN/89 15 20 10 10 10 20 10 60 10 10 10 40 40 20 10 20 160
* Harmon et al. (1988).
greatest amount of antigenic change with an eightfold drop in titre against the antiserum to B/YM/88. The B/VI/87-1ike viruses were antigenically homogeneous from 1987 to 1989. Antiserum to B/BJ/87, a virus genetically and antigenically similar to B/VI/87, was used because it had a higher homologous titre than the antiserum to B/VI/87. B/IN/89 showed little crossreactivity with any of the influenza B viruses tested. Although no other B/IN/89-1ike viruses were reported, this virus was chosen for further analysis because of its unique antigenic characteristics. To characterize the influenza type B isolates further, the nucleotide and deduced amino acid sequences were determined from purified viral R N A as described previously (Rota et al., 1990) for all of the HA1 domains and some of the HA2 domains of the viruses listed in Table 1. The HA1 domains from recently cocirculating viruses varied by as many as 83 (8.7~) nucleotide and 34 (9.1 ~ ) amino acid changes. The group of B/YM/88-1ike viruses differed by as many as 10 amino acids in HA1 and were more genetically diverse than the recent B/VI/87-1ike viruses. Sequence analysis indicated that B/IN/89 is clearly related to the B/VI/87-1ike viruses. Fig. 1 shows the deduced amino acid sequences for the HA 1-encoding regions of the recent influenza B virus isolates compared to the HA1 sequences of either B/VI/87 or B/YM/88. The HAls of the majority of the more recent influenza B viruses from both lineages had amino acid changes at positions 73, 197 and 199. At some amino acid residues, changes appeared to be limited to
one lineage. For example, all of the recent B/VI/87-1ike viruses had an amino acid change (V to I) at residue number 137 whereas the majority of recent B/YM/88like viruses had changes at positions 150, 203, 230 and 298. Viruses from both lineages had the majority of amino acid changes between positions 100 and 200; this region includes the previously proposed immunodominant region of the HA of influenza B virus (Berton & Webster, 1985). The B/YM/88-1ike viruses had two or three changes in the region of amino acids 200 to 300 in contrast to the B/VI/87-1ike viruses which had none or one. The antigenic presentation of amino acids 200 to 300 on the B/YM/88-1ike HAls may be affected by a nearby potential glycosylation site at positions 233 to 235 that is not present on any of the B/VI/87-1ike HAls. Within the H A l s of viruses from each of the main lineages, amino acid differences that could account for altered antigenic reactivity were sometimes apparent. The amino acid change that had the greatest effect on antigenicity was the glycine to arginine change at position 141 in B/IN/89. This residue has been shown to vary with the host cell used for isolation of the virus (Oxford et al., 1990, 1991). Among the B/YM/88-1ike viruses showing the greatest antigenic drift, it was observed that B/HK289 and B/PN/90 shared unique amino acid changes at positions 76 and 122, whereas B/BK/91 had unique changes at positions 149 and 217. Although amino acid changes at positions 76 and 150 were unique to recent B/HK289-1ike influenza B viruses, the histidine to glutamic acid change at position number 122 has been observed in an earlier B/YM/88-1ike virus, B/SN/88 (Fig. 3), which was antigenically identical to B/YM/88 (Rota et al., 1990). Previous sequence analysis has revealed that clinical specimens of influenza B virus passaged in mammalian cells retain a potential glycosylation site at amino acids 197 to 199 whereas virus passaged in eggs usually lose this site (Schild et al., 1983; Robertson et al., 1985, 1990). However, analysis of individual cDNA clones did reveal that a small number of egg-derived viruses retained this glycosylation signal (Robertson et al., 1990). Our data, which were obtained by sequencing R N A purified from egg-passaged virus, indicated that several of these viruses had retained the potential glycosylation site at amino acids 197 to 199 (Fig. 1). To determine whether this site was utilized in these viruses, the HAs of viruses (B/IN/89 and B/VI/89, B/NY/90 and B/TX/91, B/HK289 and B/PN/90) which had very similar HA1 sequences, but differed at this predicted glycosylation site, were analysed by SDS-PAGE and Western blotting. The HAs from only one pair of related viruses, B/IN/89 and B/VI/89, which differed by four amino acids showed differences in apparent Mr. This migrational difference was not observed after the HAs were
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HAl: VI87 BJe7 P589 IN89 VII9
YM88 GDS9 HK89 HK289 VII03 SD89 PN90 NYg0 TX90 BKgl Txgl
1 DRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPTKSHFANLKGTKTRGKLCPK•LNCTDLDVALARPKCMGTIPSAKASILHEVKPVTSG•FPIMHD Q
T T T
100
F F
DRICTGITSSNSPH~TATQGEVNVTGVIPLTTTPTKSHFANLKGTKTRGKLCPNCLNCTDLDVALARPMCMGTIPSAKASILHEVRPVTSGCFPIMHD I V V I V I I I I
T
T R
VI87 BJ87 PS89 IN89 VII9
101 RTKIR•LPNLLRGYEHIRLSTH•VINAETAPGGPYKVGTSGSCPNVTNGNGFFATMAWAVPKNDNNKTATNPLTVEVPYICTEGEDQITVWGFHSDSETQ N I I I R K I S
N A N A N N I
YM88 GD89 HK$9 HK289 VII03 SD89 PN90 NY90 TX90 BK91 TX91
RTKIR•LPNLLRGYENIRL•THNVINAERAPGGPYRLGTSGSCPNVT•RNGFFATMAWAVPRDN.K.TATNPLTVEVPYICTKGED•ITVWGFHSDDKT• S I S Q D G A S Q D S S KS S
K S S N A S N N N N N N I
VI87 BJ87 PS89 IN89 VII9
YM88 GD89 HK89 HK289 VII03 SD89 PNg0 NYg0 TX90 BK91 TX91
VI87 BJ87 PS89 IN89 VII9
YM$8 GD89 HK$9 HK289 VII03 SD89 PN90 NY90 TX90 BKgl TX91
201 MVKLYGDSKP•KFTSSANGVTTHYVSQIG•FPN•AEDGGLPQSGRIVVDYMV•KSGKTGTITY•RGILLP•KVWCASGRSKVIKGSL•LIGEADCLHEKY R a
MKKLYGDSNP~KFTSSANGVTTHYVS~IGDFPNQTEDGGLPQSGRIVVDYMVQKPGKTGTIVYQR~VLLP~KVWCASGRSKVIKGSLPLIGEADCLHAKY N N S N G N G G N G N G N G N V G N G
200
300
E E E E E E
301 347 GGLNKSKPYYTGEHAKAIGNCPIWVKTpLKLANGTKYRPPAKLLKER
GGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKER
I'1~2 : SN79 SU83 AA86 VI87 HK289
SN79 SU83 AAS6 VI8? HK289
SN79 SU83 AA86 VI8? HK289
348 GFFGAIAGFLEGGWEGMIAGWHGYTSHGAHGVAVAAD
E
447 L K S T Q E A I NK I T K N L N S LSE LEVI(NLQRLSGAMDELHNE I L E L D E K V D D L R A D T I SSQI E ~ V
O
448 547 L L S N E G I I N S E D E H L L A L E R K L K K M L G P S A V D I GNGCFETKIIKCNOTCLDR I A A G T F N A G E F S LP TFDS LNI T A A S L N D D G L D N H T I L L Y Y S T A A S S L A V
E
548 570 T LMIAI F I V Y M V S R D N V S C S I C L
Fig. 1. Changes in the deduced amino acid sequence of the HA1- and HA2-encoding regions of recent B/YM/88-1ike or B/VI/87-1ike influenza B viruses. HA 1 sequences begin with the first amino acid after signal peptide cleavage and end at the HA 1-HA2 cleavage site. A period indicates an amino acid deletion. See Table 1 for strain abbreviations.
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1
2
3
4
MD59
HAl:
VI87 -
FI49
IN89
PS90 BJ87 FI45 88 Vl89 AA86 N82
~ u
85 VI103 8 SH87
PN90 ~{--HK289 5N88
Fig. 2. Electrophoretic analysis of the HAs of influenza B viruses. Preparations of B/IN/89 (lanes I and 3) or B/VI/89 (lanes-2 and 4) viruses were subjected to electrophoresis through 8-0% 4 M-ureapolyacrylamide gels before being transferred to nitrocellulose and reacted with rabbit antiserum against influenza B virus HA (Rota et al., 1987). Lanes 3 and 4 show preparations after treatment with 4 units of endoglycosidase F (endoglycosidase F/N-glycosidase F, BoehringerMannheim) and incubation at 37 °C for 1 h.
--HK89 TX90
L\ °
U83 ~SNS7 K289 VL87
Z - -
HK73 LE40
treated with endoglycosidase F (Fig. 2). Therefore, the additional glycosylation site at amino acids 197 to 199 was apparently maintained and utilized in some eggderived viruses. The evolutionary relationships of the recent influenza B virus isolates and of several previously characterized strains are shown in Fig. 3. Within each of the two main lineages of recent influenza B virus, multiple sublineages were recognized. There are at least four sublineages among the B/YM/88-1ike viruses; however, only the B/HK289-1ike viruses showed consistent antigenic differences with polyclonal antiserum. Within the B/VI/87-1ike viruses, there are only two sub-branches that can be identified genetically, one related to the B/TX/88-1ike viruses and the other represented by B/VI/89-1ike viruses; however viruses from these subbranches are antigenically indistinguishable with polyclonal antiserum. Viruses that were genetically related to both B/TX/88 and B/VI/89 have been isolated in Finland during 1989/1990 (Kinnunen et al., 1992). Viruses that were genetically most closely related to either B/VI 103 or B/VI/89 were isolated in Russia during 1990/1991 (M. L. Hemphill & P. A. Rota, unpublished observations). The relationships observed after the sequences of influenza B viruses isolated before 1979 were included in the analysis suggested that the branchpoint between the presently cocirculating lineages occurred between 1973 and 1979. The nucleotide and deduced amino acid sequences of the HA2 domains of B/SU/83, B/AA/86, B/HK289 and
HA2:
-HK75
t
_
_
- - L E 4 0
J
s~Je:25muts~ons Fig. 3. Evolutionary relationships of the HA1 and HA2 domains of influenza B viruses from 1940to 1991. Sequence data were analysed by using version 7.0 of the sequence analysis software package of the University of Wisconsin Genetics Computer Group (Devereaux et al., 1984) and version 3.4 of the Phylogeny Inference Package (Phylip; Felsenstein, 1989). Pbenograms were based on the nucleotide differences between viruses. Sequences from previous reports are of: B/Lee/40 (LE40) (Krystal et al., 1982); B/Hong Kong/73 (HK73) and B/Maryland/59 (MD59) (Krystal et al., 1983); B/Singapore/222/79 (SN79) (Verhoeyenet aL, 1983); B/England/222/82 (EN82) (Rota et al., 1987); B/Ann Arbor/I/86 (AA86) and B/USSR/100/83 (SU83) (Bootman & Robertson, 1988); B/Singapore/7/88 (SN88), B/Shanghai/12/87 (SH87), B/Hong Kong/14/88 (B/HK/88), B/VI/89 (VI89), B/YM/88 (YM88), B/BJ/87 (BJ87) and B/TX/88 (TX88)(see Table 1) (Rota et al., 1990); B/Finland/149/90 (FI49), B/Finland/145/90 (FI45) (Kinnunen et al., 1992).
B/VI/87 were determined and compared to those of several other influenza type B viruses. As expected, few amino acid changes were detected over 10 years (Fig. 1). However, there were as many as 24 nucleotide changes between the HA2 domains of B/VI/87 and B/HK289. Phylogenetic analysis indicated that the HA2 domains of currently circulating B/YM/88- and B/VI/87-1ike viruses were also on separate lineages. For the 12-year period from 1979 to 1991, the nucleotide substitution rate for HA1 (0.236 + 0.04%/ year) was similar to the rate for HA2 (0.196 + 0.06%/
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year). However, the rate of amino acid change was 0-30~/year for HA1 compared to 0-056~/year for HA2. Although approximately 40~o of the nucleotide changes in HA1 coded for amino acid substitutions, only 8.7~ of the nucleotide changes in HA2 caused changes in the HA2 protein. The rate calculated for influenza B virus is only about 309/o less than that of influenza A viruses. However, when B/Lee/40 is used as the root virus, the rate that can be calculated from our data (0-11 ~/year) is similar to that previously described for influenza B virus ( Y a m a s h i t a et al., 1988; A i r et al., 1990). A l t h o u g h the presence of m u l t i p l e cocirculating v a r i a n t s of influenza B virus had b e e n d e m o n s t r a t e d (Lu et al., 1983; Oxford et al., 1984; Y a m a s h i t a et al., 1988; D o n a t e l l i et al., 1989; R o t a et al., 1990), our results i n d i c a t e d that two d i s t i n c t lineages of influenza B virus have persisted a n d have caused disease over a period of at least 4 years. I n the case of the type A viruses a single lineage of H A rapidly b e c a m e the d o m i n a n t e p i d e m i c v a r i a n t (Both et al., 1983; R a y m o n d et al., 1986; Cox et al., 1989). T h e a n t i g e n i c as well as genetic r e l a t i o n s h i p s a m o n g the currently circulating strains of influenza virus provide useful i n f o r m a t i o n for d e t e r m i n i n g the most a p p r o p r i a t e c o m p o s i t i o n for the influenza vaccine. T h e complexity of the e v o l u t i o n a r y p a t t e r n s of influenza type B m a k e s the choice of type B c o m p o n e n t difficult. Vaccines for use in 1991/1992 a n d 1992/1993 have b e e n u p d a t e d to include either B / P N / 9 0 or B / Y M / 8 8 as the type B c o m p o n e n t (World H e a l t h O r g a n i z a t i o n , 1991a, 1992). C o n t i n u e d surveillance of these viruses will be necessary to detect the e m e r g e n c e of n e w lineages o f virus with altered a n t i g e n i c properties a n d to ensure t h a t future vaccines c o n t a i n the most a p p r o p r i a t e strain of
virus. We are grateful to Brian Holloway, Edward George and Melissa Olsen-Rasmussen for the synthesis of the oligonucleotide sequencing primers, Sarah McKneally for assistance with computer analysis of sequence data and Victor Tsang for preparation of the monoclonal antibody used in neutralization tests, and to Nancy Cox for review of this manuscript. T.W. is a National Research Council Research Associate.
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(Received 27 March 1992; Accepted 15 June 1992)
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Antigenic and genetic characterization of the haemagglutinins of recent cocirculating strains of influenza B virus Paul A. Rota,* Mark L. Hemphill, Toni Whistler, Helen L. Regnery and Alan P. Kendal Influenza Branch, Division of Viral and Rickettsial Diseases, National Center for Infectious Diseases, MS-G17, 1600 Clifton Road, Department of Health and Human Services, Centers for Disease Control, Atlanta, Georgia 30333, U.S.A.
The antigenic and genetic characteristics of the haemagglutinins of influenza type B viruses isolated since 1988 during periods of both widespread activity (1990/1991) and sporadic activity (1989/1990) were examined using microneutralization tests and direct RNA sequencing. During 1989/1990, influenza B viruses representative of two distinct lineages antigenically and genetically related to either B/Victoria/2/87 or B/Yamagata/16/88 were isolated, and a minor drift variant of B/Yamagata/16/88, B/Hong Kong/22/89, was identified. In 1990/1991,
B/Hong Kong/22/89- or B/Yamagata/16/88-1ike viruses accounted for the majority of the influenza virus isolates in most countries. Sequence analysis of the HA 1 domains of representative viruses confirmed the continued existence of two mainlineages among recent strains of influenza B virus and identified uniqueamino acid changes that could account for the altered antigenic reactivity of some variants. Sequence analysis of the HA2 domains of some of the recent influenza B viruses allowed for a comparison of the evolutionary rates and patterns between the HA1 and HA2 domains.
Influenza type B viruses have been isolated during periods of widespread influenza activity during five of the last 10 influenza seasons in the United States. During this time, each major peak of influenza B virus activity has been associated with the emergence of a new antigenic variant of the virus. The antigenic sites on the haemagglutinin (HA) of influenza type B viruses have been delineated by sequence analysis of both circulating viruses and laboratory-derived variants (Verhoeyen et al., 1983; Webster & Berton, 1981; Krystal et al., 1982, 1983; Berton et al., 1984; Berton & Webster, 1985; Hovanec & Air, 1984; Bootman & Robertson, 1988). For recent influenza B viruses, at least two discrete lineages have co-existed since at least 1983 (Rota et al., 1990; Kanegae et al., 1990). Viruses from each of these two lineages, represented by either B/Yamagata/16/88 (B/YM/88) or B/Victoria/2/87 (B/VI/87), had as many as 27 amino acid differences between their HA 1 proteins by 1988 and were distinct antigenically. During 1989/1990, influenza B viruses antigenically related to both B/VI/87 and B/YM/88 were isolated sporadically throughout the world and altogether, influenza B viruses represented less than 1% of the total number of influenza virus isolates reported in the United
States (Centers for Disease Control, 1990). Despite this limited circulation, antigenic drift variants were identified from each lineage of influenza B virus. During 1990/1991, the majority of influenza virus isolates in many countries were of type B (World Health Organization, 1991c). Although viruses closely related to both B/VI/87 and B/YM/88 were identified, 65% of these isolates were antigenically related to a drift variant of B/YM/88, B/Hong Kong/22/89 (Centers for Disease Control, 1991; World Health Organization, 1991a). In 1991/1992, influenza type A (H3N2) viruses were the predominant strain, but infrequent isolations of B/YM/88-, B/Hong Kong/22/89- and B/VI/87-1ike viruses have been reported (World Health Organization, 1992). In this report, influenza B viruses representative of viruses isolated during the 1989/1990 and 1990/1991 influenza seasons were further characterized by molecular and serological methods. Recently isolated influenza B viruses were initially identified as being most closely related to B/YM/88 or B/VI/87 using haemagglutination inhibition assays (data not shown) (Palmer et al., 1976), and strains representative of recently circulating influenza B viruses were chosen for further analysis. The results of microneutralization tests indicated that drift variants had arisen among the B/YM/88-1ike viruses (Table 1). B/HK289, B/PN/90 and B/BK/91 showed the
New sequence data reported in this paper are available from GenBank under accession numbers M65165 to M65177. 0001-1000 © 1992 SGM
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Table 1. Antigenic drift of influenza type B viruses: 1989 to 1991 Neutralization titre* with post-infection ferret antiserum to: Strain B/Yamagata/16/88 B/Hong Kong/9/89 B/Guandong/55/89 B/Texas/l/91 B/South Dakota/5/89 B/Texas/4/90 B/New York/3/90 B/Bangkok/163/91 B/Victoria/103/89 B/Hong Kong/22/89 B/Panama/45/90 B/Victoria/2/87 B/Beijing/2/87 B/Texas/37/88 B/Victoria/19/89 B/Paris/329/90 B/India]3/89
(B/YM/88) (B/HK/89) (B/GD/89) (B/TX/91) (B/SD/89) (B/TX/90) (B/NY/90) (B/BK/91) (B/VII03) (B/HK289) (B/PN/90) (B/VI/87) (B/BJ/87) (B/TX/88) (B/VI/89) (B/PS/90) (B/IN/89)
B/YM/88
B/BJ/87
1280 1280 640 1920 480 960 480 160 640 160 160 10 10 10 10 10 10
40 80 40 80 30 80 40 40 20 10 10 640 32,0 160 160 240 60
B/IN/89 15 20 10 10 10 20 10 60 10 10 10 40 40 20 10 20 160
* Harmon et al. (1988).
greatest amount of antigenic change with an eightfold drop in titre against the antiserum to B/YM/88. The B/VI/87-1ike viruses were antigenically homogeneous from 1987 to 1989. Antiserum to B/BJ/87, a virus genetically and antigenically similar to B/VI/87, was used because it had a higher homologous titre than the antiserum to B/VI/87. B/IN/89 showed little crossreactivity with any of the influenza B viruses tested. Although no other B/IN/89-1ike viruses were reported, this virus was chosen for further analysis because of its unique antigenic characteristics. To characterize the influenza type B isolates further, the nucleotide and deduced amino acid sequences were determined from purified viral R N A as described previously (Rota et al., 1990) for all of the HA1 domains and some of the HA2 domains of the viruses listed in Table 1. The HA1 domains from recently cocirculating viruses varied by as many as 83 (8.7~) nucleotide and 34 (9.1 ~ ) amino acid changes. The group of B/YM/88-1ike viruses differed by as many as 10 amino acids in HA1 and were more genetically diverse than the recent B/VI/87-1ike viruses. Sequence analysis indicated that B/IN/89 is clearly related to the B/VI/87-1ike viruses. Fig. 1 shows the deduced amino acid sequences for the HA 1-encoding regions of the recent influenza B virus isolates compared to the HA1 sequences of either B/VI/87 or B/YM/88. The HAls of the majority of the more recent influenza B viruses from both lineages had amino acid changes at positions 73, 197 and 199. At some amino acid residues, changes appeared to be limited to
one lineage. For example, all of the recent B/VI/87-1ike viruses had an amino acid change (V to I) at residue number 137 whereas the majority of recent B/YM/88like viruses had changes at positions 150, 203, 230 and 298. Viruses from both lineages had the majority of amino acid changes between positions 100 and 200; this region includes the previously proposed immunodominant region of the HA of influenza B virus (Berton & Webster, 1985). The B/YM/88-1ike viruses had two or three changes in the region of amino acids 200 to 300 in contrast to the B/VI/87-1ike viruses which had none or one. The antigenic presentation of amino acids 200 to 300 on the B/YM/88-1ike HAls may be affected by a nearby potential glycosylation site at positions 233 to 235 that is not present on any of the B/VI/87-1ike HAls. Within the H A l s of viruses from each of the main lineages, amino acid differences that could account for altered antigenic reactivity were sometimes apparent. The amino acid change that had the greatest effect on antigenicity was the glycine to arginine change at position 141 in B/IN/89. This residue has been shown to vary with the host cell used for isolation of the virus (Oxford et al., 1990, 1991). Among the B/YM/88-1ike viruses showing the greatest antigenic drift, it was observed that B/HK289 and B/PN/90 shared unique amino acid changes at positions 76 and 122, whereas B/BK/91 had unique changes at positions 149 and 217. Although amino acid changes at positions 76 and 150 were unique to recent B/HK289-1ike influenza B viruses, the histidine to glutamic acid change at position number 122 has been observed in an earlier B/YM/88-1ike virus, B/SN/88 (Fig. 3), which was antigenically identical to B/YM/88 (Rota et al., 1990). Previous sequence analysis has revealed that clinical specimens of influenza B virus passaged in mammalian cells retain a potential glycosylation site at amino acids 197 to 199 whereas virus passaged in eggs usually lose this site (Schild et al., 1983; Robertson et al., 1985, 1990). However, analysis of individual cDNA clones did reveal that a small number of egg-derived viruses retained this glycosylation signal (Robertson et al., 1990). Our data, which were obtained by sequencing R N A purified from egg-passaged virus, indicated that several of these viruses had retained the potential glycosylation site at amino acids 197 to 199 (Fig. 1). To determine whether this site was utilized in these viruses, the HAs of viruses (B/IN/89 and B/VI/89, B/NY/90 and B/TX/91, B/HK289 and B/PN/90) which had very similar HA1 sequences, but differed at this predicted glycosylation site, were analysed by SDS-PAGE and Western blotting. The HAs from only one pair of related viruses, B/IN/89 and B/VI/89, which differed by four amino acids showed differences in apparent Mr. This migrational difference was not observed after the HAs were
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HAl: VI87 BJe7 P589 IN89 VII9
YM88 GDS9 HK89 HK289 VII03 SD89 PN90 NYg0 TX90 BKgl Txgl
1 DRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPTKSHFANLKGTKTRGKLCPK•LNCTDLDVALARPKCMGTIPSAKASILHEVKPVTSG•FPIMHD Q
T T T
100
F F
DRICTGITSSNSPH~TATQGEVNVTGVIPLTTTPTKSHFANLKGTKTRGKLCPNCLNCTDLDVALARPMCMGTIPSAKASILHEVRPVTSGCFPIMHD I V V I V I I I I
T
T R
VI87 BJ87 PS89 IN89 VII9
101 RTKIR•LPNLLRGYEHIRLSTH•VINAETAPGGPYKVGTSGSCPNVTNGNGFFATMAWAVPKNDNNKTATNPLTVEVPYICTEGEDQITVWGFHSDSETQ N I I I R K I S
N A N A N N I
YM88 GD89 HK$9 HK289 VII03 SD89 PN90 NY90 TX90 BK91 TX91
RTKIR•LPNLLRGYENIRL•THNVINAERAPGGPYRLGTSGSCPNVT•RNGFFATMAWAVPRDN.K.TATNPLTVEVPYICTKGED•ITVWGFHSDDKT• S I S Q D G A S Q D S S KS S
K S S N A S N N N N N N I
VI87 BJ87 PS89 IN89 VII9
YM88 GD89 HK89 HK289 VII03 SD89 PNg0 NYg0 TX90 BK91 TX91
VI87 BJ87 PS89 IN89 VII9
YM$8 GD89 HK$9 HK289 VII03 SD89 PN90 NY90 TX90 BKgl TX91
201 MVKLYGDSKP•KFTSSANGVTTHYVSQIG•FPN•AEDGGLPQSGRIVVDYMV•KSGKTGTITY•RGILLP•KVWCASGRSKVIKGSL•LIGEADCLHEKY R a
MKKLYGDSNP~KFTSSANGVTTHYVS~IGDFPNQTEDGGLPQSGRIVVDYMVQKPGKTGTIVYQR~VLLP~KVWCASGRSKVIKGSLPLIGEADCLHAKY N N S N G N G G N G N G N G N V G N G
200
300
E E E E E E
301 347 GGLNKSKPYYTGEHAKAIGNCPIWVKTpLKLANGTKYRPPAKLLKER
GGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKER
I'1~2 : SN79 SU83 AA86 VI87 HK289
SN79 SU83 AAS6 VI8? HK289
SN79 SU83 AA86 VI8? HK289
348 GFFGAIAGFLEGGWEGMIAGWHGYTSHGAHGVAVAAD
E
447 L K S T Q E A I NK I T K N L N S LSE LEVI(NLQRLSGAMDELHNE I L E L D E K V D D L R A D T I SSQI E ~ V
O
448 547 L L S N E G I I N S E D E H L L A L E R K L K K M L G P S A V D I GNGCFETKIIKCNOTCLDR I A A G T F N A G E F S LP TFDS LNI T A A S L N D D G L D N H T I L L Y Y S T A A S S L A V
E
548 570 T LMIAI F I V Y M V S R D N V S C S I C L
Fig. 1. Changes in the deduced amino acid sequence of the HA1- and HA2-encoding regions of recent B/YM/88-1ike or B/VI/87-1ike influenza B viruses. HA 1 sequences begin with the first amino acid after signal peptide cleavage and end at the HA 1-HA2 cleavage site. A period indicates an amino acid deletion. See Table 1 for strain abbreviations.
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1
2
3
4
MD59
HAl:
VI87 -
FI49
IN89
PS90 BJ87 FI45 88 Vl89 AA86 N82
~ u
85 VI103 8 SH87
PN90 ~{--HK289 5N88
Fig. 2. Electrophoretic analysis of the HAs of influenza B viruses. Preparations of B/IN/89 (lanes I and 3) or B/VI/89 (lanes-2 and 4) viruses were subjected to electrophoresis through 8-0% 4 M-ureapolyacrylamide gels before being transferred to nitrocellulose and reacted with rabbit antiserum against influenza B virus HA (Rota et al., 1987). Lanes 3 and 4 show preparations after treatment with 4 units of endoglycosidase F (endoglycosidase F/N-glycosidase F, BoehringerMannheim) and incubation at 37 °C for 1 h.
--HK89 TX90
L\ °
U83 ~SNS7 K289 VL87
Z - -
HK73 LE40
treated with endoglycosidase F (Fig. 2). Therefore, the additional glycosylation site at amino acids 197 to 199 was apparently maintained and utilized in some eggderived viruses. The evolutionary relationships of the recent influenza B virus isolates and of several previously characterized strains are shown in Fig. 3. Within each of the two main lineages of recent influenza B virus, multiple sublineages were recognized. There are at least four sublineages among the B/YM/88-1ike viruses; however, only the B/HK289-1ike viruses showed consistent antigenic differences with polyclonal antiserum. Within the B/VI/87-1ike viruses, there are only two sub-branches that can be identified genetically, one related to the B/TX/88-1ike viruses and the other represented by B/VI/89-1ike viruses; however viruses from these subbranches are antigenically indistinguishable with polyclonal antiserum. Viruses that were genetically related to both B/TX/88 and B/VI/89 have been isolated in Finland during 1989/1990 (Kinnunen et al., 1992). Viruses that were genetically most closely related to either B/VI 103 or B/VI/89 were isolated in Russia during 1990/1991 (M. L. Hemphill & P. A. Rota, unpublished observations). The relationships observed after the sequences of influenza B viruses isolated before 1979 were included in the analysis suggested that the branchpoint between the presently cocirculating lineages occurred between 1973 and 1979. The nucleotide and deduced amino acid sequences of the HA2 domains of B/SU/83, B/AA/86, B/HK289 and
HA2:
-HK75
t
_
_
- - L E 4 0
J
s~Je:25muts~ons Fig. 3. Evolutionary relationships of the HA1 and HA2 domains of influenza B viruses from 1940to 1991. Sequence data were analysed by using version 7.0 of the sequence analysis software package of the University of Wisconsin Genetics Computer Group (Devereaux et al., 1984) and version 3.4 of the Phylogeny Inference Package (Phylip; Felsenstein, 1989). Pbenograms were based on the nucleotide differences between viruses. Sequences from previous reports are of: B/Lee/40 (LE40) (Krystal et al., 1982); B/Hong Kong/73 (HK73) and B/Maryland/59 (MD59) (Krystal et al., 1983); B/Singapore/222/79 (SN79) (Verhoeyenet aL, 1983); B/England/222/82 (EN82) (Rota et al., 1987); B/Ann Arbor/I/86 (AA86) and B/USSR/100/83 (SU83) (Bootman & Robertson, 1988); B/Singapore/7/88 (SN88), B/Shanghai/12/87 (SH87), B/Hong Kong/14/88 (B/HK/88), B/VI/89 (VI89), B/YM/88 (YM88), B/BJ/87 (BJ87) and B/TX/88 (TX88)(see Table 1) (Rota et al., 1990); B/Finland/149/90 (FI49), B/Finland/145/90 (FI45) (Kinnunen et al., 1992).
B/VI/87 were determined and compared to those of several other influenza type B viruses. As expected, few amino acid changes were detected over 10 years (Fig. 1). However, there were as many as 24 nucleotide changes between the HA2 domains of B/VI/87 and B/HK289. Phylogenetic analysis indicated that the HA2 domains of currently circulating B/YM/88- and B/VI/87-1ike viruses were also on separate lineages. For the 12-year period from 1979 to 1991, the nucleotide substitution rate for HA1 (0.236 + 0.04%/ year) was similar to the rate for HA2 (0.196 + 0.06%/
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year). However, the rate of amino acid change was 0-30~/year for HA1 compared to 0-056~/year for HA2. Although approximately 40~o of the nucleotide changes in HA1 coded for amino acid substitutions, only 8.7~ of the nucleotide changes in HA2 caused changes in the HA2 protein. The rate calculated for influenza B virus is only about 309/o less than that of influenza A viruses. However, when B/Lee/40 is used as the root virus, the rate that can be calculated from our data (0-11 ~/year) is similar to that previously described for influenza B virus ( Y a m a s h i t a et al., 1988; A i r et al., 1990). A l t h o u g h the presence of m u l t i p l e cocirculating v a r i a n t s of influenza B virus had b e e n d e m o n s t r a t e d (Lu et al., 1983; Oxford et al., 1984; Y a m a s h i t a et al., 1988; D o n a t e l l i et al., 1989; R o t a et al., 1990), our results i n d i c a t e d that two d i s t i n c t lineages of influenza B virus have persisted a n d have caused disease over a period of at least 4 years. I n the case of the type A viruses a single lineage of H A rapidly b e c a m e the d o m i n a n t e p i d e m i c v a r i a n t (Both et al., 1983; R a y m o n d et al., 1986; Cox et al., 1989). T h e a n t i g e n i c as well as genetic r e l a t i o n s h i p s a m o n g the currently circulating strains of influenza virus provide useful i n f o r m a t i o n for d e t e r m i n i n g the most a p p r o p r i a t e c o m p o s i t i o n for the influenza vaccine. T h e complexity of the e v o l u t i o n a r y p a t t e r n s of influenza type B m a k e s the choice of type B c o m p o n e n t difficult. Vaccines for use in 1991/1992 a n d 1992/1993 have b e e n u p d a t e d to include either B / P N / 9 0 or B / Y M / 8 8 as the type B c o m p o n e n t (World H e a l t h O r g a n i z a t i o n , 1991a, 1992). C o n t i n u e d surveillance of these viruses will be necessary to detect the e m e r g e n c e of n e w lineages o f virus with altered a n t i g e n i c properties a n d to ensure t h a t future vaccines c o n t a i n the most a p p r o p r i a t e strain of
virus. We are grateful to Brian Holloway, Edward George and Melissa Olsen-Rasmussen for the synthesis of the oligonucleotide sequencing primers, Sarah McKneally for assistance with computer analysis of sequence data and Victor Tsang for preparation of the monoclonal antibody used in neutralization tests, and to Nancy Cox for review of this manuscript. T.W. is a National Research Council Research Associate.
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(Received 27 March 1992; Accepted 15 June 1992)
